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nyse:gsk GlaxoSmithKline Apr 26th, 2022 12:00AM Feb 5th, 2019 12:00AM https://www.uspto.gov?id=US11312709-20220426 Ghrelin O-acyltransferase inhibitors This invention relates to novel compounds according to Formula (I) which are inhibitors of ghrelin O-acyltransferase (GOAT), to pharmaceutical compositions containing them, to processes for their preparation, and to their use in therapy for the treatment of metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.). 11312709 1. A compound according to Formula (I) or a pharmaceutically acceptable salt thereof: wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or C(═O)NH2; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. 2. The compound or pharmaceutically acceptable salt thereof according to claim 1, represented by Formula (II): wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or C(═O)NH2; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. 3. The compound or pharmaceutically acceptable salt thereof according to claim 1, represented by Formula (III): wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or —C(═O)NH2; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. 4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is hydrogen, chloro, cyano, methyl, —CF3, or —C(═O)NH2. 5. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is hydrogen. 6. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X is CH2. 7. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X is O. 8. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R2 is chloro. 9. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R3 is hydrogen, chloro, or fluoro. 10. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R3 is hydrogen. 11. The compound according to claim 1 which is: 2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid; (R)-2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid; or (S)-2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid; or a pharmaceutically acceptable salt thereof. 12. The compound according to claim 1 which is: or a pharmaceutically acceptable salt thereof. 13. The compound according to claim 1 which is: or a pharmaceutically acceptable salt thereof. 14. The compound according to claim 13 which is: 15. A combination of a compound or pharmaceutically acceptable salt thereof according claim 1 and at least one anti-adiposity agent or anti-adiposity therapy. 16. A pharmaceutical composition comprising the compound or pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutically acceptable excipient. 17. The pharmaceutical composition of claim 16, further comprising an additional pharmaceutical agent. 18. A method of treating Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus, type 2 diabetes mellitus, dysglycemia, hyperglycemia, obesity, increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia, atherogenic dyslipidemia, hepatic steatosis, non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof. 19. A method of treating obesity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof. 20. A method of treating Prader-Willi syndrome in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof. 20 RELATED APPLICATIONS The present application is a national stage filing under 35 U.S.C. § 371 of international PCT application, PCT/EP2019/052770, filed Feb. 5, 2019, which claims priority under 35 U.S.C. § 119(a) to Indian patent application, Application Number 201811004277, filed Feb. 5, 2018, the entire contents of each of which are incorporated herein by reference. FIELD OF THE INVENTION This invention relates to compounds which inhibit ghrelin O-acyltransferase (GOAT) and thus are useful for reducing appetite and adiposity, as well as improving energy balance and glycemic control. BACKGROUND OF THE INVENTION Ghrelin is a 28-amino acid gastric hormone produced primarily in the fundus of the stomach. Two forms of the hormone are found in circulation: unacylated ghrelin and acylated ghrelin. The lone enzyme known to perform this post-translational acylation on serine 3 of ghrelin is ghrelin O-acyltransferase (GOAT). There is no other known function of GOAT. Only acyl ghrelin is capable of interacting with its receptor, growth hormone secretagogue receptor 1 (GHSR1). Binding of acyl ghrelin to GHSR1 in the brain stimulates orexigenic activity and adiposity and reduces energy expenditure. When acyl ghrelin was administered to humans, appetite and food intake were increased (covered in Cummings, Physiology & Behavior 2006, 89, 71-84). Binding of acyl ghrelin to GHSR1 in pancreatic islet cells modulates insulin release. Acute administration of acyl ghrelin to humans led to significant reductions in plasma insulin and increased glucose levels (Broglio, J. Clin. Endocrinol. Metab. 2001, 86, 5083-5086). Levels of acylated ghrelin increase in anticipation of a meal and decrease post-prandially, leading acyl ghrelin to dubbed the “hunger hormone.” If increased levels of acyl ghrelin stimulate adiposity and adversely impact glycemic control, which could contribute to the development of the metabolic syndrome, then decreasing the amount of acyl ghrelin in circulation should do the opposite: reduce appetite and adiposity, improve energy balance, and benefit glycemic control, potentially ameliorating the metabolic syndrome. Inhibition of GOAT decreases acyl ghrelin production. Indeed, as reviewed by Ariyasu and Akamizu (Endocrine Journal 2015, 62(11), 953-963) mice with ghrelin, GHSR1, or GOAT knocked out demonstrate decreased food intake on a high fat diet and increased insulin secretion. When wild type mice were administered a peptide-based GOAT inhibitor, they demonstrated reduced weight gain and improved glucose tolerance (Barnett et al., Science 2010, 330 (6011), 1689-1692). In addition, it was recently reported that ghrelin deletion is protective against age-associated hepatic steatosis, suggesting a role for GOAT inhibition in the treatment of nonalcoholic steatohepatitis (NASH) (Guillory et al., Aging Cell 2017 published ahead of print 10.1111/ace1.12688). Thus, there is strong evidence to suggest that inhibition of GOAT decreases appetite and adiposity and improves glycemic control. Accordingly, compounds that inhibit GOAT activity would be useful for the treatment of obesity. SUMMARY OF THE INVENTION The present invention relates to compounds according to Formula (I) or pharmaceutically acceptable salts thereof. wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or —C(═O)NH2; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. Exemplary compounds of Formula (I) include, but are not limited to: and pharmaceutically acceptable salts thereof. Another aspect of this invention relates to a method of treating obesity. In particular, this invention relates to a method of treating obesity caused by Prader-Willi syndrome. Prader-Willi syndrome is a well-known genetic cause of obesity and is found in people of both sexes and in all races worldwide, particularly in children. Patients suffering from Prader-Willi syndrome experience hyperphagia and typically have trouble controlling their weight. Many complications of Prader-Willi syndrome are due to obesity. Another aspect of this invention relates to a method of treating metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.). Another aspect of the invention relates to pharmaceutical preparations comprising compounds of Formula (I) and pharmaceutically acceptable excipients. In another aspect, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for use in the treatment of a disorder mediated by GOAT, such as obesity. In another aspect, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for use in the treatment of a disorder mediated by GOAT, such as metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.). In another aspect, the invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in therapy. In another aspect, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of a disorder mediated by GOAT. In another aspect, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of obesity. In another aspect, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.). In another aspect, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of Prader-Willi syndrome. In another aspect, provided herein are methods of co-administering the presently invented compounds of Formula (I) with other active ingredients. In another aspect, there is provided a combination of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and at least one anti-adiposity agent or anti-adiposity therapy for use in the treatment of a disorder mediated by GOAT. In another aspect, there is provided a combination of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and at least one lifestyle modification (e.g., a reduced-calorie diet and/or exercise), weight loss agent (e.g., orlistat, lorcaserin, liraglutide, phentermine/topimarate, or naltrexone/bupropion), hormone therapy (e.g., testosterone, estrogen, progesterone, or human growth hormone), selective serotonin reuptake inhibitors (SSRIs), or anti-diabetic therapy (e.g., insulin, miglitol, acarbose, metformin, exenatide, pramlintide) for use in the treatment of a disorder mediated by GOAT. In another aspect, there is provided a combination of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and at least one anti-adiposity agent or anti-adiposity therapy for use in the treatment of obesity. In another aspect, there is provided a combination of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and at least one lifestyle modification (e.g., a reduced-calorie diet and/or exercise), weight loss agent (e.g., orlistat, lorcaserin, liraglutide, phentermine/topimarate, or naltrexone/bupropion), hormone therapy (e.g., testosterone, estrogen, progesterone, or human growth hormone), selective serotonin reuptake inhibitors (SSRIs), and/or anti-diabetic therapy (e.g., insulin, miglitol, acarbose, metformin, exenatide, pramlintide) for use in the treatment of metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.). In another aspect, the present disclosure provides pharmaceutical compositions or preparations including a compound described herein, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical compositions described herein include a therapeutically or prophylactically effective amount of a compound described herein. The pharmaceutical composition or preparation may be useful for treating and/or preventing a disease (e.g., metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.) in a subject in need thereof. The pharmaceutical composition or preparation may be useful for inhibiting the activity of GOAT in a subject, biological sample, tissue, or cell. In another aspect, the present disclosure provides pharmaceutical compositions or preparations including a compound described herein, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical compositions described herein include a therapeutically or prophylactically effective amount of a compound described herein. The pharmaceutical composition or preparation may be useful for treating metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.) in a subject in need thereof, or inhibiting the activity of GOAT in a biological sample, tissue, or cell. In another aspect, described herein are methods for treating and/or preventing a disease (e.g., metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.) in a subject, biological sample, tissue, or cell. Another aspect relates to methods of inhibiting the activity of GOAT using a compound described herein in a biological sample (e.g., cell, or tissue). In another aspect, described herein are methods of inhibiting the activity of GOAT using a compound described herein in a subject. In another aspect, the present disclosure provides compounds of Formula (I), and pharmaceutically acceptable salts thereof, for use in the treatment of a disease (e.g., metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.) in a subject, biological sample, tissue, or cell. Another aspect of the present disclosure relates to kits comprising a container with a compound, or pharmaceutical composition or preparation thereof, as described herein. The kits described herein may include a single dose or multiple doses of the compound or pharmaceutical composition or preparation. The kits may be useful in a method of the disclosure. In certain embodiments, the kit further includes instructions for using the compound or pharmaceutical composition or preparation. A kit described herein may also include information (e.g., prescribing information) as required by a regulatory agency, such as the U.S. Food and Drug Administration (FDA). The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, Examples, Figures, and Claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an X-ray powder diffraction pattern of the compound of Example 1a. FIG. 2 shows a differential scanning calorimetry trace of the compound of Example 1a and a thermogravimetric analysis trace of the compound of Example 1a. FIG. 3 shows the dose response of Example 1a on fasting induced acyl ghrelin levels. FIG. 4 shows the effect of Example 1a on fasting-induced acyl ghrelin levels in male SD rats. FIG. 5 shows the effect of Example 1a on fasting-induced des-acyl ghrelin levels in male SD rats. FIG. 6 shows acyl ghrelin reduction after a single 10 mg/kg dose in Cynomolgus monkeys. FIG. 7 shows the effect of Example 1a and Rimonabant on plasma acyl ghrelin levels in high fat high carbohydrate fed male C57BL/6 mice. FIG. 8 shows the effect of Example 1a and Rimonabant on plasma des-acyl ghrelin levels in high fat high carbohydrate fed male C57BL/6 mice. DETAILED DESCRIPTION OF THE INVENTION The present invention provides compounds that inhibit GOAT, and pharmaceutical compositions/preparations thereof, for the treatment of a disease in a subject. The present invention further provides methods of using the compounds described herein, e.g., as biological probes to study the inhibition of GOAT or ghrelin activity, and as therapeutics, e.g., in the treatment of diseases associated with GOAT activity. In certain embodiments, the diseases include, but are not limited to, metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.) in a subject, biological sample, tissue or cell. This invention relates to compounds of the Formula (I) as defined above, or pharmaceutically acceptable salts thereof. Formula (I) contains the substituent R1. In certain embodiments, R1 is H. In certain embodiments, R1 is halogen. In certain embodiments, R1 is Cl. In certain embodiments, R1 is —CN. In certain embodiments, R1 is —(C1-C4)alkyl. In certain embodiments, R1 is -Me. In certain embodiments, R1 is -Et. In certain embodiments, R1 is —Pr. In certain embodiments, R1 is -halo(C1-C4)alkyl. In certain embodiments, R1 is —CF3. In certain embodiments, R1 is —C(═O)NH2. In certain embodiments, R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, —C(═O)N(Ra)2, wherein Ra is hydrogen, substituted or unsubstituted C1-C6 alkyl, or a nitrogen protecting group. In certain embodiments, R1 is —C(═O)N(Ra)2. In certain embodiments, Ra is hydrogen. In certain embodiments, Ra is substituted or unsubstituted C1-C6 alkyl. In certain embodiments, Ra is -Me. In certain embodiments, Ra is -Et. In certain embodiments, Ra is a nitrogen protecting group. Formula (I) contains the substituent X. In certain embodiments, X is CH2. In certain embodiments, X is O. Formula (I) contains the substituent R2. In certain embodiments, R2 is halogen. In certain embodiments, R2 is —Cl. Formula (I) contains the substituent R3. In certain embodiments, R3 is H. In certain embodiments, R3 is halogen. In certain embodiments, R3 is F. In certain embodiments, R3 is Cl. In certain embodiments, X is CH2, and R1 is methyl. In certain embodiments, R1 is Me, R2 is Cl, and R3 is H. In certain embodiments, X is CH2, R1 is methyl, R2 is Cl, and R3 is H. Exemplary compounds of Formula (I) include, but are not limited to: Example Structure 1 2 3 4 5 6 7 8 9 and pharmaceutically acceptable salts thereof. In one embodiment, this invention relates to compounds of Formula (II) represented by Formula (II): wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or —C(═O)NH2; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. In certain embodiments, this invention relates to compounds of Formula (II), wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or —C(═O)N(Ra)2, wherein Ra is hydrogen, substituted or unsubstituted C1-C6 alkyl, or a nitrogen protecting group; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. Formula (II) contains the substituent R1. In certain embodiments, R1 is H. In certain embodiments, R1 is halogen. In certain embodiments, R1 is Cl. In certain embodiments, R1 is —CN. In certain embodiments, R1 is —(C1-C4)alkyl. In certain embodiments, R1 is -Me. In certain embodiments, R1 is -halo(C1-C4)alkyl. In certain embodiments, R1 is —CF3. In certain embodiments, R1 is —C(═O)NH2. In certain embodiments, R1 is —C(═O)N(Ra)2. In certain embodiments, Ra is hydrogen. In certain embodiments, Ra is substituted or unsubstituted C1-C6 alkyl. In certain embodiments, Ra is -Me. In certain embodiments, Ra is -Et. In certain embodiments, Ra is a nitrogen protecting group. Formula (II) contains the substituent X. In certain embodiments, X is CH2. In certain embodiments, X is O. Formula (II) contains the substituent R2. In certain embodiments, R2 is halogen. In certain embodiments, R2 is —Cl. Formula (II) contains the substituent R3. In certain embodiments, R3 is H. In certain embodiments, R3 is halogen. In certain embodiments, R3 is F. In certain embodiments, R3 is Cl. In certain embodiments, X is CH2 and R1 is methyl. In certain embodiments, R1 is Me, R2 is Cl, and R3 is H. In certain embodiments, X is CH2, R1 is methyl, R2 is Cl and R3 is H. Exemplary compounds of Formula (II) include, but are not limited to: Example Structure 1a 2a 3a 4a 5a 6a 7a 8a 9a and pharmaceutically acceptable salts thereof. In certain embodiments, Formula (II) is of the formula: In certain embodiments, Formula (II) is of the formula or a pharmaceutically acceptable salt thereof. In another embodiment, this invention relates to compounds of Formula (I) represented by Formula (III): wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or —C(═O)NH2; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. In another embodiment, this invention relates to compounds of Formula (III), wherein: R1 is hydrogen, halogen, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, or —C(═O)N(Ra)2, wherein Ra is hydrogen, substituted or unsubstituted C1-C6 alkyl, or a nitrogen protecting group; X is CH2 or O; R2 is halogen; and R3 is hydrogen or halogen. Formula (III) contains the substituent R1. In certain embodiments, R1 is H. In certain embodiments, R1 is halogen. In certain embodiments, R1 is Cl. In certain embodiments, R1 is —CN. In certain embodiments, R1 is —(C1-C4)alkyl. In certain embodiments, R1 is -Me. In certain embodiments, R1 is -halo(C1-C4)alkyl. In certain embodiments, R1 is —CF3. In certain embodiments, R1 is —C(═O)NH2. In certain embodiments, R1 is —C(═O)N(Ra)2. In certain embodiments, Ra is hydrogen. In certain embodiments, Ra is substituted or unsubstituted C1-C6 alkyl. In certain embodiments, Ra is -Me. In certain embodiments, Ra is -Et. In certain embodiments, Ra is a nitrogen protecting group. Formula (III) contains the substituent X. In certain embodiments, X is CH2. In certain embodiments, X is O. Formula (III) contains the substituent R2. In certain embodiments, R2 is halogen. In certain embodiments, R2 is —Cl. Formula (III) contains the substituent R3. In certain embodiments, R3 is H. In certain embodiments, R3 is halogen. In certain embodiments, R3 is F. In certain embodiments, R3 is Cl. In certain embodiments, X is CH2 and R1 is methyl. In certain embodiments, R1 is Me, R2 is Cl, and R3 is H. In certain embodiments, X is CH2, R1 is methyl, R2 is Cl and R3 is H. Exemplary compounds of Formula (III) include, but are not limited to: Example Structure 1b 2b 3b 4b 5b 6b 7b 8b 9b and pharmaceutically acceptable salts thereof. Specific compounds of this invention include: 2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (R)-2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; (S)-2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid; 2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid; (R)-2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid; and (S)-2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid; and pharmaceutically acceptable salts thereof. Typically, but not absolutely, the salts of the present invention are pharmaceutically acceptable salts. Salts of the disclosed compounds containing a basic amine or other basic functional group may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or the like. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, phenylacetates, phenylpropionates, phenylbutrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates mandelates, and sulfonates, such as xylenesulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates and naphthalene-2-sulfonates. Salts of the disclosed compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acid such as lysine and arginine. Other salts, which are not pharmaceutically acceptable, may be useful in the preparation, isolation, or storage of the compounds of this invention, and these should be considered to form a further aspect of the invention. These salts, such as oxalic or trifluoroacetate, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. The compound of Formula (I) may exist in a crystalline or noncrystalline form, or as a mixture thereof. The skilled artisan will appreciate that pharmaceutically acceptable solvates may be formed for crystalline or non-crystalline compounds. In crystalline solvates, solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve non-aqueous solvents such as, but not limited to, ethanol, isopropanol, DMSO, acetic acid, ethanolamine, or ethyl acetate, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent incorporated into the crystalline lattice are typically referred to as “hydrates.” Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water. The invention includes all such solvates. The skilled artisan will further appreciate that the compounds of the invention that exist in crystalline form, including the various solvates thereof, may exhibit polymorphism (i.e. the capacity to occur in different crystalline structures). These different crystalline forms are typically known as “polymorphs.” The invention includes all such polymorphs. Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, which may be used for identification. The skilled artisan will appreciate that different polymorphs may be produced, for example, by changing or adjusting the reaction conditions or reagents, used in making the compound. For example, changes in temperature, pressure, or solvent may result in polymorphs. In addition, one polymorph may spontaneously convert to another polymorph under certain conditions. The present invention is further directed to crystalline forms of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid. In some embodiments, a crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern comprising at least nine diffraction angles, when measured using Cu Kα radiation, selected from a group consisting of about 5.9, 13.6, 14.0, 14.3, 21.9, 22.5, 23.1, 23.3, 24.1, 24.5, 24.7, 25.7, 26.1, 26.6, and 27.4 degrees 2θ. In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern comprising at least eight diffraction angles or at least seven diffraction angles or at least six diffraction angles or at least five diffraction angles or at least four diffraction angles, when measured using Cu Kα radiation, selected from a group consisting of about 5.9, 13.6, 14.0, 14.3, 21.9, 22.5, 23.1, 23.3, 24.1, 24.5, 24.7, 25.7, 26.1, 26.6, and 27.4 degrees 2θ. In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern comprising at least three diffraction angles, when measured using Cu Kα radiation, selected from a group consisting of about 5.9, 13.6, 14.0, 14.3, 21.9, 22.5, 23.1, 23.3, 24.1, 24.5, 24.7, 25.7, 26.1, 26.6, and 27.4 degrees 2θ. In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern comprising diffraction angles, when measured using Cu Kα radiation, of about 5.9, 13.6, 14.0, 14.3, 23.3, 24.5, and 27.4 degrees 2θ. In yet another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern substantially in accordance with FIG. 1. In further embodiments, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by a differential scanning calorimetry trace substantially in accordance with FIG. 2 and/or a thermogravimetric analysis trace substantially in accordance with FIG. 2. In still further embodiments, as a person having ordinary skill in the art will understand, (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by any combination of the analytical data characterizing the aforementioned embodiments. For example, in one embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern substantially in accordance with FIG. 1 and a differential scanning calorimetry trace substantially in accordance with FIG. 2 and a thermogravimetric analysis trace substantially in accordance with FIG. 2. In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern substantially in accordance with FIG. 1 and a differential scanning calorimetry trace substantially in accordance with FIG. 2 In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern substantially in accordance with FIG. 1 and a thermogravimetric analysis trace substantially in accordance with FIG. 2. In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern comprising diffraction angles, when measured using Cu Kα radiation, of about 5.9, 13.6, 14.0, 14.3, 23.3, 24.5, and 27.4 degrees 2θ, and a differential scanning calorimetry trace substantially in accordance with FIG. 2. In another embodiment, the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is characterized by an X-ray powder diffraction (XRPD) pattern comprising diffraction angles, when measured using Cu Kα radiation, of about 5.9, 13.6, 14.0, 14.3, 23.3, 24.5, and 27.4 degrees 2θ, and a thermogravimetric analysis trace substantially in accordance with FIG. 2. An XRPD pattern will be understood to comprise a diffraction angle (expressed in degrees 2θ) of “about” a value specified herein when the XRPD pattern comprises a diffraction angle within ±0.3 degrees 2θ of the specified value. Further, it is well known and understood to those skilled in the art that the apparatus employed, humidity, temperature, orientation of the powder crystals, and other parameters involved in obtaining an X-ray powder diffraction (XRPD) pattern may cause some variability in the appearance, intensities, and positions of the lines in the diffraction pattern. An X-ray powder diffraction pattern that is “substantially in accordance” with that of FIG. 1 provided herein is an XRPD pattern that would be considered by one skilled in the art to represent a compound possessing the same crystal form as the compound that provided the XRPD pattern of FIG. 1. That is, the XRPD pattern may be identical to that of FIG. 1, or more likely it may be somewhat different. Such an XRPD pattern may not necessarily show each of the lines of any one of the diffraction patterns presented herein, and/or may show a slight change in appearance, intensity, or a shift in position of said lines resulting from differences in the conditions involved in obtaining the data. A person skilled in the art is capable of determining if a sample of a crystalline compound has the same form as, or a different form from, a form disclosed herein by comparison of their XRPD patterns. For example, one skilled in the art can overlay an XRPD pattern of a sample of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid, with FIG. 1 and, using expertise and knowledge in the art, readily determine whether the XRPD pattern of the sample is substantially in accordance with the XRPD pattern of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid disclosed herein. If the XRPD pattern is substantially in accordance with FIG. 1, the sample form can be readily and accurately identified as having the same form as the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid disclosed herein. The compound of Formula (I) or a salt thereof may exist in stereoisomeric forms (e.g., it contains one or more asymmetric carbon atoms). The individual stereoisomers (enantiomers and diastereomers) and mixtures of these are included within the scope of the present invention. It is to be understood that the present invention includes all combinations and subsets of the particular groups defined hereinabove. The scope of the present invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerically enriched mixtures. It is to be understood that the present invention includes all combinations and subsets of the particular groups defined hereinabove. The subject invention also includes isotopically-labeled compounds, which are identical to those recited in Formula (I) and following, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, chlorine, and iodine, such as 2H, 3H, 11C, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, 123I, and 125I. Compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. 11C and 18F isotopes are particularly useful in PET (positron emission tomography), and 125I isotopes are particularly useful in SPECT (single photon emission computerized tomography), all useful in brain imaging. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of Formula (I) and following of this invention can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. The invention further provides a pharmaceutical composition (also referred to as a pharmaceutical formulation) comprising a compound of Formula (I) or pharmaceutically acceptable salt thereof and one or more excipients (also referred to as carriers and/or diluents in the pharmaceutical arts). The excipients are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof (i.e., the patient). Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the compound or compounds of the invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance. Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation. Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention. In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press). The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company). Pharmaceutical compositions may be in unit dose form containing a predetermined amount of active ingredient per unit dose. Such a unit may contain a therapeutically effective dose of the compound of Formula (I) or salt thereof or a fraction of a therapeutically effective dose such that multiple unit dosage forms might be administered at a given time to achieve the desired therapeutically effective dose. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Furthermore, such pharmaceutical compositions may be prepared by any of the methods well-known in the pharmacy art. In the present invention, tablets and capsules are preferred for delivery of the pharmaceutical composition. In accordance with another aspect of the invention there is provided a process for the preparation of a pharmaceutical composition comprising mixing (or admixing) a compound of Formula (I) or salt thereof with at least one excipient. The present invention also provides a method of treatment in a mammal, especially a human. The compounds and compositions of the invention are used to treat GOAT mediated disorders or diseases. Disease states or disorders which can be treated by the methods and compositions provided herein include, but are not limited to, obesity The present invention also provides a method of treatment in a subject (e.g., a mammal, especially a human). Disease states or disorders which can be treated by the methods and compositions or preparations provided herein include, but are not limited to, metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.). The compositions and methods provided herein are particularly deemed useful for the treatment of GOAT mediated disorders, such as obesity, increased adiposity, poor glycemic control, etc. The compositions and methods provided herein are particularly deemed useful for the treatment of GOAT mediated disorders, such as metabolic disorders (e.g. Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))), psychiatric disorders (e.g., eating disorders (e.g., bulimia nervosa, binge eating disorder, night-time eating syndrome), substance related disorders (e.g., addiction disorders (e.g., alcohol, smoking, overeating, or use of illicit drugs))), as well as disorders related to or complications of metabolic or psychiatric disorders (e.g., cardiovascular diseases (e.g., diabetic heart disease (e.g., diabetic cardiomyopathy), heart failure, or hypertension), ischemia (e.g., myocardial ischemia, cerebral ischemia, ischemic stroke), or BMI-related cancers (e.g., pancreatic cancer, gallbladder cancer, esophageal cancer, colorectal cancer, breast cancer etc.)). More particularly, diseases or disorders that may be treated by the compositions and methods of the invention include Prader-Willi syndrome, excess weight, and/or obesity (e.g., obesity caused by Prader-Willi syndrome). Weight that is higher than what is considered as a healthy weight for a given height is considered overweight or obese. In one embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 25. In one embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 26. In one embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 27. In one embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 28. In one embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 29. In one embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 30. In another embodiment, a compound of the invention is administered to a human having a body mass index (BMI) of at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, or at least about 40. In one embodiment, the obesity is extreme or severe obesity. In a particular embodiment, the obesity is caused by Prader-Willi syndrome. The instant compounds can be combined with or co-administered with other therapeutic agents, particularly agents that may enhance the activity or time of disposition of the compounds. Combination therapies according to the invention comprise the administration of at least one compound of the invention and the use of at least one other treatment method. In one embodiment, combination therapies according to the invention comprise the administration of at least one compound of the invention and surgical therapy, such as bariatric surgery. In one embodiment, combination therapies according to the invention comprise the administration of at least one compound of the invention and lifestyle modification. Lifestyle modification can include, for example, a reduced-calorie diet and/or exercise. In one embodiment, combination therapies according to the invention comprise the administration of at least one compound of the invention and a weight-loss agent, such as orlistat, lorcaserin, liraglutide, phentermine/topimarate, or naltrexone/bupropion. In one embodiment, combination therapies according to the invention comprise the administration of at least one compound of the invention and a hormone therapy (e.g., testosterone, estrogen, progesterone, or human growth hormone), selective serotonin reuptake inhibitors (SSRIs), or anti-diabetic therapy (e.g., insulin, miglitol, acarbose, metformin, exenatide, pramlintide). In yet another embodiment, the invention comprises a therapeutic regimen where the GOAT inhibitors of this disclosure are not in and of themselves active or significantly active, but when combined with another therapy, which may or may not be active as a standalone therapy, the combination provides a useful therapeutic outcome. By the term “co-administration” and derivatives thereof as used herein refers to either simultaneous administration or any manner of separate sequential administration of a GOAT inhibiting compound, as described herein, and a further active ingredient or ingredients, known to be useful in the treatment of obesity, including orlistat, lorcaserin, liraglutide, phentermine/topimarate, and naltrexone/bupropion, or a hormone therapy (e.g., testosterone, estrogen, progesterone, or human growth hormone), selective serotonin reuptake inhibitors (SSRIs), or anti-diabetic therapy (e.g., insulin, miglitol, acarbose, metformin, exenatide, pramlintide). The term further active ingredient or ingredients, as used herein, includes any compound or therapeutic agent known to or that demonstrates advantageous properties when administered to a patient in need of treatment for obesity. Preferably, if the administration is not simultaneous, the compounds are administered in a close time proximity to each other. Furthermore, it does not matter if the compounds are administered in the same dosage form, e.g. one compound may be administered topically and another compound may be administered orally. Typically, any weight loss agent may be co-administered in the treatment of obesity in the present invention. Typically, any weight loss agent, hormone therapy, or anti-diabetic therapy may be co-administered in the methods and uses of the present invention. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the obesity involved. Typical weight loss agents useful in the present invention include, but are not limited to, appetite-suppressing agents and lipase inhibitors. Examples of a further active ingredient or ingredients for use in combination or co-administered with the present GOAT inhibiting compounds are weight-loss agents. Examples of weight-loss agents include, but are not limited to, orlistat, lorcaserin, liraglutide, phentermine/topimarate, and naltrexone/bupropion. Orlistat is a lipase inhibitor which prevents some of the fat in foods eaten from being absorbed in the intestines. The unabsorbed fat is removed from the body in the stool. Lorcaserin (BELVIQ) is a serotonin receptor agonist. Lorcaserin targets the 5HT2C receptor and alters body weight by regulating satiety. Liraglutide (SAXENDA) is a glucagonlike peptide-1 (GLP-1) receptor agonist. Liraglutide is an anti-diabetic agent that has been approved for weight loss. Phentermine/Topimarate (QYSMIA) is a combination product. Phentermine is an anorectic and topiramate is an anticonvulsant. Phentermine/Topimarate decreases appetite and causes feelings of fullness to last longer after eating. Naltrexone/Bupropion (CONTRAVE) is a combination product. Naltrexone is an opiate antagonist and Bupropion is an antidepressant. Naltrexone/Bupropion regulates brain activity to reduce appetite. Pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Such a unit may contain, for example, 0.5 mg to 1 g, preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg of a compound of the Formula (I), depending on the condition being treated, the route of administration and the age, weight and condition of the patient, or pharmaceutical compositions may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage compositions are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Furthermore, such pharmaceutical compositions may be prepared by any of the methods well known in the pharmacy art. Pharmaceutical compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association a compound of formula (I) with the carrier(s) or excipient(s). Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages. Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of a compound of Formula (I). Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added. Where appropriate, dosage unit pharmaceutical compositions for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like. Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or as enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. It should be understood that in addition to the ingredients particularly mentioned above, the pharmaceutical compositions may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. A therapeutically effective amount of a compound of the present invention will depend upon a number of factors including, for example, the age and weight of the intended recipient, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant prescribing the medication. However, an effective amount of a compound of Formula (I) for the treatment of obesity will generally be in the range of 0.001 to 100 mg/kg body weight of recipient per day, suitably in the range of 0.01 to 10 mg/kg body weight per day. For a 70 kg adult mammal, the actual amount per day would suitably be from 7 to 700 mg and this amount may be given in a single dose per day or in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt or solvate, etc., may be determined as a proportion of the effective amount of the compound of Formula (I) per se. It is envisaged that similar dosages would be appropriate for treatment of the other conditions referred to above. In certain embodiments, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid wherein at least 10% by weight of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is present as the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid described herein. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid wherein at least 20% by weight, or at least 30% by weight, or at least 40% by weight, or at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is present as the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid described herein. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid wherein at least 95% by weight, or at least 96% by weight, or at least 97% by weight, or at least 98% by weight, or at least 99% by weight, or at least 99.5% by weight, or at least 99.8% by weight, or at least 99.9% by weight of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is present as the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid described herein. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid, wherein not more than 90% by weight of the compound is amorphous. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid, wherein not more than 80% by weight, or not more than 70% by weight, or not more than 60% by weight, or not more than 50% by weight, or not more than 40% by weight, or not more than 30% by weight, or not more than 20% by weight, or not more than 10% by weight of the compound is amorphous. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid, wherein not more than 5% by weight, or not more than 4% by weight, or not more than 3% by weight, or not more than 2% by weight, or not more than 1% by weight, or not more than 0.5% by weight, or not more than 0.2% by weight, or not more than 0.1% by weight of the compound is amorphous. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid wherein not more than 90% by weight of the (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is present in a form other than the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid described herein. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid wherein not more than 80% by weight, or not more than 70% by weight, or not more than 60% by weight, or not more than 50% by weight, or not more than 40% by weight, or not more than 30% by weight, or not more than 20% by weight, or not more than 10% by weight of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is present in a form other than the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid described herein. In another embodiment, this invention relates to a pharmaceutical composition comprising (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid wherein not more than 5% by weight, or not more than 4% by weight, or not more than 3% by weight, or not more than 2% by weight, or not more than 1% by weight, or not more than 0.5% by weight, or not more than 0.2% by weight, or not more than 0.1% by weight of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid is present in a form other than the crystalline form of (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid described herein. Definitions Terms are used within their accepted meanings. The following definitions are meant to clarify, but not limit, the terms defined. As used herein, the term “alkyl” represents a saturated, straight or branched hydrocarbon moiety having the specified number of carbon atoms. The term “(C1-C4)alkyl” refers to an alkyl moiety containing from 1 to 4 carbon atoms. Exemplary alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, and t-butyl. The term “halo(C1-C4)alkyl” is intended to mean a radical having one or more halogen atoms, which may be the same or different, at one or more carbon atoms of an alkyl moiety containing from 1 to 4 carbon atoms, which is a straight or branched-chain carbon radical. Examples of “halo(C1-C4)alkyl” groups useful in the present invention include, but are not limited to, —CF3 (trifluoromethyl), —CCl3 (trichloromethyl), 1,1-difluoroethyl, 2-fluoro-2-methylpropyl, 2,2-difluoropropyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl. The terms “halogen” and “halo” represent fluoro, chloro, bromo, or iodo substituents. As used herein, the term “cyano” refers to the group —CN. In certain embodiments, the substituent present on the nitrogen atom is an nitrogen protecting group (also referred to herein as an “amino protecting group”). Nitrogen protecting groups include, but are not limited to, —OH, —ORaa, —N(Rcc)2, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRcc)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as described herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by reference. For example, nitrogen protecting groups such as amide groups (e.g., —C(═O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide. Nitrogen protecting groups such as carbamate groups (e.g., —C(═O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate. Nitrogen protecting groups such as sulfonamide groups (e.g., —S(═O)2Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide. Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys). In certain embodiments, a nitrogen protecting group is benzyl (Bn), tert-butyloxycarbonyl (BOC), carbobenzyloxy (Cbz), 9-flurenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethyloxycarbonyl (Troc), triphenylmethyl (Tr), tosyl (Ts), brosyl (Bs), nosyl (Ns), mesyl (Ms), triflyl (Tf), or dansyl (Ds). “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the term “pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the subject compound. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, phenylacetates, phenylpropionates, phenylbutrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates mandelates, and sulfonates, such as xylenesulfonates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates and naphthalene-2-sulfonates. Salts of the disclosed compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acid such as lysine and arginine. As used herein, the term “compound(s) of the invention” means a compound of Formula (I) (as defined above) in any form, i.e., any salt or non-salt form (e.g., as a free acid or base form, or as a pharmaceutically acceptable salt thereof) and any physical form thereof (e.g., including non-solid forms (e.g., liquid or semi-solid forms), and solid forms (e.g., amorphous or crystalline forms, specific polymorphic forms, solvates, including hydrates (e.g., mono-, di- and hemi-hydrates)), and mixtures of various forms. As used herein, the terms “treatment”, “treat,” and “treating” refer to reversing, alleviating the specified condition, eliminating or reducing one or more symptoms of the condition, delaying the onset of, slowing or eliminating the progression of the condition, and delaying the reoccurrence of a condition in a previously afflicted or diagnosed patient or subject. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., paediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog). The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease. The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. The terms “condition,” “disease,” and “disorder” are used interchangeably. As used herein, the term “effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. The term “therapeutically effective amount” of a compound described herein is any amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition as compared to a corresponding subject who has not received such amount, resulting in improved treatment, healing, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts of a compound of Formula (I), as well as salts thereof, may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition or preparation. Additionally, the active ingredient or salt thereof may be presented as a pharmaceutical composition or preparation. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibition of GOAT in a subject, biological sample, tissue, or cell. As used herein the term “inhibit” or “inhibition” in the context of proteins, for example, in the context of GOAT, refers to a reduction in the activity of the enzyme. In some embodiments, the term refers to a reduction of the level of activity, e.g., GOAT activity, to a level that is statistically significantly lower than an initial level, which may, for example, be a baseline level of activity. In some embodiments, the term refers to a reduction of the level of enzyme activity, e.g., GOAT activity, to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of enzyme activity. Pharmaceutical compositions or preparations described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit. The term “metabolic disorder” refers to any disorder that involves an alteration in the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination thereof. A metabolic disorder is associated with either a deficiency or excess in a metabolic pathway resulting in an imbalance in metabolism of nucleic acids, proteins, lipids, and/or carbohydrates. Factors affecting metabolism include, and are not limited to, the endocrine (hormonal) control system (e.g., the insulin pathway, the enteroendocrine hormones including GLP-1, PYY or the like), the neural control system (e.g., GLP-1 in the brain), or the like. Examples of metabolic disorders include, but are not limited to, Prader-Willi syndrome, metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, diabetes mellitus (e.g., type II diabetes mellitus), dysglycemia (e.g., hyperglycemia), obesity (e.g., obesity caused by Prader-Willi syndrome), increased adiposity, poor glycemic control, hyperphagia, impaired satiety, dyslipidemia (e.g., atherogenic dyslipidemia), hepatic steatosis (e.g., non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis))). A “diabetic condition” refers to diabetes and pre-diabetes. Diabetes refers to a group of metabolic diseases in which a person has high blood sugar, either because the body does not produce enough insulin, or because cells do not respond to the insulin that is produced. This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger). There are several types of diabetes. Type I diabetes results from the body's failure to produce insulin, and presently requires the person to inject insulin or wear an insulin pump. Type II diabetes results from insulin resistance a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency. Gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes develop a high blood glucose level. Other forms of diabetes include congenital diabetes, which is due to genetic defects of insulin secretion, cystic fibrosis-related diabetes, steroid diabetes induced by high doses of glucocorticoids, and several forms of monogenic diabetes, e.g., mature onset diabetes of the young (e.g., MODY 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Pre-diabetes indicates a condition that occurs when a person's blood glucose levels are higher than normal but not high enough for a diagnosis of diabetes. All forms of diabetes increase the risk of long-term complications. These typically develop after many years, but may be the first symptom in those who have otherwise not received a diagnosis before that time. The major long-term complications relate to damage to blood vessels. Diabetes doubles the risk of cardiovascular disease and macrovascular diseases such as ischemic heart disease (angina, myocardial infarction), stroke, and peripheral vascular disease. Diabetes also causes microvascular complications, e.g., damage to the small blood vessels. Diabetic retinopathy, which affects blood vessel formation in the retina of the eye, can lead to visual symptoms, reduced vision, and potentially blindness. Diabetic nephropathy, the impact of diabetes on the kidneys, can lead to scarring changes in the kidney tissue, loss of small or progressively larger amounts of protein in the urine, and eventually chronic kidney disease requiring dialysis. Diabetic neuropathy is the impact of diabetes on the nervous system, most commonly causing numbness, tingling and pain in the feet and also increasing the risk of skin damage due to altered sensation. Together with vascular disease in the legs, neuropathy contributes to the risk of diabetes-related foot problems, e.g., diabetic foot ulcers, that can be difficult to treat and occasionally require amputation. The term “psychiatric disorder” refers to a disease of the mind and includes diseases and disorders listed in the Diagnostic and Statistical Manual of Mental Disorders—Fourth Edition (DSM-IV), published by the American Psychiatric Association, Washington D.C. (1994). Psychiatric disorders include, but are not limited to, eating disorders (e.g., night eating syndrome), substance-related disorders (e.g., alcohol dependence, amphetamine dependence, cannabis dependence, cocaine dependence, hallucinogen dependence, inhalant dependence, nicotine dependence, opioid dependence, phencyclidine dependence, and sedative dependence). An “obesity-related condition” includes, but is not limited to, Prader-Willi syndrome, obesity, undesired weight gain (e.g., from medication-induced weight gain, from cessation of smoking) and an over-eating disorder (e.g., binge eating, bulimia, compulsive eating, or a lack of appetite control each of which can optionally lead to undesired weight gain or obesity). “Obesity” and “obese” refers to class I obesity, class II obesity, class III obesity, and pre-obesity (e.g., being “over-weight”) as defined by the World Health Organization. Reduction of storage fat is expected to provide various primary and/or secondary benefits in a subject (e.g., in a subject diagnosed with a complication associated with obesity) such as, for example, an increased insulin responsiveness (e.g., in a subject diagnosed with Type II diabetes mellitus); a reduction in elevated blood pressure; a reduction in elevated cholesterol levels; and/or a reduction (or a reduced risk or progression) of ischemia (e.g., ischemic heart disease, cerebral ischemia, or ischemic stroke) arterial vascular disease, angina, myocardial infarction, stroke, migraines, congestive heart failure, deep vein thrombosis, pulmonary embolism, gall stones, gastroesophagael reflux disease, obstructive sleep apnea, obesity hypoventilation syndrome, asthma, gout, poor mobility, back pain, erectile dysfunction, urinary incontinence, liver injury (e.g., fatty liver disease, liver cirrhosis, alcoholic cirrhosis, endotoxin mediated liver injury) or chronic renal failure. Thus, the method of this invention is applicable to obese subjects, diabetic subjects, and alcoholic subjects. Abbreviations Compound Preparation AcOEt ethyl acetate AcOH acetic acid ADDP 1,1′-(azodicarbonyl)dipiperidine Ar Ar gas aq aquaeous BBr3 boron tribromide Boc tert-butyloxycarbonyl Bu4NCl tetrabutylammonium chloride CDCl3 deuterochloroform CHAPS 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate hydrate CH3CN acetonitrile Cs2CO3 cesium carbonate DCE 1,2-dichloroethane DCM dichloromethane DIAD diisopropyl azodicarboxylate DIPEA N,N-diisopropylethylamine DM water demineralized water DMA dimethylacetamide DME 1,2-dimethoxyethane DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DMPU N,N′-dimethylpropylene urea EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA ethylenediaminetetraacetic acid ES electrospray Et3N triethylamine Et2O diethyl ether EtOAc ethyl acetate EtOH ethanol h hour(s) H2 hydrogen gas HCl hydrochloric acid H2O water HOAt 1-hydroxy-7-azabenzotriazole H2SO4 sulfuric acid HPLC high-performance liquid chromatography HTRF homogeneous time resolved fluorescence KOtBu potassium tert-butoxide K2CO3 potassium carbonate KMnO4 potassium permanganate LCMS liquid chromatography mass spectrometry LiAlH4 lithium aluminum hydride LiOH lithium hydroxide MeI methyl iodide MeOH methanol MeSO3H methanesulfonic acid MgSO4 magnesium sulfate MOPS 3-(N-morpholino)propanesulfonic acid min minute(s) M molar MS mass spectrometry MTBE methyl tert-butyl ether N normal N2 nitrogen gas NaBH4 sodium borohydride NaBH(OAc)3 sodium triacetoxyborohydride Na2CO3 sodium carbonate NaH sodium hydride NaHCO3 sodium bicarbonate NaHMDS sodium bis(trimethylsilyl)amide NaOH sodium hydroxide NaOMe sodium methoxide Na2SO4 sodium sulfate (n-Bu)3P tri-n-butylphosphine NBS N-bromosuccinimide NH4Cl ammonium chloride NH4OAc ammonium acetate NH4OH ammonium hydroxide NMM N-methylmorpholine Pd-C palladium on carbon [PdCl(allyl)]2 allylpalladium(II) chloride dimer Pd(OAc)2 palladium(II) acetate Pd(PPh3)4 tetrakis(triphenylphosphine)palladium(0) Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0) PLM polarized light microscopy Pet ether petroleum ether P(o-to1)3 tri(o-tolyl)phosphine POBr3 phosphorus(V) oxybromide RB round bottom RT or r.t. room temperature RuCl[R,R)- [N-[(1R,2R)-2-(Amino-κN)-1,2- diphenylethyl]- Tsdpen](mesitylene) 4-methylbenzenesulfonamidato-κN]chloro [(1,2,3,4,5,6-η)-1,3,5-trimethylbenzene]- ruthenium sat. saturated SFC supercritical fluid chromatography SOCl2 thionyl chloride tBuOH tert-butanol TBME tert-butyl methyl ether TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TRF time resolved fluorescence xantphos 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene Zn(CN)2 zinc cyanide Generic Synthesis Schemes The compounds of this invention may be made by a variety of methods, including well-known standard synthetic methods. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the working examples. The skilled artisan will appreciate that if a substituent described herein is not compatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group that is stable to the reaction conditions. The protecting group may be removed at a suitable point in the reaction sequence to provide a desired intermediate or target compound. In all of the schemes described below, protecting groups for sensitive or reactive groups are employed where necessary in accordance with general principles of synthetic chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T. W. Green and P. G. M. Wuts, (1991) Protecting Groups in Organic Synthesis, John Wiley & Sons, incorporated by reference with regard to protecting groups). These groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art. The selection of processes as well as the reaction conditions and order of their execution shall be consistent with the preparation of compounds of the present invention. Starting materials are commercially available or are made from commercially available starting materials using methods known to those skilled in the art. Certain compounds of Formula (I) can be prepared according to Scheme-10 or analogous methods. Alkylation of a substituted 6-hydroxybenzo[b]thiophene with an optionally substituted 5-halo-6,7-dihydro-5H-cyclopenta[b]pyridine or an optionally substituted 3-chloro-2,3-dihydrofuro[2,3-b]pyridine is followed by saponification of the intermediate ester to afford compounds of Formula (I). Certain compounds of Formula (I) can be prepared according to Scheme-11 or analogous methods. A Mitsunobu reaction involving a substituted 6-hydroxybenzo[b]thiophene and an optionally substituted 6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol or an optionally substituted 2,3-dihydrofuro[2,3-b]pyridin-3-ol is followed by saponification of the intermediate ester to afford compounds of Formula (I). Certain compounds of Formula (I) can be prepared according to Scheme-12 or analogous methods. Alkylation of a substituted 6-hydroxybenzo[b]thiophene with 2,5-dihalo-6,7-dihydro-5H-cyclopenta[b]pyridine or 3,6-dihalo-2,3-dihydrofuro[2,3-b]pyridine followed by a palladium-mediated cyanation provides the nitrile. Saponification of the intermediate ester followed by hydrolysis of the nitrile affords compounds of Formula (I). EXPERIMENTALS Intermediates a) (3-Chloro-5-methoxyphenyl)(4-methoxybenzyl)sulfane To a mixture of DIPEA (369 mL, 2113 mmol), xantphos (20.38 g, 35.2 mmol), 1-bromo-3-chloro-5-methoxybenzene (156 g, 704 mmol), (4-methoxyphenyl)methanethiol (109 g, 704 mmol) in toluene (500 mL) was added Pd2(dba)3 (32.2 g, 35.2 mmol) at room temperature and reaction mixture was refluxed for 5 h. After cooling, water was added to the mixture and extracted with EtOAc. The organic layer was separated, washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane) to give the title compound (yield 170.0 g) as a pale yellow liquid. 1H NMR (400 MHz, CDCl3) δ 7.25-7.22 (m, 2H), 6.87-6.82 (m, 3H), 6.69-6.67 (m, 2H), 4.08 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H). LCMS (ES) m/z 293 [M+H]+. b) 3-Chloro-5-methoxybenzenethiol TFA (180 ml, 2336 mmol) was added to the solution of (3-chloro-5-methoxyphenyl)(4-methoxybenzyl)sulfane (180 g, 611 mmol) in anisole (180 mL) at 0° C. The reaction was stirred at 85° C. for 2 h under nitrogen atmosphere. Reaction mixture was quenched with 6N NaOH solution and extracted with EtOAc. The aqueous layer was acidified with 2N HCl and extracted with EtOAc. The EtOAc layer was washed with water and brine, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated under reduced pressure to get crude residue. The residue was purified by silica gel column chromatography (EtOAc/hexane) to give the title compound (yield 80.0 g) as pale yellow liquid. LCMS (ES) m/z 172.88 [M+H]+. c) Ethyl 4-((3-chloro-5-methoxyphenyl)thio)-3-oxobutanoate To a mixture of 3-chloro-5-methoxybenzenethiol (80 g, 458 mmol) and dry DMF (500 mL) were added K2CO3 (63.3 g, 458 mmol) and ethyl 4-chloro-3-oxobutanoate (75 g, 458 mmol) at 0° C. The mixture was stirred at room temperature for 3 h. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed successively with water and brine, dried over MgSO4, and concentrated in vacuo to get crude product (100 g). This was used for the next step without any further purification. d) Ethyl 2-(4-chloro-6-methoxybenzo[b]thiophen-3-yl)acetate and Ethyl 2-(6-chloro-4-methoxybenzo[b]thiophen-3-yl)acetate To ethyl 4-((3-chloro-5-methoxyphenyl)thio)-3-oxobutanoate (100 g, 330 mmol) was added methanesulfonic acid (500 mL) at 0° C. The mixture was stirred at 0° C. under nitrogen atmosphere for 15 min. The mixture was poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane) to give the mixture of isomers (60 g, ratio 2.5:1) as colorless oil and used for the next step. LCMS (ES) m/z 285.16 [M+H]+. e) Ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate and ethyl 2-(6-chloro-4-hydroxybenzo[b]thiophen-3-yl)acetate To a solution of ethyl 2-(4-chloro-6-methoxybenzo[b]thiophen-3-yl)acetate (60 g, 211 mmol) in DCM (500 mL) was added BBr3 (29.9 mL, 316 mmol) at 0° C. The mixture was warmed to room temperature and continued stirring for 6 h at RT. The mixture was quenched with water and NaHCO3 solution, and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane) to give 27 g of ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate) as white solid and 5.8 g of ethyl 2-(6-chloro-4-hydroxybenzo[b]thiophen-3-yl)acetate). Ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (Desired Compound): 1H NMR (300 MHz, CDCl3) δ 7.07 (s, 1H), 6.95 (d, J=2.4 Hz, 1H), 6.77 (d, J=2.4 Hz, 1H), 5.72 (s, 1H), 4.24 (q, J=9.2 Hz, 2H), 4.08 (s, 2H), 1.30 (t, J=6.9 Hz, 3H). LCMS (ES) m/z 271.12 (M+H)+. Ethyl 2-(6-chloro-4-hydroxybenzo[b]thiophen-3-yl)acetate (regioisomer Compound): 1H NMR (300 MHz, CDCl3) δ 7.07 (s, 1H), 7.01 (d, J=0.9 Hz, 1H), 6.81 (d, J=1.2 Hz, 1H), 5.45 (s, 1H), 4.23 (q, J=7.2 Hz, 2H), 4.08 (s, 2H), 1.29 (t, J=7.2 Hz, 3H). LCMS (ES) m/z 270.93 (M+H)+. a) 1-Bromo-2,5-dichloro-3-methoxybenzene To a solution of potassium KO′Bu (20.7 g, 185 mmol)) suspended in toluene (270 mL) and DMPU (90 mL, 746 mmol) was added methanol (30 mL). The mixture was placed in an oil bath at 80° C. under N2 with a reflux condenser for 25 minutes to obtain a solution. The solution was then allowed to cool to room temperature under N2, after which 1-bromo-2,5-dichloro-3-fluorobenzene (15 g, 61.5 mmol) was added dropwise to the solution and the resulting suspension was placed in an oil bath at 80° C. under N2. After 4 h, the reaction mixture was allowed to cool to room temperature and was then diluted with hexanes (200 mL) and water. The layers were separated and the aqueous layer was extracted with hexanes. The combined organic portions were washed with water, dried (MgSO4), filtered and concentrated to afford crude. The crude was purified by silica gel chromatography using 30% EtOAc/pet ether as an eluent to afford 1-bromo-2,5-dichloro-3-methoxybenzene (13 g, 81% yield) as off white solid. 1H NMR (300 MHz, CDCl3) δ 7.26 (d, J=2.8 Hz, 1H), 6.87 (d, J=2.8 Hz, 1H), 3.93 (s, 3H). b) (2,5-dichloro-3-methoxyphenyl)(4-methoxybenzyl)sulfane To an argon purged solution of 1-bromo-2,5-dichloro-3-methoxybenzene (13 g, 50.8 mmol), (4-methoxyphenyl)methanethiol (9.40 g, 61.0 mmol) and DIPEA (17.74 mL, 102 mmol) in toluene (200 mL), xantphos (2.94 g, 5.08 mmol) and Pd2(dba)3 (4.65 g, 5.08 mmol) were added at ambient temperature and heated to 90° C. for 4 h under argon atmosphere. After 4 h the reaction mixture was cooled to RT and passed through a pad of Celite® and the filtrate was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude. The crude was purified by silica gel chromatography using 10% EtOAc/pet ether as an eluent to afford (2,5-dichloro-3-methoxyphenyl)(4-methoxybenzyl)sulfane (13 g, 61% yield) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.30-7.25 (m, 2H), 6.87-6.84 (m, 3H), 6.73-6.72 (m, 1H), 4.10 (s, 2H). 3.87 (s, 3H), 3.79 (s, 3H). c) 2,5-Dichloro-3-methoxybenzenethiol To a stirred solution of (2,5-dichloro-3-methoxyphenyl)(4-methoxybenzyl)sulfane (13 g, 39.5 mmol) in anisole (70 mL) was added TFA (70 mL, 909 mmol) at ambient temperature and heated to 100° C. for 2 h. After 2 h the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with 2N NaOH solution. The aqueous layer was washed twice with EtOAc, finally acidified with conc. HCl and extracted with EtOAc. The organic layer washed with water and evaporation to afford 2,5-dichloro-3-methoxybenzenethiol (5.5 g, 54.1% yield) as a yellow liquid. 1H NMR (300 MHz, DMSO-d6) δ 7.28 (s, 1H), 6.99 (s, 1H), 6.02 (brs, 1H). 3.86 (s, 3H). d) Ethyl 4-((2,5-dichloro-3-methoxyphenyl)thio)-3-oxobutanoate To an ice cooled solution of 2,5-dichloro-3-methoxybenzenethiol (5.5 g, 26.3 mmol) and K2CO3 (10.91 g, 79 mmol) in DMF (50 mL) was slowly added ethyl 4-chloro-3-oxobutanoate (8.66 g, 52.6 mmol) and the reaction mass was allowed to stir at ambient temperature. After 2 h the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude which was used as such for next step. e) Ethyl 2-(4,7-dichloro-6-methoxybenzo[b]thiophen-3-yl)acetate To the above crude ethyl 4-((2,5-dichloro-3-methoxyphenyl)thio)-3-oxobutanoate (6 g, 17 mmol), methane sulfonic acid (5 mL, 77 mmol) was added and stirred at ambient temperature for 1 h. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude. The crude was purified by silica gel chromatography using 30% EtOAc/pet-ether as an eluent to afford ethyl 2-(4,7-dichloro-6-methoxybenzo[b]thiophen-3-yl)acetate (4 g, 47.2% yield) as an off white solid. 1H NMR (300 MHz, CDCl3) δ 7.19 (s, 1H), 7.13 (s, 1H), 4.25-4.15 (q, J=4 Hz, 2H), 4.09 (s, 2H), 3.98 (s, 3H), 1.30 (t, J=4.5 Hz, 3H). LCMS (ES) m/z 318.8 (M+H)+ f) Ethyl 2-(4,7-dichloro-6-hydroxybenzo[b]thiophen-3-yl)acetate To a stirred solution of ethyl 2-(4,7-dichloro-6-methoxybenzo[b]thiophen-3-yl)acetate (1 g, 3.13 mmol) in DCM (10 mL) was slowly added boron trifluoride methyl sulfide complex (5 mL, 3.13 mmol) at ambient temperature and allowed to stir for 12 h. The reaction mixture was diluted with water and basified with saturated NaHCO3, extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude. The crude was triturated with ethers to afford ethyl 2-(4,7-dichloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (800 mg, 73.6% yield) as off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 7.51 (s, 1H), 7.10 (s, 1H), 4.12-4.05 (q, J=4 Hz, 2H), 3.96 (s, 2H), 1.19 (t, J=4.0 Hz, 3H). a) (5-Chloro-2-fluoro-3-methoxyphenyl)(methyl)sulfane To a stirred solution of 4-chloro-1-fluoro-2-methoxybenzene (8.0 g, 49.8 mmol) in THF (150 mL) was added dropwise (over a period of 20 min) sec-butyllithium (80 mL, 112 mmol) at −78° C. and stirred for 30 min. Dimethyl disulfide (9.74 mL, 110 mmol) was added to the reaction mixture at same temperature. The reaction mixture was stirred at −78° C. under argon atmosphere for 1.5 h. The reaction mixture was quenched with sat NH4Cl solution and partitioned between water and EtOAc. The EtOAc layer was washed with water and brine, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated under reduced pressure to get crude material. The resultant residue was purified by column chromatography (100-200 silica mesh and eluent was 2% EtOAc in pet ether) to afford (5-chloro-2-fluoro-3-methoxyphenyl)(methyl)sulfane (5.0 g, 48.6% yield) as an off-white solid. 1H NMR (500 MHz, CDCl3): δ 6.79-6.76 (m, 2H), 3.86 (s, 3H), 2.36 (s, 3H). b) 5-Chloro-2-fluoro-1-methoxy-3-(methylsulfinyl)benzene To a stirred solution of (5-chloro-2-fluoro-3-methoxyphenyl)(methyl)sulfane (5.0 g, 24.19 mmol) in methanol (200 mL) and water (40 mL) was added sodium periodate (7.76 g, 36.3 mmol) at 0° C. The reaction mixture was stirred at 26° C. for 16 h. The reaction mixture was evaporated under reduced pressure the residue was partitioned between water and EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated under reduced pressure to get crude material. The resultant residue was purified by column chromatography (100-200 silica mesh and eluent was 15% EtOAc in pet ether) to afford 5-chloro-2-fluoro-1-methoxy-3-(methylsulfinyl)benzene (4.0 g, 74.3% yield) as an off-white solid. 1H NMR (300 MHz, DMSO-d6): δ 7.39-7.36 (m, 1H), 7.06 (dd, J=2.5, 7.5 Hz, 1H), 3.93 (s, 3H), 2.83 (s, 3H). LCMS (ES) m/z 223.16 (M+H)+ c) 5-Chloro-2-fluoro-3-methoxybenzenethiol To a stirred solution of 5-chloro-2-fluoro-1-methoxy-3-(methylsulfinyl)benzene (1.50 g, 6.74 mmol) in acetonitrile (60 mL) was added trifluoroacetic anhydride (1.9 mL, 13.47 mmol) at 0° C. and stirred at same temperature for 1 h. Then the reaction mixture was stirred at RT for 1 h. The reaction mixture was concentrated. The residue was dissolved in a mixture of methanol (10 mL) and TEA (10.0 mL) at 0° C. and stirred for 10 min and concentrated in vacuum. The mixture was diluted with sat NH4Cl and extracted with EtOAc. The organic layer was washed with 1N NaOH. The aqueous layer was acidified with 1N HCl and extracted with EtOAc. The organic layer washed with brine dried over anhydrous Na2SO4, filtered and the filtrate was evaporated under reduced pressure to get crude material. The resulted residue was purified by column chromatography (100-200 silica mesh, eluent was 10% EtOAc in pet ether) to obtain 5-chloro-2-fluoro-3-methoxybenzenethiol (0.75 g, 57.8% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6): δ 6.84 (m, 1H), 6.74 (dd, J=2.4, 6.8 Hz, 1H), 3.88 (s, 3H), 3.81 (s, H) d) Ethyl 4-((5-chloro-2-fluoro-3-methoxyphenyl)thio)-3-oxobutanoate To the stirred suspension of 5-chloro-2-fluoro-3-methoxybenzenethiol (750 mg, 3.89 mmol) and potassium carbonate (538 mg, 3.89 mmol) in DMF (10 mL) was added ethyl 4-chloro-3-oxobutanoate (705 mg, 4.28 mmol) at 0° C. The reaction mixture was stirred at RT for 2 h. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was separated and dried over anhydrous Na2SO4, filtered and the filtrate was evaporated under reduced pressure to get crude product ethyl 4-((5-chloro-2-fluoro-3-methoxyphenyl)thio)-3-oxobutanoate (750 mg, 60.1% yield) as a brown liquid. 1H NMR 400 MHz, CDCl3): δ 6.94-6.92 (m, 1H) 6.86 (dd, J=2.4, 7.2 Hz, 1H), 4.23 (q, 2H), 3.87 (s, 3H), 3.82 (s, 2H), 3.64 (s, 2H), 1.27 (t, J=2.4 Hz, 3H). e) Ethyl 2-(4-chloro-7-fluoro-6-methoxybenzo[b]thiophen-3-yl)acetate To a stirred solution of ethyl 4-((5-chloro-2-fluoro-3-methoxyphenyl)thio)-3-oxobutanoate (750 mg, 2.338 mmol) was added methanesulfonic acid (3.0 ml, 46.2 mmol) at 0° C. and mixture was stirred at RT for 1 h. The reaction mixture was partitioned between EtOAc and water, the separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure to get crude material. The resultant residue was purified by column chromatography (100-200 silica mesh and eluent was 15% EtOAc in pet ether) to afford ethyl 2-(4-chloro-7-fluoro-6-methoxybenzo[b]thiophen-3-yl)acetate (450 mg, 58.5% yield) as a pale yellow liquid. 1H NMR 500 MHz, CDCl3): δ 7.16 (s, 1H) 7.07 (d, J=7.0 Hz, 1H), 4.17 (q, 2H), 4.07 (s, 2H), 3.95 (s, 3H), 1.26 (t, J=5.6 Hz, 3H). LCMS (ES) m/z 303.25 (M+H)+. f) Ethyl 2-(4-chloro-7-fluoro-6-hydroxybenzo[b]thiophen-3-yl)acetate To a solution of ethyl 2-(4-chloro-7-fluoro-6-methoxybenzo[b]thiophen-3-yl)acetate (350 mg, 1.156 mmol) in DCM (10 mL) was added BBr3 (0.164 mL, 1.734 mmol) at −50° C. The reaction mixture was cool to room temperature for 1 h under nitrogen atmosphere. The Reaction mixture was quenched with saturated NaHCO3 solution and partitioned between water and DCM. The DCM layer was washed with water and brine, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated under reduced pressure to get crude material. The resulted residue was purified by column chromatography (100-200 silica mesh, eluent was 10% EtOAc in pet ether) to obtain ethyl 2-(4-chloro-7-fluoro-6-hydroxybenzo[b]thiophen-3-yl)acetate (300 mg, 80% yield) as an off-white solid. 1H NMR 500 MHz, CDCl3): δ 7.15 (s, 1H) 7.05 (d, J=7.0 Hz, 1H), 4.2 (q, J=5.6 Hz, 2H), 4.08 (s, 2H), 1.26 (t, J=5.6 Hz, 3H). LCMS (ES) m/z 289.19 (M+H)+. a) Methyl 2-bromo-6-methylnicotinate Phosphorus oxybromide (21.53 g, 75 mmol) was added to the stirred solution of 2-hydroxy-6-methylnicotinic acid (5 g, 32.7 mmol), pyridine (0.475 mL, 5.88 mmol) in chlorobenzene (100 mL) at room temperature under nitrogen. The reaction mixture was refluxed for 1 h and concentrated under vacuum before treating with an excess of cold methanol. The solution was stirred for an additional 1 h and again concentrated under vacuum. The residue was dissolved in water and pH was adjusted to ˜8.0 by adding K2CO3 before extraction of the product with CH2C12. The organic layer was washed with water and brine solution, dried over anhydrous Na2SO4. Filtrate was evaporated completely under reduced pressure to give crude residue. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 30% EtOAc in pet ether) to obtained methyl 2-bromo-6-methylnicotinate (6.1 g, 79% yield) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.00 (d, J=7.6 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 3.94 (s, 3H), 2.59 (s, 3H); LCMS (ES) m/z 230.0 (M+H)+ b) Methyl (E)-2-(3-methoxy-3-oxoprop-1-en-1-yl)-6-methylnicotinate Na2CO3 (8.43 g, 80 mmol) was added to a solution of methyl 2-bromo-6-methylnicotinate (6.1 g, 26.5 mmol) and methyl acrylate (6.08 mL, 67.1 mmol) in mixture of DMA (16.99 mL, 181 mmol) and toluene (55 mL) at room temperature. Then the reaction mixture was degassed for 15 min. Tri-o-tolylphosphine (0.807 g, 2.65 mmol) and allylpalladium chloride dimer (0.4850 g, 1.326 mmol) were added and the reaction mixture was stirred at 115° C. in sealed tube for 5 h. Filtered through pad of Celite®, and the filtrate was concentrated under reduced pressure. The resultant crude compound was purified by flash column chromatography on 100-200 mesh silica gel using 20% EtOAc/pet-ether as an eluent to obtained (E)-methyl 2-(3-methoxy-3-oxoprop-1-en-1-yl)-6-methylnicotinate (3.30 g, 43.0% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.53 (dd, J=1.2, 15.2 Hz, 1H), 8.22 (d, J=6.8 Hz, 1H), 7.20-7.11 (m, 2H), 3.94 (s, 3H), 3.82 (s, 3H), 2.60 (s, 3H). LCMS (ES) m/z 236.09 (M+H)+ c) Methyl 2-(3-methoxy-3-oxopropyl)-6-methylnicotinate 10% Pd—C (300 mg, 2.82 mmol) was added to a solution of (E)-methyl 2-(3-methoxy-3-oxoprop-1-en-1-yl)-6-methylnicotinate (3.30 g, 14.03 mmol) in methanol (120 mL) at 25° C. The reaction mixture was stirred for 3 h at 25° C. under hydrogen atmospheric pressure of 50 psi. The reaction mixture was filtered and filtrate was evaporated under pressure to get methyl 2-(3-methoxy-3-oxopropyl)-6-methylnicotinate (2.8 g, 74.3% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.08 (d, J=8.0 Hz, 1H), 7.06 (d, J=7.6 Hz, 1H), 3.90 (s, 3H), 3.67 (s, 3H), 3.50 (t, J=7.6 Hz, 2H), 2.81 (t, J=8.0 Hz, 2H), 2.55 (s, 3H). LCMS (ES) m/z 238.10 (M+H)+ d) 2-Methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one Sodium methoxide (0.956 g, 17.70 mmol) was added to a solution of methyl 2-(3-methoxy-3-oxopropyl)-6-methylnicotinate (2.8 g, 11.80 mmol) in THF (30 mL) under nitrogen atmosphere. The reaction mixture was warmed to reflux during 2 h. The solvent was removed under vacuo and HCl (20 ml, 90 mmol) 4.5 M was added, the mixture was stirred 2 h at reflux. The reaction mixture was dissolved in water and pH was adjusted to ˜8.0 by adding K2CO3 before extraction of the product with CH2Cl2. The organic layer was washed with water and brine solution, dried over anhydrous Na2SO4. Filtrate was evaporated completely under reduced pressure to give crude residue. The crude residue was purified by silica gel column chromatography by using EtOAc in hexane as eluent, the product was eluted at 40% EtOAc/Pet-ether to get 2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (1.3 g, 66.1% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.91 (d, J=8.4 Hz, 1H), 7.19 (d, J=7.6 Hz, 1H), 3.24 (t, J=6.0 Hz, 2H), 2.78 (t, J=8.0 Hz, 2H), 2.67 (s, 3H). LCMS (ES) m/z 147.98 (M+H)+ e) 2-Methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol NaBH4 (0.334 g, 8.83 mmol) was added lot wise to the stirred solution of 2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (1.3 g, 8.83 mmol) in methanol (30 ml) at 0° C. and the mixture was stirred at 0° C. for 2 h. The reaction mixture was diluted with water and mixture was concentrated under reduced pressure. The resulted residue was partitioned between EtOAc and water, the separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 70% EtOAc in pet ether) to obtained 2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (0.900 g, 67.6% yield) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 7.59 (d, J=7.5 Hz, 1H), 7.01 (d, J=7.5 Hz, 1H), 5.25 (s, 1H), 3.18-3.08 (m, 1H), 2.95-2.84 (m, 1H), 2.59 (s, 4H), 2.04-1.94 (m, 2H). LCMS (ES) m/z 150.3 [M+H]+ f) 5-Chloro-2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridine To solution of 2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (20.66 g, 139 mmol) in DCM (200 mL) was added thionyl chloride (6.74 mL, 92 mmol) at RT and stirred for 20 min, solvents were evaporated under reduced pressure to afford crude product. The crude product was used for the next step without further purification. a) 6,7-Dihydro-5H-cyclopenta[b]pyridin-5-one KMnO4 (53.0 g, 336 mmol) dissolved in water (2000 mL) was added to the stirred solution of 6,7-dihydro-5H-cyclopenta[b]pyridine (20 g, 168 mmol) and MgSO4.7H2O (40.4 g, 336 mmol) in tert-butanol (500 mL) at 25° C. and the reaction mixture was stirred at 30° C. for 3 h. The reaction mixture was filtered through a Celite® bed, partitioned between EtOAc and water, the separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure. The resulted crude compound was purified by flash column chromatography on 100-200 silica gel, using 20-30% EtOAc-Pet ether as an eluent to obtained 6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (10 g, 44.7% yield) as an off white solid. LCMS (ES) m/z 134.01 [M+H]+. b) 6,7-Dihydro-5H-cyclopenta[b]pyridin-5-ol NaBH4 (25.6 g, 676 mmol) was added portion wise to the stirred solution of 6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (60 g, 451 mmol) in methanol (600 mL) at 0° C. and the reaction mixture was stirred at 25° C. for 1 h under nitrogen atmosphere. The reaction mixture was quenched with water and then solvent was distilled off. The residue was partitioned between EtOAc and water, the separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure. The resulted crude compound was purified by flash column chromatography on 100-200 silica gel, using EtOAc-Pet ether as an eluent to obtained 6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (40 g, 62.5% yield) as an off white solid. LCMS (ES) m/z 136.11 [M+H]+. c) 5-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridine To a solution of 6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (5 g, 37.0 mmol) in DCM (50 mL) was added thionyl chloride (4.05 mL, 55.5 mmol) at 0° C. and the reaction mixture was stirred at 25° C. for 1 h under nitrogen atmosphere. Reaction mixture was concentrated under reduced pressure to get crude product 5-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (5.5 g) as a brown gummy liquid. Crude was used for the next step without further purification. LCMS (ES) m/z 154.19 [M+H]+. a) Ethyl (E)-1-(3-amino-3-oxoprop-1-en-1-yl)-2-oxocyclopentane-1-carboxylate Ethyl 2-oxocyclopentanecarboxylate (150 g, 960 mmol) was added to the stirred solution of propiolamide (113 g, 1633 mmol) and Na2CO3 (112 g, 1056 mmol) in water (1500 mL) at 0° C. and the mixture was stirred at RT for 6 h. The reaction mixture was diluted with water and extracted with DCM, the organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure to get crude product. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 80% EtOAc in pet ether) to obtained (E)-ethyl 1-(3-amino-3-oxoprop-1-en-1-yl)-2-oxocyclopentanecarboxylate (150 g, 68.8% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3): δ 6.58 (d, J=10.4 Hz, 1H), 5.97 (dd, J=2.0, 10.0 Hz, 1H), 5.89 (brs, 2H), 4.26 (q, J=4.8 Hz, 2H), 2.50-2.42 (m, 1H), 2.26-2.19 (m, 1H), 2.08-1.88 (m, 3H), 1.76-1.72 (m, 1H), 1.31 (t, J=7.2 Hz, 3H). LCMS (ES) m/z 226.23 [M+H]+ b) 6,7-Dihydro-5H-cyclopenta[b]pyridin-2-ol Conc. HCl (209 ml, 6882 mmol) was added to the (E)-ethyl 1-(3-amino-3-oxoprop-1-en-1-yl)-2-oxocyclopentanecarboxylate (155 g, 688 mmol) at room temperature and the mixture was stirred at 130° C. for 5 h. The reaction mixture was concentrated and poured into ice. The pH was adjusted to ˜7.0 by dropwise addition of saturated aqueous NaHCO3 solution, and filtered the precipitated solid. The solid was washed with water to get 6,7-dihydro-5H-cyclopenta[b]pyridin-2-ol (80 g, 86% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6): δ 7.71 (s, 1H), 6.62 (s, 1H), 2.89-2.85 (m, 2H), 2.75-2.72 (m, 2H), 2.11-2.04 (m, 2H). LCMS (ES) m/z 136.07 [M+H]+ c) 2-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridine A mixture of 6,7-dihydro-5H-cyclopenta[b]pyridin-2-ol (70 g, 518 mmol), POCl3 (200.0 ml, 2146 mmol) and DMF (10 mL) was stirred under nitrogen atmosphere at 120° C. for 3 h. After cooling, the mixture was poured into ice water, basified with 8 M NaOH aqueous solution and extracted with AcOEt. The extract was washed with brine, dried over anhydrous Na2SO4, and concentrated. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 10% EtOAc in pet ether) to obtained 2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (35 g, 43.7% yield) as an off white solid. 1H NMR (400 MHz, CDCl3): δ 7.44 (d, J=7.6 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 3.01 (t, J=7.2 Hz, 2H), 2.92 (t, J=7.2 Hz, 2H), 2.18-2.10 (m, 2H). LCMS (ES) m/z 154.09 [M+H]+ d) 2-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one KMnO4 (72.0 g, 456 mmol) dissolved in water (3.5 L) was added to the stirred solution of 2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (35.00 g, 228 mmol) and magnesium sulfate heptahydrate (68.2 g, 456 mmol) in tert-butanol (875 mL) and the reaction mixture was stirred at RT for 2 h under nitrogen atmosphere. The reaction mixture was filtered through a Celite® pad, partitioned between EtOAc and water. The separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was concentrated under reduced pressure. The resulted crude compound was purified by flash column chromatography on 100-200 silica gel, using 20-30% EtOAc-Pet ether as an eluent to obtained 2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (25 g, 65.4% yield) as an off white solid. 1H NMR (400 MHz, CDCl3): δ 7.97 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 3.28-3.25 (m, 2H), 2.82-2.79 (m, 2H); LCMS (ES) m/z 168.08 [M+H]+ e) 2-Chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol Sodium borohydride (108 mg, 2.86 mmol) was added lot wise to the stirred solution of 2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (480 mg, 2.86 mmol) in methanol (100 mL) at 0° C. and the mixture was stirred at 0° C. for 1 h. The reaction mixture was diluted with water and mixture was concentrated under reduced pressure. The resulted residue was partitioned between EtOAc and water, the separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 30% EtOAc in pet ether) to obtained 2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (450 mg, 91% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3): δ 7.67 (d, J=8.0 Hz, 1H), 7.19 (d, J=8.0 Hz, 1H), 5.28-5.26 (m, 1H), 3.13-3.10 (m, 1H), 2.95-2.92 (m, 1H), 2.05-2.02 (m, 1H) 1.89 (m, 1H). LCMS (ES) m/z 170.16 [M+H]+ f) 2,5-Dichloro-6,7-dihydro-5H-cyclopenta[b]pyridine SOCl2 (0.232 ml, 3.18 mmol) was added to the stirred solution of 2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (450.00 mg, 2.65 mmol) in DCM (50 ml) and the mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure and crude used for the next step. LCMS (ES) m/z 188.15 [M+H]+. a) Butoxy-1,1,1-trifluorobut-3-en-2-one To a stirred solution oft-(vinyloxy)butane (50 g, 499 mmol), pyridine (40.4 mL, 499 mmol) in Chloroform (500 mL) at 0° C. 1,1,1,5,5,5-hexafluoropentane-2,4-dione (104 g, 499 mmol) in chloroform (200 ml) was added and stirred for 16 h After completion of reaction, mixture was poured into cool water. The solution was extracted by DCM and washed with water followed by brine. The organic layer was dried over anhydrous sodium sulphate and solvent was removed under reduced pressure. The crude was purified by flash column chromatography on silica gel (100-200 mesh), eluting with 0-30% gradient of EtOAc in hexane to afford (E)-1-ethoxy-5,5,5-trifluoropent-1-en-3-one (70 g, 73% yield) as a liquid. 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J=12 Hz, 1H), 5.86 (d, J=2.4 Hz, 1H), 4.03 (t, J=6.4, 2H), 1.77-1.70 (m, 2H), 1.48-1.39 (m, 2H), 0.95 (t, J=7.6 Hz, 3H). b) 2-Oxo-6-(trifluoromethyl)-1,2-dihydropyridine-3-carboxamide To a stirred solution of malonamide (39.2 g, 384 mmol) in methanol (300 mL) at 0° C. (E)-1-ethoxy-5,5,5-trifluoropent-1-en-3-one (70 g, 384 mmol) in methanol (300 mL) was added and reaction mixture was stirred at reflux temperature for 6 h, After completion of reaction mixture was concentrated, poured into cool water and acidified with dil.HCl (pH 2) to get solid. Solid was filtered and dried to get pure compound 2-oxo-6-(trifluoromethyl)-1,2-dihydropyridine-3-carboxamide (60 g, 73.1% yield) as off white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 13.66 (brs, 1H), 8.46 (brs, 2H), 8.07 (brs, 1H), 7.38 (brs, 1H). LCMS (ES+) m/z 207.11 [M+H]+. c) 2-Oxo-6-(trifluoromethyl)-1,2-dihydropyridine-3-carboxylic acid To a stirred solution of 2-oxo-6-(trifluoromethyl)-1,2-dihydropyridine-3-carboxamide (60 g, 291 mmol) in methanol (300 mL), water (100 mL) LiOH (20.91 g, 873 mmol) was added at room temperature and reaction mixture was stirred at reflux temperature for 24 h. After completion of the reaction, mixture was poured into cool water and acidified with 1N HCl. to get solid. Solid was filtered and dried to get pure compound 2-oxo-6-(trifluoromethyl)-1,2-dihydropyridine-3-carboxylic acid (52 g, 86% yield) as off white solid. 1H NMR (400 MHz, CDCl3) δ ppm 12.74 (brs, 1H), 8.72 (d, J=6.5 Hz, 1H), 7.10 (d, J=5.5 Hz, 1H). LCMS (ES) m/z 208.08 (M+H)+. d) Methyl 2-bromo-6-(trifluoromethyl)nicotinate To the stirred solution of 2-hydroxy-6-(trifluoromethyl)nicotinic acid (23 g, 111 mmol) and pyridine (8.98 mL, 111 mmol) in chlorobenzene (250 mL) phosphorus oxybromide (63.7 g, 222 mmol) was added small portions wise at room temperature and the mixture was stirred at 120° C. for 16 h. After completion, the reaction mixture was concentrated under vacuum. The residue was cooled 0° C. and added excess cold methanol slowly. The solution stirred additional 1 h and again concentrated under vacuum. The residue dissolved in water and pH adjusted to −8 using K2CO3 before extraction with EtOAc. The organic layer was separated and dried over anhydrous Na2SO4, filtered and the filtrate was concentrated under reduced pressure to obtain as a brown liquid. The crude was purified by flash column chromatography on silica gel (100-200 mesh), eluting with 0-10% gradient of EtOAc in hexane to afford 4-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole in (400 g, 42.1%) yields. 1H NMR (500 MHz, CDCl3) δ 8.20 (d, J=8 Hz, 1H), 7.71 (d, J=8.0 Hz, 1H), 4.01 (s, 3H). e) Methyl (E)-2-(3-methoxy-3-oxoprop-1-en-1-yl)-6-(trifluoromethyl)nicotinate To a stirred solution of methyl 2-bromo-6-(trifluoromethyl)nicotinate (21 g, 73.9 mmol), methyl acrylate (16.75 mL, 185 mmol) and sodium carbonate (23.51 g, 222 mmol) in N,N-dimethylacetamide (DMA) (100 mL), toluene (400 mL), allylpalladium chloride dimer (1.353 g, 3.70 mmol), tri-o-tolylphosphine (2.250 g, 7.39 mmol) was added at room temperature in a sealed tube. The resulting reaction mixture was stirred for 16 h at 120° C. After completion, the reaction mixture was filtered through a Celite® bed and was washed with EtOAc thoroughly. The filtrate was concentrated to get crude residue The crude compound was purified by column chromatography (100-200 mesh silica gel) using 10% EtOAc in pet-ether as an eluent to get (E)-methyl 2-(3-methoxy-3-oxoprop-1-en-1-yl)-6-(trifluoromethyl)nicotinate (13 g, 60.8% yield) as yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J=15.6 Hz, 1H), 8.39 (dd, J=0.4, 8.0 Hz, 1H), 7.71 (d, J=8.4 Hz, 1H), 7.26 (d, J=2.8 Hz, 1H), 4.01 (s, 3H), 3.84 (s, 3H). LCMS (ES) m/z 290.22 [M+H]+. f) Methyl 2-(3-methoxy-3-oxopropyl)-6-(trifluoromethyl)nicotinate To a stirred solution of (E)-methyl 2-(3-methoxy-3-oxoprop-1-en-1-yl)-6-(trifluoromethyl)nicotinate (6.0 g, 20.75 mmol) in ethanol (180 mL), Pd/C (2.65 g) was added at room temperature. The mixture was stirred at room temperature for 1 h under hydrogen balloon pressure, filtered through pad of Celite®, and filtrate was concentrated under vacuo to afford methyl 2-(3-methoxy-3-oxopropyl)-6-(trifluoromethyl)nicotinate (4.5 g, 14.88 mmol, 71.7% yield) as a colorless liquid. 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J=8.0 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H), 3.99 (s, 3H), 3.70 (s, 3H), 3.60 (t, J=6.5 Hz, 2H), 2.88 (t, J=7.0 Hz, 2H). LCMS (ES) m/z 292.08 [M+H]+. g) 2-(Trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one To a stirred solution of methyl 2-(3-methoxy-3-oxopropyl)-6-(trifluoromethyl)nicotinate (8 g, 27.5 mmol) in dry methanol (100 mL), sodium methoxide (2.226 g, 41.2 mmol) was added at room temperature and the mixture was stirred for 8 h at 80° C. under argon. The solvent was removed under reduced pressure and the resultant residue was dissolved in HCl (12.50 mL, 411 mmol) and stirred at 80° C. for 8 h. After completion, the reaction mixture was cooled to 0° C. and basified with 3N NaOH solution and partitioned between EtOAc and water. The separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure to get the crude residue 2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (4.0 g, 45.9% yield) as brown colored gum. 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J=8.0 Hz, 1H), 7.72 (d, J=8.0 Hz, 1H), 3.39 (t, J=5.0 Hz 2H), 2.88 (t, J=6.0 Hz, 2H); LCMS (ES) m/z 202.27 [M+H]+. h) 2-(Trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol To a stirred solution of 2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-one (4.0 g, 19.89 mmol) in methanol (50 mL), sodium borohydride (0.752 g, 19.89 mmol) was added lot wise at 0° C. and the mixture was stirred at room temperature for 2 h. After completion, the reaction mixture was diluted with water and was concentrated under reduced pressure. The resulted residue was partitioned between EtOAc and water. The separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure. The crude obtained was purified by column chromatography on silica gel (100-200 mesh) eluted with 20-50% gradient of EtOAc in hexanes to afford 2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (3.1 g, 13.84 mmol, 69.6% yield) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J=8 Hz, 1H), 7.56 (d, J=7.6 Hz, 1H), 5.36-5.31 (m, 1H), 3.29-3.25 (m, 1H), 3.10-2.90 (m, 1H), 2.7-2.6 (m, 1H), 2.05-1.90 (m, 1H); LCMS (ES) m/z 204.22 [M+H]+. i) 5-Chloro-2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridine To a stirred solution of 2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (56.3 mg, 0.277 mmol) in DCM (15 mL) was added SOCl2 (0.020 mL, 0.277 mmol) at 0° C. The reaction mixture was stirred at room temperature for 30 min. and then evaporated under reduced pressure to get residue. The crude compound used directly for the next step. a) Ethyl 3-oxo-6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridine-2-carboxylate To a solution of ethyl 2-hydroxyacetate (19.50 g, 187 mmol) in 1,2-dimethoxyethane (DME) (200 mL) was added NaH (4.49 g, 187 mmol) at 0° C. and then solution of ethyl 2-chloro-6-(trifluoromethyl)nicotinate (19 g, 74.9 mmol) in 1,2-dimethoxyethane (DME) (200 mL) was added to the reaction mixture at RT. The resulting reaction mixture was stirred at 75° C. for 2 h. The reaction mixture was quenched with saturated sodium bicarbonate, extracted with EtOAc. The organic layer was washed successively with water and brine, dried over MgSO4, and concentrated in vacuo to get crude. The residue was purified by silica gel column chromatography (EtOAc/Pet ether) to afford the title compound (9.6 g) as yellow solid. LCMS (ES) m/z 276.07 [M+H]+. b) 6-(Trifluoromethyl)furo[2,3-b]pyridin-3(2H)-one To a solution of ethyl 3-oxo-6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridine-2-carboxylate (4 g, 14.54 mmol) in 1,4-dioxane (40 mL) was added HCl (11.04 mL, 363 mmol) at RT. The reaction mixture was heated to 100° C. for 24 h. The reaction mixture was quenched with saturated sodium bicarbonate and extracted with EtOAc. The organic layer was washed successively with water and brine, dried over MgSO4, and concentrated in vacuo to give the title compound (1.5 g) as yellow solid. The crude compound used for the next step without further purification. LCMS (ES) m/z 203.78 [M+H]+. d) 6-(Trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-ol The title compound was prepared as a white solid according to the procedures of Scheme 7, Step h, LCMS (ES) m/z 206.10 [M+H]+. a) Ethyl 2-chloronicotinate To a stirred solution of 2-chloronicotinic acid (25 g, 159 mmol) in DMF (300 mL) was added Mel (11.91 mL, 190 mmol), K2CO3 (54.8 g, 397 mmol) at rt. The reaction mixture was stirred at RT for 3 h. The Reaction mixture was diluted with EtOAc (500 mL) washed with water (4×500 mL) and brine (500 mL). Organic layer was dried over anhydrous sodium sulphate filtered and concentrated to afford ethyl 2-chloronicotinate (26 g, yield 95%) as off white solid. 1H NMR (400 MHz, CDCl3): δ 8.53-8.51 (m, 1H), 8.18-15 (m, 1H), 7.34-7.31 (m, 1H), 4.0 (s, 3H); LCMS (ES) m/z 171.94 [M+H]±. b) Ethyl 3-oxo-2,3-dihydrofuro[2,3-b]pyridine-2-carboxylate To a stirred suspension of NaH (10.91 g, 455 mmol) in 1,2-dimethoxyethane (1200 mL) was added ethyl 2-hydroxyacetate (39.4 g, 379 mmol) at 0° C. Reaction mixture was stirred at RT for 30 min. After that methyl 2-chloronicotinate (26 g, 152 mmol) in DME (150 mL) was added to the reaction mixture and the resulting mixture was heated at 75° C. for 2 h. Reaction mixture was concentrated. The crude was basified with saturated sodium bicarbonate and washed with EtOAc (1×500 mL). Aqueous layer was acidify with acetic acid, extracted with DCM (2×500 mL), washed with water (500 mL) and brine (500 mL). Organic layer was dried over anhydrous sodium sulphate filtered and concentrated. The crude residue was purified by column chromatography (100-200 mesh silica). Compound Eluted at 12% EtOAc in hexane. The eluents were concentrated at reduced pressure and to affording ethyl 3-oxo-2,3-dihydrofuro[2,3-b]pyridine-2-carboxylate (16 g, yield 35.2%) as off white solid. LCMS (ES) m/z 207.96 [M+1]+. c) Furo[2,3-b]pyridin-3(2H)-one To a stirred solution of ethyl 3-oxo-2,3-dihydrofuro[2,3-b]pyridine-2-carboxylate (12 g, 57.9 mmol) in HCl (9.65 ml, 57.9 mmol) was stirred at 100° C. for 1 h. Reaction mixture was basify with sat sodium bicarbonate diluted with EtOAc (500 mL) washed with water (200 mL) and brine (200 mL). Organic layer was dried anhydrous sodium sulphate filtered and concentrated. Crude was purified by column chromatography (100-200 mesh silica) to afford furo[2,3-b]pyridin-3(2H)-one (8 g, yield 84%) as off white solid. 1H NMR (500 MHz, CDCl3): δ 8.59-8.58 (m, 1H), 8.06-8.04 (m, 1H), 7.16-7.14 (m, 1H), 4.75 (s, 2H). LCMS (ES) m/z 136.07 [M+H]+. d) 2,3-Dihydrofuro[2,3-b]pyridin-3-ol To a stirred solution of furo[2,3-b]pyridin-3(2H)-one (8 g, 59.2 mmol) in methanol (80 mL) was added NaBH4 (2.24 g, 59.2 mmol) at 0° C. Reaction mixture was stirred at RT for 3 h. The reaction mixture was diluted with EtOAc (200 mL) washed with water (200 mL) and brine (200 mL). Organic layer was dried anhydrous sodium sulphate filtered and concentrated. Crude was purified by column chromatography (100-200 mesh silica gel) and the compound eluted at 80% EtOAc in hexane. Eluents were concentrated to affording 2,3-dihydrofuro[2,3-b]pyridin-3-ol (4 g, 47.7%) as off white solid. 1H NMR (500 MHz, DMSO-d6) δ 8.06-8.05 (m, 1H), 7.77-7.75 (m, 1H), 6.95-6.92 (m, 1H), 5.77-5.76 (d, J=6 Hz, 1H), 5.30-5.27 (m, 1H), 4.57-4.54 (m, 1H), 4.24-4.21 (m, 1H). LCMS (ES) m/z 138.12 [M+H]+. Example 1 Preparation of 2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid a) Ethyl 2-(4-chloro-6-(6,7-dihydro-5H-cyclopenta[b]pyridin-5-yloxy)benzo[b]thiophen-3-yl)acetate To a stirred solution of ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (5 g, 18.47 mmol) and 5-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine (4.26 g, 27.7 mmol) in DMF (50 mL) was added K2CO3 (12.76 g, 92 mmol) at RT. The reaction mixture was heated to 80° C. for 1 h. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed successively with water and brine, dried over MgSO4, and concentrated in vacuo to get crude. The residue was purified by silica gel column chromatography (EtOAc/hexane) to give the title compound (6.5 g) as brown gummy liquid. LCMS (ES) m/z 388.17 [M+H]+. b) 2-(4-Chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid To a stirred solution of ethyl 2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (40 g, 103 mmol) in THF (200 mL), methanol (200 mL) and water (100 mL) was added lithium hydroxide (monohydrate) (12.35 g, 516 mmol) at RT and the reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was neutralized with dilute HCl carefully and the precipitated compound was filtered and dried under reduced pressure to obtained 2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (32 g, 86% yield) as an off-white solid. 1H NMR (500 MHz, DMSO-d6) δ 12.5 (brs, 1H), 8.50 (dd, J=1.2, 4.5 Hz, 1H), 7.78-7.82 (m, 2H), 7.47 (s, 1H), 7.22-7.20 (m, 1H), 7.13 (d, J=2.1 Hz, 1H), 6.00-6.04 (m, 1H), 4.00 (s, 2H), 3.06-3.11 (m, 1H), 2.89-2.99 (m, 1H), 2.62-2.72 (m, 1H), 2.06-2.15 (m, 1H). LCMS (ES) m/z 360.05 [M+H]+. Chiral HPLC: 49.92%: 50.08% Analytical SFC Condition Column/dimensions:Chiralpak AD-H (250×4.6)mm, 5μ % CO2: 60.0% % Co solvent: 40.0% (100% MeOH) Total Flow: 3.0 g/min Back Pressure: 100 bar Temperature: 30.0° C. UV: 237 nm Preparative SFC Condition Column/dimensions: Chiralpak AD-H (250×21) mm, 5μ % CO2: 65.0% % Co solvent: 35.0% (100% Methanol) Total Flow: 60.0 g/min Back Pressure: 100.0 bar UV: 284 nm Stack time: 8.8 min Load/Inj: 15.0 mg Retention time: Peak 1—3.15 min, Peak 2-3.73 min. Purity: Peak 1—99.73%, Peak 2-98.00%. Solubility: Methanol+ACN+DCM+THF Chiral Separation of Example 1 Example 1a (First Eluted Enantiomer) (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid (11 g, 34.3% yield). LCMS (ES) m/z 360.18 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 12.4 (brs, 1H), 8.50 (dd, J=1.2, 4.8 Hz, 1H), 7.78-7.83 (m, 2H), 7.49 (s, 1H), 7.23-7.20 (m, 1H), 7.13 (d, J=2.1 Hz, 1H), 6.00-6.04 (m, 1H), 4.01 (s, 2H), 3.06-3.11 (m, 1H), 2.95-2.98 (m, 1H), 2.66-2.68 (m, 1H), 2.09-2.12 (m, 1H). Chiral HPLC: 99.73%. Absolute stereochemistry was determined by vibrational circular dichroism (VCD). Example 1b (Second Eluted Enantiomer) (R)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (10.3 g, 32.1% yield) as an off white solids. LCMS (ES) m/z 360.15 (M+H)±. 1H NMR (300 MHz, DMSO-d6): δ 12.4 (brs, 1H), 8.50 (dd, J=1.2, 4.8 Hz, 1H), 7.78-7.83 (m, 2H), 7.49 (s, 1H), 7.23-7.20 (m, 1H), 7.13 (d, J=2.1 Hz, 1H), 6.00-6.04 (m, 1H), 4.01 (s, 2H), 3.06-3.11 (m, 1H), 2.95-2.98 (m, 1H), 2.66-2.68 (m, 1H), 2.09-2.12 (m, 1H). Chiral HPLC: 98.00%. Chiral Synthesis of Example 1a, (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) (3-Chloro-5-methoxyphenyl)(4-methoxybenzyl)sulfane A 250 L reactor was charged with 1-bromo-3-chloro-5-methoxybenzene (7.5 kg, 33864.6 mmol) and (4-methoxyphenyl)methanethiol (5.74 kg, 37217.3 mmol). Toluene (30 L) was charged to the reaction mass. DIPEA (11.83 L, 67701.9 mmol) was added at 25° C. slowly into the above reaction mixture. The reaction mixture was degassed with N2 for 20 min. Pd2(dba)3 (1.55 kg, 1692.66 mmol) and xantphos (0.980 kg, 1692.66 mmol) were added slowly into above reaction mixture (Note: The reaction mass color changes pale yellow to dark color). The reaction mixture was again degassed with N2 for 15 min. The reaction mass was stirred at 110° C. for 3 h. Completion of the reaction was monitored by TLC (5% EtOAc in pet ether, Rf value of the product is 0.5). After completion of reaction, the reaction mass was cooled to 25° C. and filtered on a Celite® bed. The Celite® bed was washed with EtOAc. DM water was added to the filtrate and stirred at 25-30° C. for 5-10 min. The combined layers were transferred to a 250 L reactor. The Aqueous and EtOAc layers were separated. Sodium chloride solution was added to the EtOAc and stirred at 25-30° C. for 5-10 min. The combined layers were transferred to 250 L reactor. The aqueous and EtOAc layers were separated. The EtOAc layer was dried over anhydrous Na2SO4 and filtered. Na2SO4 washed with EtOAc. The EtOAc was transferred to a 250 L reactor and evaporated below 40-45° C. under vacuum. After completion of evaporation, the thick yellow liquid was subjected to drying by rotary evaporation at 40-45° C. for 1.0 h. drying was terminated and the thick yellow liquid was obtained (13.5 kg, crude). A chromatography column was packed with silica gel (20.0 kg, 100-200 mesh). The crude compound dissolved in DCM and loaded into the column. Run the mobile phase with hexane (50 L). Then followed by increasing the polarity from 2-5% EtOAc in hexane (500 L). All pure fractions (by TLC) collected and concentrated under reduced pressure at 40-45° C. (8.7 kg, yield 87.17%). 1H NMR (400 MHz, CDCl3) δ 7.25-7.22 (m, 2H), 6.87-6.82 (m, 3H), 6.69-6.67 (m, 2H), 4.08 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H). b) 3-Chloro-5-methoxybenzenethiol A 100 L reactor was charged with a solution of 3-chloro-5-methoxyphenyl(4-methoxybenzyl)sulfane (4.6 kg, 15604.01 mmol) in DCM (46 L). Anisole (15.35 kg, 141945.6 mmol) was added to the reaction mass and cooled to 0° C. Trifluoromethanesulfonic acid (1.38 L, 15604 mmol) was added dropwise to the reaction mass at 0-5° C. for 20 min (Note: The reaction mass color changed from pale yellow to red color). The reaction mass was stirred at 25° C. for 16 h under N2 atmosphere. The reaction was monitored by TLC (5% EtOAc in pet ether, Rf value of the product is 0.6). After completion of reaction, the reaction mass was cooled to 0° C. 2N NaOH solution was added dropwise to the reaction mass at 0-10° C. until the pH of the reaction mass was ˜13. The resulting mixture stirred at 25° C. for 30 min and settled for 10 min. The aqueous and organic layers were separated. The aqueous layer was cooled to 0° C. and acidified to pH˜2 with 2M HCl. EtOAc was added and resulting mixture stirred at 25° C. for 30 min. EtOAc layer was separated and the aqueous layer again extracted with EtOAc. The combined EtOAc layers were washed with DM water and the organic layer was separated. The organic layer was washed with sodium chloride solution (1.79 kg of NaCl in 17.94 L of water). The EtOAc layer was dried over anhydrous Na2SO4 and filtered. The EtOAc layer was evaporated below 40° C. After completion of evaporation, the thick yellow liquid was subjected to drying by rotary evaporation at 40-45° C. for 1.0 h. Drying was terminated and a pale yellow liquid was obtained (2.6 kg, yield 95%). 1H NMR (300 MHz, CDCl3) δ 6.85 (d, J=2.4 Hz, 1H), 6.70 (s, 2H), 3.78 (s, 3H), 3.50 (s, 1H). c) Ethyl 4-((3-chloro-5-methoxyphenyl)thio)-3-oxobutanoate A 100 L reactor was charged with a solution of 3-chloro-5-methoxybenzenethiol (2.6 kg, 1489 mmol) in acetonitrile (19.5 L). K2CO3 (3.09 kg, 2235 mmol) was added to the reaction mass at 0° C. under N2 atmosphere and stirred at same temperature for 10 min (Note: After addition of K2CO3, the reaction mass color changed from pale yellow to white color). Ethyl 4-chloroacetoacetate (2.7 kg, 1545 mmol) was added dropwise to the reaction mass at 0-10° C. for 20 min. The reaction mass was stirred at 25° C. for 1 h under N2 atmosphere (Note: After 1 h stirring, the reaction mass color changed from white to brown color). The reaction was monitored by TLC (10% EtOAc in pet ether, Rf value of the product is 0.3). After completion of the reaction, the reaction mass was cooled to 0° C. DM water was added slowly to the reaction mass at 0-10° C. EtOAc was added and the resulting mixture stirred at 25° C. for 10 min. The aqueous and organic layers were separated. The EtOAc layer was washed with 10% sodium chloride solution. The aqueous and organic layers were separated. The EtOAc layer was dried over anhydrous Na2SO4 and filtered. The Na2SO4 was washed with EtOAc. The EtOAc layer was evaporated below 40° C. Drying was terminated and a dark color liquid was obtained (4.1 kg, yield 91%). 1H NMR (400 MHz, CDCl3) δ 6.90 (d, J=2.4 Hz, 1H), 6.75 (s, 2H), 4.20-4.18 (m, 2H), 3.82 (s, 2H), 3.78 (s, 3H), 3.61 (s, 2H), 1.26 (m. 3H). d) Ethyl 2-(4-chloro-6-methoxybenzo[b]thiophen-3-yl)acetate and ethyl 2-(6-chloro-4-methoxybenzo[b]thiophen-3-yl)acetate A 20 L 4-neck round bottom flask was charged with methanesulfonic acid (9.52 L) and cooled to 0° C. Ethyl 4-(3-chloro-5-methoxyphenylthio)-3-oxobutanoate (4.0 kg, 13211 mmol) was added at 0° C. slowly dropwise into the above reaction mixture under nitrogen atmosphere for 50 min and stirred at 0° C. for 20 min (Note: The reaction mixture turns a dark black color). The reaction mass was slowly allowed to attain 25° C. The reaction mass was stirred at 25° C. for 1 h. Completion of the reaction was monitored by TLC. (10% EtOAc in pet ether, Rf value of the product is 0.4). After completion of the reaction, the reaction mass was poured into ice cold water. EtOAc was added and the resulting mixture stirred at 25° C. for 10 min. The aqueous and EtOAc layers were separated. The aqueous layer was again extracted with EtOAc. The combined EtOAc layers were washed with DM water and the organic layer was separated. The EtOAc layer was washed with 10% sodium chloride solution. The EtOAc layer was dried over anhydrous Na2SO4 and filtered. The Na2SO4 was washed with EtOAc. The EtOAc was evaporated below 40-45° C. under vacuum. After completion of evaporation, the thick yellow liquid was subjected to drying by rotary evaporation at 40-45° C. for 1.0 h. Drying was terminated and a thick black liquid was obtained (3.5 kg). A chromatography column was packed with silica gel (12 kg, 100-200 mesh). The crude compound was dissolved in DCM and loaded onto the column. The mobile phase was run with hexane (50 L) followed by increasing the polarity from 2-10% EtOAc in hexane (100 L). All pure fractions (by TLC) collected and concentrated under reduced pressure at 40-45° C. to afford a mixture of two regioisomeric compounds (2.0 kg, yield 54% as a mixture, ratio 3:1). LCMS (ES) m/z 285.12 [M+H]+. e) Ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate A 20 L 4-neck round bottom flask was charged with a mixture of ethyl 2-(4-chloro-6-methoxybenzo[b]thiophen-3-yl)acetate and ethyl 2-(6-chloro-4-methoxybenzo[b]thiophen-3-yl)acetate (mixture 1.0 kg, 3511 mmol) in DCM (9 L). The reaction mass was cooled to −78° C. BBr3 (neat, 663.85 mL, 6605.54 mmol) was added at −78° C. slowly dropwise into the above reaction mixture under nitrogen atmosphere for 45 min and stirred at −78° C. for 20 min (Note: The reaction mixture turned brick red and precipitation was observed on the walls of RB flask). The reaction mixture was allowed to attain 0° C. and stirred for 2 h (Note: The reaction mixture turned brick red and precipitation to wine red color liquid observed). Progress of the reaction was monitored by TLC. (10% EtOAc in pet ether, Rf value of the product is 0.3). After completion of the reaction, the reaction mass was poured into ice cold water slowly dropwise. (Note: Exothermic reaction while quenching of reaction). DCM was added to the above reaction mixture and the resulting mixture was stirred at 25° C. for 10 min. The aqueous and DCM layers were separated. The aqueous layer was again extracted with DCM. The combined organic layers were washed with DM water and the organic layer was separated and again washed with DM water. The combined organic layers were washed with sodium chloride solution. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated below 35-40° C. under vacuum. After completion of evaporation, the orange red color solid was subjected to drying by rotary evaporation at 40-45° C. for 1.0 h. Drying was terminated and an orange red color solid was obtained (800 g, crude) as a mixture of isomers with a ratio of 7:2. The crude solid (mixture of isomers) was taken up in MTBE (2000 mL) and stirred at 25° C. for 30 min then cooled to 0° C. for 20 min. The solid was collected by filtration, washed with cold MTBE (500 mL), and dried by suction to afford ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate as an off-white solid (500 g, yield 52%). 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H) 7.39 (s, 1H), 7.31 (d, J=2.4 Hz, 1H), 6.87 (d, J=2.4 Hz, 1H), 4.24 (q, J=9.2 Hz, 2H), 4.08 (s, 2H), 1.30 (t, J=6.9 Hz, 3H). LCMS (ES) m/z 271.15 [M+H]+. f) Ethyl (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate A 5 L 4-neck round bottom flask was charged with a mixture of (R)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol (107 g, 791.79 mmol, see Scheme-14 for preparation) in THF (2.14 L). Ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (214 g, 791.79 mmol) was added at 25° C. (Note: The reaction mixture turns a brown color). Tri-n-butylphosphine (480 g, 2374.93 mmol) and DIAD (480 g, 2374.93 mmol) were added at 25° C. The reaction mass was stirred at 25° C. for 16 h (Note: Reaction mixture turns a brown color liquid). Completion of the reaction was monitored by TLC. (50% EtOAc in pet ether, Rf value of the product is 0.4). After completion of the reaction, the reaction mass was quenched with ice cold water slowly. EtOAc was added to the above reaction mixture and the resulting mixture stirred at 25° C. for 10 min. The Aqueous and EtOAc layers were separated. The aqueous layer was again extracted with EtOAc. The aqueous and EtOAc layers were separated. The combined organic layers were washed with DM water and the organic layer was separated. The organic layer was washed with sodium chloride solution. The EtOAc layer was dried over anhydrous Na2SO4 and filtered. EtOAc was evaporated below 40-45° C. under vacuum. After completion of evaporation, the black color liquid was subjected to drying by rotary evaporation at 40-45° C. for 1.0 h. Drying was terminated and a brown color gum was obtained (400 g). A chromatography column was packed with silica gel (700 g, 100-200 mesh). The crude compound was dissolved in DCM and adsorbed onto silica gel (300 g) and loaded onto the column. The mobile phase was run with n-hexane (20 L) followed by increasing the polarity from 2-10% EtOAc in hexane (60 L). All pure fractions (by TLC) were collected and concentrated under reduced pressure at 40-45° C. to obtain pure product (210 g, yield 68%). 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J=4.8 Hz, 1H), 7.70 (d, J=4.8 Hz, 1H), 7.49 (s, 1H), 7.23-7.28 (m, 2H), 7.13 (d, J=2.1 Hz, 1H), 5.81-5.80 (m, 1H), 4.21-4.20 (q, 2H), 4.01 (s, 2H), 3.30-3.20 (m, 1H), 3.10-3.00 (m, 1H), 2.66-2.68 (m, 1H), 2.12-2.10 (m, 1H), 1.30 (t, J=6.9 Hz, 3H). LCMS (ES) m/z 388.08 [M+H]+. g) (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid A 10 L 4-neck round bottom flask was charged with (S)-ethyl 2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (400 g, 1031 mmol) in THF (2 L), methanol (2 L), and DM water (1 L) at 25° C. Lithium hydroxide (123 g, 5156 mmol) was added at 25° C. slowly into the above reaction mixture (Note: The reaction mixture turned a brown color). The reaction mass was stirred at 25° C. for 2 h. The reaction was monitored by TLC (80% EtOAc in pet ether, Rf value of the product is 0.2). After completion of the reaction, the reaction mass was poured into ice cold water. The reaction mass was acidified with 10% NaHSO4 pH˜6 solution and a white precipitate formed. The solid was collected by filtration, washed with DM water, and dried by suction. The solid was washed with diethyl ether and dried for 2 h. The solid was further dried by rotary evaporation below 45-50° C. under vacuum for 10 h. The solid compound was further triturated with MTBE, filtered, and dried to obtain the desired compound as an off-white solid (190 g, yield 51%). 1H NMR (300 MHz, DMSO-d6) δ 12.3-12.4 (br, 1H), 8.50-8.52 (dd, J=1.2, 4.8 Hz, 1H), 7.78-7.83 (m, 2H), 7.49 (s, 1H), 7.23-7.28 (m, 1H), 7.13 (d, J=2.1 Hz, 1H), 6.00-6.04 (m, 1H), 4.01 (s, 2H), 3.06-3.11 (m, 1H), 2.95-2.98 (m, 1H), 2.66-2.68 (m, 1H), 2.09-2.12 (m, 1H). LCMS (ES) m/z 359.98 [M+H]+. Chiral HPLC: 99.70%. a) 6,7-Dihydro-5H-cyclopenta[b]pyridin-5-one A 50 L glass reactor was charged KMnO4 (1857 g, 11748.0 mmol). Water (35 L) was charged to the reaction mass. The reaction mass was stirred for 30 min (Note: KMnO4 completely soluble in water). Another 100 L reactor was charged 6,7-dihydro-5H-cyclopenta[b]pyridine (700 g, 5874 mmol) in tert-butanol (17.5 L) at 25° C. MgSO4 (1414 g, 11748 mmol) was charged to the above reaction mixture. The reaction mixture was cooled to 20-25° C. with ice water. KMnO4 solution was added dropwise for 2 h (Note: Slight exothermic was observed and temperature maintained below 30° C. with ice water). The reaction mixture was maintained at 30° C. for 3 h. Progress of the reaction was monitored by TLC/LCMS (50% EtOAc in pet ether, Rf value of the product is 0.3). After completion of the reaction, EtOAc was added to the above reaction mixture and the resulting mixture stirred at 25° C. for 10 min. The aqueous and EtOAc layers were separated. The aqueous layer was again extracted with EtOAc. The combined organic layers were washed with DM water and the organic layer was separated. The combined organic layers were washed with sodium chloride solution. The EtOAc layer was dried over anhydrous Na2SO4 and filtered. EtOAc was evaporated below 40-45° C. under vacuum. After completion of evaporation, the brown color liquid was subjected to drying by rotary evaporation at 40-45° C. for 1.0 h. Drying was terminated and a brown color gum was obtained (600 g). A chromatography column was packed with silica gel (4.0 kg, 100-200 mesh). The crude compound was dissolved in DCM and adsorbed onto silica gel (1.0 kg) and loaded onto the column. The mobile phase was run with n-hexane (25 L) followed by increasing the polarity from 2-10% EtOAc in hexane (100 L). All pure fractions (TLC) were collected and concentrated under reduced pressure at 40-45° C. to give 6,7-dihydro-5H-cyclopenta[b]pyridin-5-one as a brown thick gum (270 g, yield 34%). 1H NMR (400 MHz, CDCl3) δ 8.45 (dd, J=1.5, 4.5 Hz, 1H), 8.02 (dd, J=2.0, 8.0 Hz, 1H), 7.46-7.44 (m, 1H), 3.19-3.16 (m, 2H), 2.73-2.71 (m, 2H). LCMS (ES) m/z 134.07 [M+H]+. b) (R)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol A 5 L 4-neck round bottom flask was charged 6,7-dihydro-5H-cyclopenta[b]pyridine-5-one (100 g, 751.04 mmol) in EtOAc (2 L). TEA (523 mL, 3755.2 mmol) was added at 25° C. slowly into the above reaction mixture. The reaction mass was cooled to 0° C. and formic acid (346 g, 7510.4 mmol) was added dropwise over 30 min (Note: thick white fumes were observed). The above reaction mixture was stirred at 0° C. for 30 min. RuCl[(R,R)-Tsdpen](mesitylene) (9.36 g, 15.02 mmol) was added at 0° C. The reaction mixture was maintained at 45° C. for 16 h. Progress of the reaction was monitored by TLC. (50% EtOAc in pet ether, Rf value of the product is 0.4). After completion of the reaction, the reaction mass was directly evaporated by rotary evaporation below 40-45° C. under vacuum. A chromatography column was packed with silica gel (500 g, 100-200 mesh). The crude compound was directly loaded onto the column. The mobile phase was run with n-hexane (25 L) followed by increasing the polarity from 2-80% EtOAc in hexane (50 L). All pure fractions (by TLC) were collected and concentrated under reduced pressure at 40-45° C. to give a gummy liquid, which was triturated with diethyl ether (2×100 mL) and filtered by suction to afford (R)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-ol as a pale brown solid (80 g, yield 79%). 1H NMR (300 MHz, CDCl3) δ 8.45 (dd, J=1.2, 3.6 Hz, 1H), 7.71 (d, J=7.6 Hz, 1H), 7.14 (q, J=4.8 Hz, 1H), 5.29 (d, J=5.6 Hz, 1H), 3.20-3.12 (m, 2H), 2.98-2.89 (m, 1H), 2.62-2.54 (m, 1H), 2.03-1.98 (m, 1H). LCMS (ES) m/z 136.17 [M+H]+. Crystalline Compound of Example 1a, (S)-2-(4-chloro-6-((6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid The X-ray powder diffraction (XRPD) pattern of this material is shown in FIG. 1 and a summary of the diffraction angles and d-spacings is given in Table I below. The XRPD analysis was conducted on a PANanalytical X'Pert Pro Diffractometer on Si zero-background wafers using X'celerator™ RTMS (Real Time Multi-Strip) detector. The acquisition conditions included: Cu Kα radiation, generator tension: 45 kV, generator current: 40 mA, step size: 0.0167° 2θ. Configuration on the incidental beam side: 10 mm programmable divergence slit, 0.02 rad Soller slits, anti-scatter slit (0.5°), and 10 mm beam mask. Configuration on the diffracted beam side: 10 mm programmable anti-scatter slit assembly (X'celerator module) and 0.02 rad Soller slit. TABLE I Diff. Angle [°2θ] d-spacing [Å] 5.876 15.0412 13.6014 6.51043 14.0485 6.30422 14.3468 6.17377 21.8816 4.06196 22.461 3.95847 23.0524 3.85824 23.3301 3.81294 24.1261 3.6889 24.5102 3.63196 24.6985 3.6047 25.6652 3.47107 26.0846 3.41621 26.6086 3.35011 27.4121 3.25371 The differential scanning calorimetry (DSC) thermogram of this material was recorded on a TA Instruments Discovery Differential Scanning calorimeter equipped with an autosampler and a refrigerated cooling system under 40 mL/min N2 purge and is shown in FIG. 2. The experiments were conducted using a heating rate of 10° C./min to final temperature of 350° C. in a lightly crimped aluminum pan. The thermogravimetric analysis (TGA) thermogram of this material was recorded on a TA Instruments Discovery Thermogravimetric Analyzer and is shown in FIG. 2. The experiments were conducted under N2 purge and a heating rate of 10° C./min to final temperature of 350° C. in an open aluminum pan. This compound has a simple single melting event in DSC, with onset temperature of 220.8 C, peak temperature of 223.4 C and melting enthalpy of 120 J/g. The determination of melting enthalpy is not reliable due to the immediate thermal decomposition post melting. The compound exhibited negligible weight loss by loss by TGA prior to the decomposition event. A person skilled in the art would recognize that the onset temperature, peak temperature, and enthalpy of the endotherm may vary depending on the experimental conditions. Example 2 Preparation of 2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) Ethyl 2-(4-chloro-6-(2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yloxy)benzo[b]thiophen-3-yl)acetate The crude 5-Chloro-2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridine (25 g) was dissolved in DMF (250 mL) at RT and to this ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (25 g, 92 mmol) and K2CO3 (63.8 g, 462 mmol) were added at RT. The reaction mixture was heated to 80° C. for 2 h. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed successively with water and brine, dried over MgSO4 and concentrated in vacuo to get crude. The crude residue was purified by silica gel column chromatography (EtOAc/hexane) to give the title compound (25 g) as an off white solid. LCMS (ES) m/z 402.17 [M+H]+. b) 2-(4-Chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid The title compound was obtained in a same manner as the procedure in Example 1, Step b by using ethyl 2-(4-chloro-6-(2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yloxy)benzo[b]thiophen-3-yl)acetate as an off white solid. 1H NMR (300 MHz, DMSO-d6): δ 7.76 (d, J=2.1 Hz, 1H), 7.68 (d, J=8.1 Hz, 1H), 7.47 (s, 1H), 7.11-7.08 (m, 2H), 5.98-5.95 (m, 1H), 4.00 (s, 2H), 3.17-3.01 (m, 1H), 2.93-2.83 (m, 1H), 2.67-2.59 (m, 1H), 2.47 (s, 3H), 2.13-2.04 (m, 1H); LCMS (ES) m/z 374.09 [M+H]+. Chiral HPLC: 49.85%: 50.14%. Analytical SFC Condition Column/dimensions: Chiralpak AD-H (250×4.6) mm, 5μ % CO2: 60.0% % Co solvent: 40.0% (100% Methanol) Total Flow: 4.0 g/min Back Pressure: 100 bar Temperature: 30.0° C. UV: 235 nm Preparative SFC Condition Column/dimensions: Lux Amylose-1 (250×30) mm, 5μ % CO2: 55.0% % Co solvent: 45.0% (100% Methanol) Total Flow: 90.0 g/min Back Pressure: 100.0 bar UV: 235 nm Stack time: 5.3 min Load/Inj: 82.0 mg Retention time: Peak 1—3.02 min, Peak 2—4.93 min. Purity: Peak 1—99.91%, Peak 2—99.24%. Solubility: Methanol (660 mL)+12 ml DEA Instrument details: Make/Model: Thar SFC-200-002 Example 2a (S)-2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (5.1 g, 30.7% yield). 1H NMR (300 MHz, DMSO-d6): δ 7.76 (d, J=2.4 Hz, 1H), 7.68 (d, J=7.8 Hz, 1H), 7.47 (s, 1H), 7.11-7.08 (m, 2H), 5.98-5.95 (m, 1H), 4.00 (s, 2H), 3.06-2.89 (m, 2H), 2.64-2.62 (m, 1H), 2.47 (s, 3H), 2.10-2.06 (m, 1H). LCMS (ES) m/z 374.09 [M+H]+. Chiral HPLC: 99.91%. Example 2b (R)-2-(4-chloro-6-((2-methyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (3.0 g, 18.24% yield). 1H NMR (300 MHz, DMSO-d6): δ 7.76 (d, J=2.4 Hz, 1H), 7.68 (d, J=7.8 Hz, 1H), 7.47 (s, 1H), 7.11-7.08 (m, 2H), 5.98-5.95 (m, 1H), 4.00 (s, 2H), 3.06-2.89 (m, 2H), 2.64-2.62 (m, 1H), 2.47 (s, 3H), 2.10-2.06 (m, 1H). LCMS (ES) m/z 374.24 [M+H]+. Chiral HPLC: 99.24%. Example 3 Preparation of 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) Ethyl 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate To the crude 2,5-dichloro-6,7-dihydro-5H-cyclopenta[b]pyridine (100 mg) dissolved in DMF was added to a stirred solution of ethyl 2-(4,7-dichloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (100 mg, 0.328 mmol) and K2CO3 (181 mg, 1.311 mmol) in DMF (5 mL) at ambient temperature and then heated to 100 for 2 h. After TLC analysis the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude. The crude was purified by silica gel chromatography using 30% EtOAc/pet ether as an eluent to afford ethyl 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (60 mg, 40.0% yield) as an oily liquid. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J=8.0 Hz, 1H), 7.31-7.18 (m, 3H), 5.78-5.75 (m, 1H), 4.22 (q, J=8 Hz, 2H), 4.09 (s, 2H), 3.35-3.27 (m, 1H), 3.08-3.00 (m, 1H), 2.69-2.64 (m, 1H), 2.42-2.39 (m, 1H), 1.29 (t, J=8 Hz, 3H). LCMS (ES) m/z 456.79 [M+H]+. b) 2-(4,7-Dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid To a stirred solution of ethyl 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (60 mg, 0.131 mmol) in methanol (2 mL), THF (2 mL) and water (2 mL), LiOH (6.29 mg, 0.263 mmol) was added at ambient temperature and stirred for 4 h. After TLC analysis the reaction mixture was evaporated to remove solvents and the crude was cooled to 0° C., acidified with saturated citric acid solution (pH˜5). Obtained solids were filtered and dried well to afford 2-(4,7-dichloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (46 mg, 81% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.47 (brs, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.65-7.61 (m, 2H), 7.39 (d, J=8.0 Hz, 1H) 6.10-6.08 (m, 1H), 4.03 (s, 2H), 3.19-3.11 (m, 1H), 2.99-2.92 (m, 1H), 2.71-2.62 (m, 1H), 2.20-2.10 (m, 1H). LCMS (ES) m/z 428 [M+H]+. Chiral HPLC: 48.83%: 51.16%. Analytical SFC Conditions Column/dimensions: Chiralpak AD-H (4.6×250 mm), 5μ % CO2: 60.0% % Co solvent: 40.0% (100% MeOH) Total Flow: 4.0 g/min Back Pressure: 100 bar Temperature: 30° C. UV: 235 nm Preparative SFC Conditions Column/dimensions: Chiralpak AD-H (30×250 mm), 5μ % CO2: 50.0% % Co solvent: 50.0% (100% MeOH) Total Flow: 90.0 g/min Back Pressure: 100.0 bar UV: 235 nm Stack time: 15.3 min Load/Inj: 48.3 mg Retention time: Peak 1—3.59 min, Peak 2—13.34 min. Purity: Peak 1—99.06%, Peak 2—99.73%. Solubility: 20 ml MeOH+few drops of methanolic ammonia solution Instrument details: Make/Model: SFC-200-003 Chiral Separation of Example 3 Example 3a (First Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6) δ 12.47 (brs, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.65-7.61 (m, 2H), 7.39 (d, J=8.0 Hz, 1H) 6.10-6.08 (m, 1H), 4.03 (s, 2H), 3.19-3.11 (m, 1H), 2.99-2.92 (m, 1H), 2.71-2.62 (m, 1H), 2.20-2.10 (m, 1H). LCMS (ES) m/z 427.9 [M+H]+. Chiral HPLC: 99.06%. Example 3b (Second Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6) δ 12.47 (brs, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.65-7.61 (m, 2H), 7.39 (d, J=8.0 Hz, 1H) 6.10-6.08 (m, 1H), 4.03 (s, 2H), 3.19-3.11 (m, 1H), 2.99-2.92 (m, 1H), 2.71-2.62 (m, 1H), 2.20-2.10 (m, 1H). LCMS (ES) m/z 427.99 [M+H]+. Chiral HPLC: 99.77% Example 4 Preparation of 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) Ethyl 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate To the crude 5-Chloro-2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridine (120 mg) dissolved in DMF was added to a stirred solution of ethyl 2-(4,7-dichloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (100 mg, 0.328 mmol) and K2CO3 (181 mg, 1.311 mmol) in DMF (5 mL) at ambient temperature and then heated to 100° C. for 2 h. After TLC analysis the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude. The crude was purified by silica gel chromatography using 30% EtOAc/pet. ether as an eluent to afford ethyl 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (50 mg, 30.9% yield) as an oily liquid. 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J=8.0 Hz, 1H), 7.58 (d, J=8 Hz, 1H), 7.26 (s, 1H), 7.20 (s, 1H), 5.82-5.78 (m, 1H), 4.22 (q, J=8 Hz, 2H), 4.10 (s, 2H), 3.42-3.35 (m, 1H), 3.18-3.10 (m, 1H), 2.75-2.69 (m, 1H), 2.46-2.41 (m, 1H), 1.27 (t, J=8 Hz, 3H). LCMS (ES) m/z 490.67 (M+H)+. b) 2-(4,7-Dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid To a stirred solution of ethyl 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (50 mg, 0.102 mmol) in methanol (2 mL), THF (2 mL) and water (2 mL), LiOH (4.88 mg, 0.204 mmol) was added at ambient temperature and stirred for 4 h. After TLC analysis the reaction mixture was evaporated to remove solvents and the crude was cooled to 0° C., acidified with saturated citric acid solution (pH˜5). Obtained solids were filtered and dried well to afford 2-(4,7-dichloro-6-((2-(trifluoromethyl)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (40 mg, 85% yield) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.48 (brs, 1H), 8.08 (d, J=7.5 Hz, 1H), 7.80 (d, J=7.8 Hz, 1H), 7.70 (s, 1H), 7.63 (s, 1H), 6.21-6.17 (m, 1H), 4.04 (s, 2H), 3.31.3.11 (m, 1H), 3.08-3.01 (m, 1H), 2.79-2.67 (m, 1H), 2.28-2.18 (m, 1H). LCMS (ES) m/z 462.17 [M+H]+. Chiral HPLC 47.52%: 52.47%. Analytical SFC Condition Column/dimensions: Chiralpak AD-H (4.6×250 mm), 5μ % CO2: 60.0% % Co solvent: 40.0% (100% methanol) Total Flow: 4.0 g/min Back Pressure: 100 bar Temperature: 30° C. UV: 234 nm Preparative SFC Condition Column/dimensions: Chiralpak AD-H (30×250 mm), 5μ % CO2: 60.0% % Co solvent: 40.0% (100% methanol) Total Flow: 90.0 g/min Back Pressure: 100.0 bar UV: 234 nm Stack time: 8.5 min Load/Inj: 50.0 mg Retention time: Peak 1—1.82 min, Peak 2—5.75 min. Purity: Peak 1—99.68%, Peak 2—99.82%. Solubility: Methanol+ACN Instrument details: Make/Model: SFC-PIC SOLUTION Chiral Separation of Example 4 Example 4a (First Eluted Enantiomer) 1H NMR (500 MHz, CDCl3): δ 7.88 (d, J=8.0 Hz, 1H), 7.58 (d, J=7.5 Hz, 1H), 7.30 (s, 1H), 7.22 (s, 1H), 5.84-5.82 (m, 1H), 4.17 (s, 2H), 3.41-3.36 (m, 1H), 3.18-3.11 (m, 1H), 2.75-2.69 (m, 1H), 2.46-2.40 (m, 1H). LCMS (ES) m/z 462.14 [M+H]+, HPLC: 99.69%. Chiral HPLC: 99.81%. Example 4b (Second Eluted Enantiomer) 1H NMR (500 MHz, CDCl3): δ 7.88 (d, J=8.0 Hz, 1H), 7.58 (d, J=7.5 Hz, 1H), 7.30 (s, 1H), 7.22 (s, 1H), 5.83-5.81 (m, 1H), 4.15 (s, 2H), 3.40-3.35 (m, 1H), 3.17-3.11 (m, 1H), 2.73-2.70 (m, 1H), 2.44-2.39 (m, 1H); LCMS (ES) m/z 462.11 [M+H]+, HPLC: 99.94%, Chiral HPLC: 99.82% Example 5 Preparation of 2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) Ethyl 2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate To a stirred solution of ethyl 2-(4-chloro-7-fluoro-6-hydroxybenzo[b]thiophen-3-yl)acetate (0.141 g, 0.487 mmol), 6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-ol (0.100 g, 0.487 mmol) and ADDP (0.123 g, 0.487 mmol) in THF (10 mL) was added tri-n-butylphosphine (0.120 mL, 0.487 mmol) at RT. The reaction mixture was stirred at room temperature for 48 h, filtered through Celite® and evaporated under reduced pressure to afford crude product as yellow liquid, which was purified by flash column chromatography on 100-200 silica gel, using 30% EtOAc-Pet ether as an eluent to obtained ethyl 2-(4-chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate (0.090 g, 37.5% yield) as pale yellow liquid. LCMS (ES) m/z 476.05 [M+H]+. b) 2-(4-Chloro-7-fluoro-6-((6-(trifluoromethyl)-2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid The title compound was prepared as a white solid according to the procedures of examples XX as white solid. 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, J=7.6 Hz, 1H), 7.60 (d, J=6.8 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.46 (s, 1H), 6.31-6.30 (m, 1H), 4.94-4.83 (m, 2H), 3.77 (s, 2H). LCMS (ES) m/z 448.16 [M+H]+. Chiral HPLC: 50.55%: 49.45%. Analytical SFC Condition Column/dimensions: Chiralcel OJ-H (4.6×250 mm), 5μ % CO2: 80.0% % Co solvent: 20.0% (100% MeOH) Total Flow: 4.0 g/min Back Pressure: 100 bar Temperature: 30° C. UV: 214 nm Preparative SFC Condition Column/dimensions: Chiralcel OJ-H (21×250 mm), 5μ % CO2: 90.0% % Co solvent: 10.0% (100% MeOH) Total Flow: 60.0 g/min Back Pressure: 100.0 bar UV: 214 nm Stack time: 4.3 min Load/Inj: 2.5 mg Retention time: Peak 1—2.93 min, Peak 2—4.89 min. Purity: Peak 1—99.59%, Peak 2—99.30%. Solubility: MeOH Instrument details: Make/Model: SFC-80 Chiral Separation of Example 5 Example 5a (First Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, J=7.6 Hz, 1H), 7.60 (d, J=6.8 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.46 (s, 1H), 6.31-6.30 (m, 1H), 4.94-4.83 (m, 2H), 3.77 (s, 2H). LCMS (ES) m/z 447.82 [M+H]+. Chiral HPLC: 99.59%. Example 5b (Second Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6): δ 8.09 (d, J=7.6 Hz, 1H), 7.60 (d, J=6.8 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 7.46 (s, 1H), 6.31-6.30 (m, 1H), 4.94-4.83 (m, 2H), 3.77 (s, 2H). LCMS (ES) m/z 448.26 [M+H]+. Chiral HPLC: 99.30%. Example 6 Preparation of 2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) Ethyl 2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate The title compound was obtained in a same manner as the procedure in Example 5, Step a by using 2,3-dihydrofuro[2,3-b]pyridin-3-ol and 5-chloro-6,7-dihydro-5H-cyclopenta[b]pyridine, LCMS (ES) m/z 390.34 (M+H)+. b) 2-(4-Chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid To a solution of ethyl 2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate (1.7 g, 4.36 mmol) in Methanol (16 mL), THF (8 mL) and Water (16 mL) was added LiOH (0.522 g, 21.80 mmol) at rt and stirred for 2 h at same temperature. Reaction mixture was acidified with citric acid solution (nearly pH=6-7), filtered the solid precipitated and dried under vacuum to get 2-(4-chloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (1 g, 2.73 mmol, 62.7% yield) as an white solid. 1H NMR (400 MHz, DMSO-d6): δ 12.39 (brs, 1H), 8.19 (dd, J=2.0, 5.2 Hz, 1H), 7.92 (dd, J=1.6, 7.2 Hz, 1H), 7.79 (d, J=2.4 Hz, 1H), 7.51 (s, 1H), 7.15 (d, J=2.8 Hz, 1H), 7.00 (dd, J=4.8, 7.4 Hz, 1H), 6.23-6.21 (m, 1H), 4.85-4.81 (m, 1H), 4.62-4.59 (m, 1H), 4.01 (s, 2H). ESI-MS m/z 362.13 [M+H]+. Chiral HPLC: 48.09%: 50.68%. Analytical SFC Condition Column/dimensions: Chiralpak AS-H (4.6×250 mm), 5μ % CO2: 65.0% % Co solvent: 35.0% (100% methanol) Total Flow: 3.0 g/min Back Pressure: 100 bar Temperature: 30° C. UV: 235 nm Preparative SFC Condition Column/dimensions: Chiralpak AS-H (30×250 mm), 5μ % CO2: 65.0% % Co solvent: 35.0% (100% methanol) Total Flow: 100.0 g/min Back Pressure: 100.0 bar UV: 235 nm Stack time: 6.5 min Load/Inj: 18.0 mg Retention time: Peak 1—3.41 min, Peak 2—4.92 min. Purity: Peak 1—99.82%, Peak 2—99.50%. Solubility: Methanol+ACN Instrument details: Make/Model: SFC-200-004 (PIC-Solution) Chiral Separation of Example 6 Example 6a (First Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6): δ 12.39 (brs, 1H), 8.19 (dd, J=2.0, 5.2 Hz, 1H), 7.92 (dd, J=1.6, 7.2 Hz, 1H), 7.79 (d, J=2.4 Hz, 1H), 7.51 (s, 1H), 7.15 (d, J=2.8 Hz, 1H), 7.00 (dd, J=4.8, 7.4 Hz, 1H), 6.23-6.21 (m, 1H), 4.87-4.80 (m, 1H), 4.64-4.57 (m, 1H), 4.01 (s, 2H). LCMS (ES) m/z 362.13 [M+H]+. Chiral HPLC purity: 99.80%. Example 6b (Second Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6): δ 12.39 (brs, 1H), 8.19 (dd, J=2.0, 5.2 Hz, 1H), 7.92 (dd, J=1.6, 7.2 Hz, 1H), 7.79 (d, J=2.4 Hz, 1H), 7.51 (s, 1H), 7.15 (d, J=2.8 Hz, 1H), 7.00 (dd, J=4.8, 7.4 Hz, 1H), 6.23-6.21 (m, 1H), 4.87-4.80 (m, 1H), 4.64-4.57 (m, 1H), 4.01 (s, 2H). LCMS (ES) m/z 362.13 [M+H]+. Chiral HPLC purity: 99.50%. Example 7 Preparation of 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid a) Ethyl 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate To a stirred solution of ADDP (827 mg, 3.28 mmol) in dry THF (2 mL), tri-n-butylphosphine (1.079 mL, 4.38 mmol) was slowly added at ambient temperature. After decolorisation was observed, 2,3-dihydrofuro[2,3-b]pyridin-3-ol (300 mg, 2.188 mmol) was added and stirred for 5 min. Finally ethyl 2-(4,7-dichloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (668 mg, 2.188 mmol) was added. The reaction mass was stirred at the same temperature for 24 h. After TLC analysis the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure to afford crude. The crude was purified by silica gel chromatography using 30% EtOAc/pet ether as an eluent to afford ethyl 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate (120 mg) as a colorless gummy liquid. LCMS (ES) m/z 424.14 [M+H]+ b) 2-(4,7-Dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid To a stirred solution of ethyl 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetate (120 mg, 0.283 mmol) in methanol (1 mL), THF (1 mL) and water (1.000 mL), lithium hydroxide (20.32 mg, 0.848 mmol) was added at ambient temperature and stirred for 4 h. After TLC analysis the reaction mixture was evaporated to remove solvents and the crude was cooled to 0° C. and acidified with saturated citric acid solution (pH˜5). Obtained solids were filtered and dried well to afford 2-(4,7-dichloro-6-((2,3-dihydrofuro[2,3-b]pyridin-3-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (72 mg, 63.7% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.21-8.20 (m, 1H), 7.85-7.84 (m, 1H), 7.63 (s, 1H), 7.54 (s, 1H), 7.02-6.99 (m, 1H), 6.31 (d, J=4.5 Hz, 1H), 4.83-4.74 (m, 1H), 4.67-4.64 (m, 1H), 3.92 (s, 2H). LCMS (ES) m/z 396.25 [M+H]+. Chiral HPLC: 48.99%: 49.56%. Analytical SFC Condition Column/dimensions: Chiralpak AD-H (4.6×250 mm), 5μ % CO2: 60.0% % Co solvent: 40.0% (100% MeOH) Total Flow: 4.0 g/min Back Pressure: 100 bar Temperature: 30° C. UV: 214 nm Preparative SFC Condition Column/dimensions: Chiralpak AD-H (30×250 mm), 5μ % CO2: 60.0% % Co solvent: 40.0% (100% MeOH) Total Flow: 90.0 g/min Back Pressure: 90.0 bar UV: 214 nm Stack time: 8.0 min Load/Inj: 25.0 mg Retention time: Peak 1-2.73 min, Peak 2—5.34 min. Purity: Peak 1-99.81%, Peak 2—98.63% Solubility: MeOH+Acetonitrile Chiral Separation of Example 7 Example 7a (First Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6) δ 12.89 (brs, 1H), 8.21-8.20 (m, 1H), 7.85-7.84 (m, 1H), 7.63 (s, 1H), 7.54 (s, 1H), 7.02-6.99 (m, 1H), 6.31 (d, J=4.5 Hz, 1H), 4.83-4.74 (m, 1H), 4.67-4.64 (m, 1H), 3.92 (s, 2H). LCMS (ES) m/z 396.15 [M+H]+. Chiral purity: 99.81%. Example 7b (Second Eluted Enantiomer) 1H NMR (400 MHz, DMSO-d6) δ 12.89 (brs, 1H), 8.21-8.20 (m, 1H), 7.85-7.84 (m, 1H), 7.63 (s, 1H), 7.54 (s, 1H), 7.02-6.99 (m, 1H), 6.31 (d, J=4.5 Hz, 1H), 4.83-4.74 (m, 1H), 4.67-4.64 (m, 1H), 3.92 (s, 2H). LCMS (ES) m/z 396.25 [M+H]+. Chiral purity: 98.63% Examples 8 and 9 Preparation of 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid (Example 8) a) Ethyl 2-(4-chloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate 2,5-Dichloro-6,7-dihydro-5H-cyclopenta[b]pyridine (556 mg, 2.95 mmol) was added to the stirred solution of K2CO3 (1225 mg, 8.86 mmol) and ethyl 2-(4-chloro-6-hydroxybenzo[b]thiophen-3-yl)acetate (800 mg, 2.95 mmol) in DMF (20 mL) at 0° C. and the mixture was stirred at 60° C. for 2 h. The reaction mixture was diluted with water and mixture was concentrated under reduced pressure. The resulted residue was partitioned between EtOAc and water, the separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 20% EtOAc in pet ether) to obtained ethyl 2-(4-chloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (600 mg, 43.0% yield) as a colorless liquid. LCMS (ES) m/z 422.10 [M+H]+. b) Ethyl 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate Tetrakis (164 mg, 0.142 mmol) was added to a degassed solution of ethyl 2-(4-chloro-6-((2-chloro-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (600 mg, 1.421 mmol) and dicyanozinc (167 mg, 1.421 mmol) in DMF (10 mL). The mixture was further degassed for 10 min and heated to 120° C. for 1 h under microwave condition. The reaction mixture was filtered through a pad of Celite® and filtrate was partitioned between EtOAc and water. The separated organic layer was washed with brine solution, dried over anhydrous Na2SO4, filtered and filtrate was evaporated under reduced pressure to get the crude. The crude was purified by silica gel column chromatography by using EtOAc in hexane as eluent. The product was eluted at 40% EtOAc-Pet ether to get ethyl 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (380 mg, 64.4% yield) as an off-white solids. 1H NMR (500 MHz, DMSO-d6): δ 8.06 (d, J=8.0 Hz, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.82 (d, J=2.0 Hz, 1H), 7.53 (s, 1H), 7.17 (d, J=2.0 Hz, 1H), 6.10-6.07 (m, 1H), 4.13 (q, J=7.0 Hz 2H), 4.01 (s, 2H), 3.20-3.13 (m, 1H), 3.07-3.03 (m, 1H), 2.77-2.72 (m, 1H), 2.16-2.12 (m, 1H), 1.20 (t, J=7.5 Hz, 3H). LCMS (ES) m/z 413.25 [M+H]+. c) 2-(4-Chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic Acid 3N HCl (10 mL, 30.0 mmol) was added to a stirred solution of ethyl 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetate (0.35 g, 0.848 mmol) in THF (50 mL) at 0° C. The reaction mixture was stirred and heated to 70° C. for 8 h. Evaporated the excess of solvents under reduced pressure and water was added to the reaction mixture. The precipitated solid was filtered and dried under vacuum to get crude material. The resulted crude compound was purified by flash column chromatography (100-200 silica mesh, eluent was 3% MeOH-DCM to obtained 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (0.2000 g, 58.8% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6): δ 12.36 (brs, 1H), 8.06 (d, J=7.6 Hz, 1H), 7.91 (d, J=7.6 Hz, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.50 (s, 1H), 7.17 (d, J=2.0 Hz, 1H), 6.10-6.07 (m, 1H), 4.01 (s, 2H), 3.20-3.13 (m, 1H), 3.07-3.03 (m, 1H), 2.77-2.72 (m, 1H), 2.16-2.12 (m, 1H). LCMS (ES) m/z 385.10 [M+H]+. d) 2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic Acid: (Example 9) H2O2 (0.064 mL, 2.079 mmol) was added to a stirred solution of 2-(4-chloro-6-((2-cyano-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)benzo[b]thiophen-3-yl)acetic acid (400 mg, 1.039 mmol) in KOH (117 mg, 2.079 mmol) and ethanol (50 mL) at 0° C. The reaction mixture was stirred at RT for 1 h. The reaction mixture was concentrated under reduced pressure to get crude. Water (10 mL) was added and adjusted acidic pH by using 2N citric acid solution and then filtered the precipitated solid. The solid was washed with n-pentane to get 2-(6-((2-carbamoyl-6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl)oxy)-4-chlorobenzo[b]thiophen-3-yl)acetic acid (200 mg, 47.7% yield) as an off-white solid. 1H NMR (500 MHz, DMSO-d6): δ 12.35 (brs, 1H), 8.06 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.64 (s, 1H), 7.49 (s, 1H), 7.16 (d, J=2.0 Hz, 1H), 6.08-6.06 (m, 1H), 4.01 (s, 2H), 3.20-3.14 (m, 1H), 3.05-3.00 (m, 1H), 2.76-2.72 (m, 1H), 2.17-2.13 (m, 1H). LCMS (ES) m/z 402.82 [M+H]+. Chiral HPLC: 49.44%: 50.55%. Analytical SFC Condition Column/dimensions: Chiralpak-IG (4.6×250 mm), 5μ % CO2: 50.0% % Co solvent: 50.0% (100% MeOH) Total Flow: 4.0 g/min Back Pressure: 100 bar Temperature: 30° C. UV: 214 nm Preparative SFC Condition Column/dimensions: Chiralpak-IG (30×250 mm), 5μ % CO2: 50.0% % Co solvent: 50.0% (100% MeOH) Total Flow: 90.0 g/min Back Pressure: 90.0 bar UV: 214 nm Stack time: 15.5 min Load/Inj: 15.0 mg Retention time: Peak 1-11.41 min, Peak 2—14.44 min. Purity: Peak 1-99.61%, Peak 2—99.07%. Solubility: Few drops of H2O+THF+MeOH Instrument details: Make/Model: SFC-200-003 Chiral Separation of Examples 9 Examples 9a (First Eluted Enantiomer) 1H NMR (500 MHz, DMSO-d6): δ 12.35 (brs, 1H), 8.06 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.64 (s, 1H), 7.49 (s, 1H), 7.16 (d, J=2.0 Hz, 1H), 6.08-6.06 (m, 1H), 4.01 (s, 2H), 3.20-3.14 (m, 1H), 3.05-3.00 (m, 1H), 2.76-2.72 (m, 1H), 2.17-2.13 (m, 1H). LCMS (ES) m/z 403.22 [M+H]+. Chiral HPLC: 99.61%. Examples 9b (Second Eluted Enantiomer) 1H NMR (500 MHz, DMSO-d6): δ 12.35 (brs, 1H), 8.06 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.64 (s, 1H), 7.49 (s, 1H), 7.16 (d, J=2.0 Hz, 1H), 6.08-6.06 (m, 1H), 4.01 (s, 2H), 3.20-3.14 (m, 1H), 3.05-3.00 (m, 1H), 2.76-2.72 (m, 1H), 2.17-2.13 (m, 1H). LCMS (ES) m/z 403.28 [M+H]+. Chiral HPLC: 99.07% Assay Protocol Compounds contained herein were evaluated for their ability to inhibit the activity of GOAT. GOAT activity was assessed using a time-resolved fluorescence energy transfer (TR-FRET) assay in a 384-well format. His-tag human GOAT enzyme was in the form of a cell membrane preparation from sf9 cells infected with hGOAT-V5-His baculovirus. Varying concentrations of test compound with final DMSO concentration kept to 0.5% were added to membrane solution. Human GOAT membrane activity was established in a buffer having final concentration 0.25 mg/mL in 50 mM MOPS, pH7.5; 50 mM KCl; 0.1 mg/mL BSA; 50 μM CHAPS; and 2 mM EDTA. Substrate solution consisting of biotinylated ghrelin peptide (final concentration 100 nM), octanoyl coA (final concentration 2 μM) and palmitoyl CoA (final concentration 50 μM) was added to initiate the reaction. Plates were sealed, centrifuged for 1 minute at 2000 rpm, then incubated at 30° C. for 80 minutes with gentle shaking on an Eppendorf mix plate. Reaction termination and detection mix consisting of chicken anti-active ghrelin antibody (final concentration of 10 nM), Europium W1024-labeled streptavidin (final concentration of 4 nM), GOAT anti-chicken Dylight (final concentration of 12.5 nM), and GS[DAP-oc]-FL-amide inhibitor (final concentration of 1 μM) was added before further incubation for 40 minutes at 30° C. The plate was then read on an Envision in HTRF mode with excitation filter UV (TRF) 340 and first emission filter of APC 665 and a second emission filter of Europium 615. HTRF readings were acquired as per instrument defined LANCE-DELFIA protocol with a delay and window times of 50 μs for both; number of sequential windows: 1; time between flashes: 2000 μs between each of 100 flashes and 10 flashes for the second detector. The HTRF ratio was calculated directly by the instrument as the ratio of 665 window/615 window. Percent inhibition was calculated as 100−(100×(U−NC)/(PC−NC)) where U was the unknown value HTRF ratio (test compound value), NC was the negative control (100% inhibition value generated from a potent inhibitor), and PC was the positive control (100% activity generated from 0.5% DMSO vehicle). IC50 values were generated in GraphPad Prism (Version 4.03) using non-linear regression curve fit and sigmoidal dose response variable slope analysis. Results The exemplified compounds were generally tested according to the above or an analogous assay and were found to be inhibitors of GOAT. Specific biological activities tested according to such assays are listed in the following table as follows (IC50): A<50 nM, B: <500 nM, C: <5000 nM. As variability in such assays is inevitable, repeating the assay run(s) may result in slightly different IC50 values. hGOAT IC50 Example (nM) 1a A 1b C 2a A 2b C 3a B 3b A 4a B 4b A 5a C 5b A 6a A 6b A 7a C 7b A 8  A 9a B 9b A Evaluation in Animal Models The activity of Example 1a was evaluated in vivo in three preclinical species by assessing the level of reduction of acyl ghrelin in circulation after treatment. Acyl Ghrelin Reduction in Mice Normal mice were administered various oral doses of Example 1a on a bid basis for two days (4 doses). Food was withdrawn the evening of the second day after the fourth dose, and then the animals were administered a final dose the morning of the third day. Three hours after this fifth and final dose, blood was collected for ghrelin and acyl ghrelin measurement by ELISA. Dose-dependent decreases in acyl ghrelin and increases in des-acyl ghrelin were observed. As seen in FIG. 3, acyl ghrelin reductions were statistically significant (p<0.05) at both 1 and 10 mg/kg Example 1a. Acyl Ghrelin Reduction in Rats The same experimental design was conducted in rats administered 10 mg/kg of Example 1a. The GOAT inhibitor significantly reduced acyl ghrelin levels (FIG. 4) and increased des-acyl ghrelin levels (FIG. 5) in rats. Acyl Ghrelin Reduction in Cynomolgus Monkeys A single-dose PK/PD study was conducted in cynomolgus monkeys after a 10 mg/kg dose of Example 1a. On day zero, after an overnight fast, three monkeys were administered an oral dose of vehicle, and blood was collected pre-dose and 1, 3, 8, and 24 hours later. The 24 hour time point served as the pre-dose measurement for day 1 when, after an overnight fast, the monkeys were administered a single oral dose of 10 mg/kg Example 1a. Blood was collected at various time points (15 minutes, 30 minutes, 1 hour, 3 hours, 8 hours, 24 hours, 48 hours, 96 hours, and 168 hours) with overnight fasting throughout the study. Levels of acyl ghrelin were measured using a Millipore metabolic panel that did not include des-acyl ghrelin. Over the first 24 hours, treatment with Example 1a caused a 51% reduction in the acyl ghrelin AUC relative to vehicle treatment (FIG. 6). Acyl Ghrelin Reduction in Mice on a High Fat, High Carb Diet Normal mice were acclimated to individual housing for two weeks on normal chow diet (20% calories from protein, 35% from carbohydrate, and 45% from fat). All mice except a control group were then switched to a high fat, high carb (HFHC) diet to cause obesity. Mice on the HFHC diet were administered 3 mg/kg Rimonabant once daily for seven days to cause weight loss except for one group that initiated 10 mg/kg Example 1a immediately. Mice that had been treated with Rimonabant were then administered various treatments in the evening for 21 days: vehicle, 3 mg/kg Rimonabant, or 10 mg/kg Example 1a. Mice were fasted overnight and administered a final treatment in the morning. Three hours later, blood was collected for plasma acyl ghrelin and des-acyl ghrelin determination. As seen in FIG. 7, by the end of the study, mice fed the HFHC diet, regardless of other treatment, had lower acyl ghrelin levels than mice fed normal chow. Continuous treatment with Example 1a and treatment with Example 1a after Rimonabant produced statistically significant 57% and 61% reductions in acyl ghrelin levels compared to Rimonabant followed by vehicle. Continuous treatment of Example 1a produced a 28% reduction in acyl ghrelin compared to vehicle treatment alone. As seen in FIG. 8, des-acyl ghrelin levels were statistically significantly elevated by both Example 1a treatments relative to the Rimonabant followed by vehicle treatment. Rimonabant treatment alone also led to increased acyl ghrelin levels, but not as much as that seen for treatment with Example 1a with or without Rimonabant pretreatment. 16967262 glaxosmithkline intellectual property development limited USA B2 Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001. Open Apr 27th, 2022 09:14AM Apr 27th, 2022 09:14AM GlaxoSmithKline Health Care Pharmaceuticals & Biotechnology
nyse:gsk GlaxoSmithKline Apr 19th, 2022 12:00AM May 22nd, 2020 12:00AM https://www.uspto.gov?id=US11304999-20220419 Dried composition of saponin in a liposomal formulation with a neutral lipid, a sterol, and a cryoprotectant Composition are described, which are dried under reduced pressure from a liquid mixture comprising an adjuvant comprising a saponin (e.g., such as QS21) in a liposomal formulation wherein the liposomes contain a neutral lipid (e.g., such as a phosphatidylcholine) and a sterol (e.g., such as cholesterol), and, a cryoprotectant that is an amorphous sugar. The adjuvant may further comprises a TLR-4 agonist. The compositions may further comprising an antigen, such as an antigen derived from Plasmodium falciparum, Mycobacterium tuberculosis, HIV, Moraxella, ntHi or Varicella Zoster Virus. The cryoprotectant is an amorphous sugar or mixture of amorphous sugars, and preferably is a combination of at least two cryoprotectants selected from sucrose, trehalose and dextran. The compositions may further comprise a buffer and/or a surfactant. 11304999 1. A composition dried under reduced pressure from a liquid mixture comprising an adjuvant which comprises a saponin in a liposomal formulation wherein the liposomes contain a neutral lipid and a sterol, and, a cryoprotectant that is an amorphous sugar. 2. The composition of claim 1, wherein the neutral lipid is a phosphatidylcholine selected from eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine, preferably DOPC. 3. The composition of claim 1, wherein the saponin is QS21. 4. The composition of claim 1, wherein the sterol is cholesterol. 5. The composition of claim 1, wherein the adjuvant further comprises a TLR-4 agonist. 6. The composition of claim 1 further comprising an antigen. 7. The composition of claim 6, wherein the antigen is derived from Plasmodium falciparum, Mycobacterium tuberculosis, HIV, Moraxella, ntHi or Varicella Zoster Virus. 8. The composition of claim 6, wherein the antigen is selected from RTS,S, M72, UpsA, PiLa, PE-Pila and gE VZV or truncated form thereof. 9. The composition of claim 1, wherein the cryoprotectant is an amorphous sugar or mixture of amorphous sugars. 10. The composition of claim 1, wherein the cryoprotectant is present in an amount of 3 to 10% (w/v) of the liquid mixture. 11. The composition of claim 1, wherein the cryoprotectant is a combination of at least two cryoprotectants selected from sucrose, trehalose and dextran. 12. The composition of claim 1 further comprising a buffer. 13. The composition of claim 1 having a pH of at least 4 and less than 9. 14. The composition of claim 1 further comprising a buffer selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. 15. The composition of claim 14, wherein the buffer is present in the liquid mixture in an amount of at least 6 mM and less than 100 mM. 16. The composition of claim 1 further comprising a surfactant. 17. A method of making the composition of claim 1, comprising the steps of: i. admixing: a. a liquid liposomal preparation comprising liposomes containing a neutral lipid and a sterol, and optionally the lipo-polysaccharide; b. the saponin; c. the cryoprotectant; d. optionally the antigen; e. optionally the buffer; f. optionally a surfactant; and, ii. drying the liquid mixture provided by step (i) under reduced pressure. 18. The method of claim 17, wherein the liquid liposomal preparation does not contain a cryoprotectant. 19. The method of claim 17, wherein the drying under step ii is done by lyophilisation. 20. The method of claim 17, wherein the order for admixing is first mixing the cryoprotectant and the buffer, followed by the addition of the liquid liposomal preparation, the saponin, the surfactant and the antigen respectively. 21. The composition of claim 1 having a pH of at least 5 and less than 9. 22. The composition of claim 1 having a pH of at least 5.5 and less than 9. 23. The composition of claim 1 having a pH of at least 5.8 and less than 9. 24. The composition of claim 1 having a pH of at least 6 and less than 9. 25. The composition of claim 14, wherein the buffer is present in the liquid mixture in an amount of at least 10 mM and less than 100 mM. 26. The composition of claim 14, wherein the buffer is present in the liquid mixture in an amount of at least 40 mM and less than 100 mM. 26 CROSS-REFERENCE TO RELATED APPLICATIONS This application is a Continuation of U.S. application Ser. No. 16/061,137, filed on Jun. 11, 2018, which was filed as PCT International Application No. PCT/EP2016/080814 on Dec. 13, 2016, which claims the benefit under 35 U.S.C. § 119(a) to Patent Application No. 1522068.4, filed in Great Britain on Dec. 15, 2015, all of which are hereby expressly incorporated by reference into the present application. REFERENCE TO SEQUENCE LISTING The Sequence Listing concurrently submitted herewith as a text file named “28010303PUS2 Sequence Listing.txt,” created on Aug. 8, 2020, and having a size of 4,636 bytes is herein incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD OF THE INVENTION The present invention relates to the formulation of immunogenic or vaccine compositions comprising neutral lipid liposome based adjuvants, where the composition is suitable for lyophilisation. In particular, the invention relates to lyophilised forms of such immunogenic or vaccine compositions wherein both the immunogen or vaccine antigen and the adjuvant are present in one and the same vial, as well as to the formulation and manufacture of lyophilised forms of such immunogenic or vaccine composition. TECHNICAL BACKGROUND Christensen et al. (2007) [Biochim. Biophys. Acta 1768(9):2120-2129—Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying.] studied the ability of the disaccharides trehalose and sucrose to stabilise a non-phospholipid-based liposomal adjuvant composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) upon freeze-drying. Trehalose in concentrations of 211 mM and above was found to protect and preserve DDA/TDB liposomes during freeze-drying, whilst sucrose had to be used in concentrations above 396 mM. The protective effect was not observed in liposomes without TDB. Ingvarsson et al. (2013) [J. Controlled Release 167:256-264, Designing CAF-adjuvanted dry powder vaccines: Spray drying preserves the adjuvant activity] studied spray-drying of the cationic liposome adjuvant DDA/TDB using mannitol, lactose or trehalose. Mohammed et al. (2006) [Methods 40(1):30-8, Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products] is also concerned with lyophilisation of cationic liposome adjuvanted vaccines. It is highlighted that in order to effectively protect liposomes from fusion the cryoprotectant should be present both internally within the liposome and in the external phase and that the intra and extra-liposomal media should have the same osmolarity. To that end, the protocol disclosed provides for the cryoprotectant to be included in the liposomes during liposome formation. Orr et al. (2014) [J. Control Release, 177:20-6 (published electronically 2013) Elimination of the cold chain dependence of a nanoemulsion adjuvanted vaccine against tuberculosis by lyophilisation] relates to co-lyophilisation of emulsion-based adjuvant and antigen. WO99/65465 relates to a method for agent entrapment in liposomes in the presence of a sugar. SUMMARY OF THE INVENTION The inventors surprisingly found that neutral lipid liposome based adjuvants can successfully be lyophilised, thus conferring thermo stabilisation of its components and allowing co-vialling of the adjuvant and antigen in dry form. The invention therefore provides compositions dried under reduced pressure from a liquid mixture comprising an adjuvant which comprises a saponin in a liposomal formulation wherein the liposomes contain a neutral lipid and a sterol, and, a cryoprotectant that is an amorphous sugar. In addition, the invention provides methods for making such compositions. It has surprisingly been found that in order to provide for such compositions, formation of the liposomes is not required to done in the presence of the cryoprotectant. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1-A illustrates with the solid line the freeze-drying cycle used for the samples of example 1; the dotted lines delineate the process acceptable range for the freeze drying of a vaccine composition comprising RTS,S antigen. FIG. 1-B illustrates an alternative freeze-drying cycle used for the lyophilisation of vaccine compositions comprising AS01 and antigen, as exemplified in example 2; the dotted lines delineate the process acceptable range for the freeze drying of a vaccine composition. FIG. 2-A and FIG. 2-B illustrate the preclinical immunogenicity data as obtained in example 1: FIG. 2-A. Anti-CSP cellular immune response; FIG. 2-B. Anti-CSP antibodies; I. Mosquirix™; II. Co-lyophilised RTS,S/AS01 reconstituted with 150 mM NaCl. FIG. 3 illustrates the nephelometry data as obtained in example 1. FIG. 4 illustrates the amino acid sequence of VZV gE as used in example 2. FIG. 5 illustrates the SDS-page analysis of integrity of VZV gE before and after lyophilisation under different circumstances in example 2; NR refers to non-reducing conditions, R refers to reducing conditions, the legend for lanes 1 to 12 is provided in example 2. FIG. 6 shows the results of the analysis of the impact of lyophilisation on QS21 haemolytic activity in example 2; FB refers to the composition before lyophilisation, FC refers to the composition after lyophilisation. DETAILED DESCRIPTION While lyophilisation of vaccines containing protein, live-attenuated or inactivated virus, or bacteria-has been reported, to date, successful lyophilisation (or drying under reduced pressure in general) and thermostability characterization of neutral liposome based adjuvants has not been reported. Apart from the effect on thermostability, lyophilisation of such adjuvant could allow for co-vialing of adjuvant and antigen. Development of vaccines that reduce the need for cold-chain maintenance would reduce the cost and technological hurdles of implementation of new vaccines. Co-vialing of adjuvant and antigen might further reduce cost, logistical and technological hurdles in the distribution of vaccines worldwide. The present invention describes the vacuum drying, such as lyophilisation, of a composition comprising neutral liposome based adjuvants, the formulation of such composition suitable for lyophilisation as well as methods for lyophilising. The inventors found that an adjuvant comprising a saponin, liposomes and optionally a lipopolysaccharide, wherein the liposomes are neutrallipid based, can be lyophilised from a mixture further comprising a cryoprotectant selected from amorphous sugars such as sucrose and trehalose. The composition may further comprise an immunogen or antigen. In particular, the inventors found that for the claimed liposomal adjuvant composition, the liposomes are not required to be formed in the presence of cryoprotectant in order for the adjuvant to retain its structural integrity and its adjuvant or immune-potentiating properties upon drying or lyophilisation. It is a further advantage of the invention that drying confers thermostability to the composition. Following lyophilisation as described herein, the composition can be stored up to 12, to 24, or to 36 months at 30° C.; up to 6 months, or up to 12 months or 1 year at 37° C.; or, up to three months at 45° C. Suitability for storage can be based on either or both of retention of immunogenicity and retention of structural integrity of the components to an acceptable level. Structural integrity of the liposomes may be assessed by methods such as dynamic light scattering (DLS) measuring the size and polydisperity of the liposomes, or, by electron microscopy for analysis of the structure of the liposomes. In one embodiment the average particle size (by photon correlation spectroscopy) is between 95 and 120 nm, and/or, the polydispersity index (by photon correlation spectroscopy) is not more than 0.2. From a functional perspective, antigenicity of the antigen can be measured by ELISA. Preclinical assays are available for assessing the overall immunogenicity of the compositions described herein. Immunological assays may quantify a range of responses such as CD4 T cells and/or CD8 T cells. Immunogenicity refers to the effect of the composition on the immune response upon administration of the composition to a subject. The adjuvant in accordance with the invention comprises a saponin in a liposomal formulation and optionally a TLR-4 agonist. Definitions By “liposomal formulation” is meant the saponin (and optionally TLR-4 agonist) formulated with liposomes, or, stated alternatively, presented in a liposome based composition. The liposomes intended for the present invention contain a neutral lipid or consist essentially of neutral lipid, i.e. “neutral liposomes”. By “neutral lipid” is understood that the overall net charge of the lipid is (approximately) zero. The lipid may therefore be non-ionic overall or may be zwitterionic. In one embodiment the liposomes comprises a zwitterionic lipid. Examples of suitable lipids are phospholipids such as phosphatidylcholine species. In one embodiment the liposomes contain phosphatidylcholine as a liposome forming lipid which is suitably non-crystalline at room temperature. Examples of such non-chrystalline phosphatidylcholine lipids include egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine (DLPC). In a particular embodiment, the liposomes of the present invention contain DOPC, or, consist essentially of DOPC. The liposomes may also contain a limited amount of a charged lipid which increases the stability of the liposome-saponin structure for liposomes composed of saturated lipids. In these cases the amount of charged lipid is suitably 1-20% w/w, preferably 5-10% w/w of the liposome composition. Suitable examples of such charged lipids include phosphatidylglycerol and phosphatidylserine. Suitably, the neutral liposomes will contain less than 5% w/w charged lipid, such as less than 3% w/w or less than 1% w/w. The liposomes intended for the present invention further comprise a sterol. Suitable sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In one particular embodiment, the liposomal formulation comprises cholesterol as sterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11th Edn., page 341, as a naturally occurring sterol found in animal fat. The ratio of sterol to phospholipid is 1-50% (mol/mol), suitably 20-25%. Where the active saponin fraction is QS21, the ratio of QS21:sterol will typically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10 to 1:1 (w/w), and preferably 1:5 to 1:1 (w/w). Suitably excess sterol is present, the ratio of QS21:sterol being at least 1:2 (w/w). In one embodiment, the ratio of QS21:sterol is 1:5 (w/w). In one embodiment, the sterol is cholesterol. The term ‘liposome’ is well known in the art and defines a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more lipid and/or phospholipid bilayers and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or entrap water-soluble material, lipid-soluble material, and/or amphiphilic compounds. As used herein, a ‘neutral liposome based adjuvant’ means the adjuvant comprises neutral liposomes for the presentation of the immune-potentiating agents included. As used herein, ‘consisting essentially of’ means additional components may be present provided they do not alter the overall properties or function. As used herein, a ‘vial’ refers to a container suitable for use in packaging, distributing, and using vaccines or immunogenic compositions. A vial may be ‘single dose’ vial (i.e., a vial containing a quantity of immunogenic or vaccine composition equal to a single dose, such as a single human dose; the specific dosage will vary depending on factors as will be apparent to one skilled in the art, such as the specific composition and the intended recipient). Alternatively, the vial may contain more than one dose (‘multi-dose’ vial). As used herein, ‘co-vialing’ means placing at least two different components, ingredients, or compositions, in a single vial. The vial may be a single-dose vial (containing a single dose of each component, ingredient or composition), or a multi-dose vial. As used herein, the terms ‘mixture’ and ‘admixture’ are used interchangeably. Saponins A suitable saponin for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quillaja Saponaria Molina and was first described as having adjuvant activity by Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. für die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p 243-254). Purified fragments of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21). QS-21 is a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2a antibody response and is a preferred saponin in the context of the present invention. In a suitable form of the present invention, the saponin adjuvant within the immunogenic composition is a derivative of saponaria molina quil A, preferably an immunologically active fraction of Quil A, such as QS-7, QS-17, QS-18 or QS-21, suitably QS-21. The saponin is provided in its less reactogenic composition where it is quenched with an exogenous sterol, such as cholesterol, and as provided in the liposomal formulation as defined herein above. Several particular forms of less reactogenic compositions wherein QS21 is quenched with an exogenous cholesterol exist. The saponin/sterol is presented in a liposomal formulation structure. Methods for obtaining saponin/sterol in a liposomal formulation are described in WO 96/33739, in particular Example 1. TLR4 Agonsits In one embodiment of the present invention, the adjuvant comprises a TLR-4 agonist. A suitable example of a TLR-4 agonist is a lipopolysaccharide, suitably a non-toxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly 3-Deacylated monophoshoryl lipid A (3D-MPL). 3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and is referred throughout the document as MPL or 3D-MPL See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4+ T cell responses with an IFN-g (Th1) phenotype. 3D-MPL can be produced according to the methods described in GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. In the compositions of the present invention small particle 3D-MPL may be used to prepare the aqueous adjuvant composition. Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.22 μm filter. Such preparations are described in WO 94/21292. Preferably, powdered 3D-MPL is used to prepare the aqueous adjuvant compositions of the present invention. Other TLR-4 ligands which can be used are alkyl Glucosaminide phosphates (AGPs) such as those described in WO98/50399 or U.S. Pat. No. 6,303,347 (processes for preparation of AGPs are also described), suitably RC527 or RC529 or pharmaceutically acceptable salts of AGPs as described in U.S. Pat. No. 6,764,840. Some AGPs are TLR-4 agonists, and some are TLR-4 antagonists. Both are thought to be useful as adjuvants. Other suitable TLR-4 ligands are as described in WO2003/011223 and in WO 2003/099195, such as compound I, compound II and compound III described on pages 4-5 of WO2003/011223 or on pages 3-4 of WO2003/099195 and in particular those compounds described in WO2003/011223 as ER803022, ER803058, ER803732, ER804053, ER804057m ER804058, ER804059, ER804442, ER804680 and ER804764. For example, one suitable TLR-4 ligand is ER804057. Other TLR-4 ligands which may be of use in the present invention include Glucopyranosyl Lipid Adjuvant (GLA) such as described in WO2008/153541 or WO2009/143457 or the literature articles Coler R N et al. (2011) Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant. PLoS ONE 6(1): e16333. doi:10.1371/journal.pone.0016333 and Arias M A et al. (2012) Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140. PLoS ONE 7(7): e41144. doi:10.1371/journal.pone.0041144. WO2008/153541 or WO2009/143457 are incorporated herein by reference for the purpose of defining TLR-4 ligands which may be of use in the present invention. In a specific embodiment, the adjuvant comprises both saponin and a TLR4 agonist. In a specific example, the aqueous adjuvant composition comprises QS21 and 3D-MPL In an alternative embodiment the aqueous adjuvant composition comprises QS21 and GLA. A TLR-4 ligand such as a lipopolysaccharide, such as 3D-MPL, can be used at amounts between 1 and 100 μg per human dose of the adjuvant composition. 3D-MPL may be used at a level of about 50 μg, such as at least 40 μg, at least 45 μg or at least 49 μg, or, less than 100 μg, less than 80 μg, less than 60 μg, less than 55 μg or less than 51 μg. Examples of suitable ranges are between 40-60 μg, suitably between 45-55 μg or between 49 and 51 μg or 50 μg. In a further embodiment, the human dose of the adjuvant composition comprises 3D-MPL at a level of about 25 μg, such as at least 20 μg, at least 21 μg, at least 22 μg or at least 24 μg, or, less than 30 μg, less than 29 μg, less than 28 μg, less than 27 μg or less than 26 μg. Examples of lower ranges include between 20-30 μg, suitably between 21-29 μg or between 22-28 μg or between 28 and 27 μg or between 24 and 26 μg, or 25 μg. A saponin, such as QS21, can be used at amounts between 1 and 100 μg per human dose of the adjuvant composition. QS21 may be used at a level of about 50 μg, such as at least 40 μg, at least 45 μg or at least 49 μg, or, less than 100 μg, less than 80 μg, less than 60 μg, less than 55 μg or less than 51 μg. Examples of suitable ranges are between 40-60 μg, suitably between 45-55 μg or between 49 and 51 μg or 50 μg. In a further embodiment, the human dose of the adjuvant composition comprises QS21 at a level of about 25 μg, such as at least 20 μg, at least 21 μg, at least 22 μg or at least 24 μg, or, less than 30 μg, less than 29 μg, less than 28 μg, less than 27 μg or less than 26 μg. Examples of lower ranges include between 20-30 μg, suitably between 21-29 μg or between 22-28 μg or between 28 and 27 μg or between 24 and 26 μg, or 25 μg. When both a TLR4 agonist and a saponin are present in the adjuvant, then the weight ratio of TLR4 agonist to saponin is suitably between 1:5 to 5:1, suitably 1:1. For example, where 3D-MPL is present at an amount of 50 μg or 25 μg, then suitably QS21 may also be present at an amount of 50 μg or 25 μg per human dose of the adjuvant. Liposomes The liposomal formulations as intended for the present invention is defined herein above. WO2013/041572 (also published as US20140234403, incorporated herein by reference in its entirety), in particular examples 3 and 4, describes methods for making a liposome preparation of DOPC liposomes further containing cholesterol and optionally 3D-MPL, for further mixing with QS21, thereby obtaining an adjuvant in accordance with the present invention. Antigen The composition of the present invention may further comprise an immunogen or antigen. The antigen may be selected from bacterial, viral or cancer antigens. In one embodiment the antigen is a recombinant protein, such as a recombinant prokaryotic protein. In one embodiment, the antigen is derived from Plasmodium falciparum, Mycobacterium tuberculosis (TB), Human Immunodeficienty Virus (HIV), Moraxella, nontypable Hoemophilus influenzae (ntHi) or Varicella Zoster Virus (VZV). The antigen may comprise or consist of preparations derived from parasites that cause Malaria such as Plasmodium falciparum or Plasmodium vivax. Suitable antigens derived from Plasmodium falciparum: include circumsporozoite protein (CS protein), RTS, PfEMP-I, Pfs 16 antigen, MSP-I, MSP-3, LSA-I, LSA-3, AMA-I and TRAP. Other P. falciparum antigens include EBA, GLURP, RAPI, RAP2, Sequestrin, Pf332, STARP, SALSA, PfEXPI, Pfs25, Pfs28, PFS27/25, Pfs48/45, Pfs230 and their analogues in other Plasmodium spp. The antigen may be an entire protein or an immunogenic fragment thereof. Alternatively the antigen may be presented as a fusion protein. An antigen derived from Plasmodium falciparum CS protein may be in the form of a hybrid fusion protein. The fusion protein may contain protein derived from P. falciparum CS protein fused to another protein or fragment thereof. The fusion protein may contain an N-terminal or C-terminal fragment from the CS protein of P. falciparum. Alternatively, or in addition, the fusion protein may comprise one or more repeat units (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or more repeat units) from the central region of P. falciparum CS protein. In one embodiment, the fusion protein is a hybrid fusion protein comprising an antigen derived from CS protein together with a surface antigen from hepatitis B (HBsAg) or an immunogenic fragment thereof. Typically, the surface antigen from Hepatitis B comprises the major surface protein known as the S antigen, for example, S antigen derived from an adw serotype. In particular, the fusion protein may comprise substantially all the C-terminal portion of the CS protein of P. falciparum, four or more tandem repeats of the CS protein immunodominant region, and the surface antigen from hepatitis B (HBsAg). In one aspect the fusion protein comprises a sequence of at least 160 contiguous amino acids having at least 99%, 98%, 95%, 90% sequence similarity to the C-terminal portion of the CS protein (Caspers et al. (1989) Mol. Biochem. Parasitol 35, 185-190; Gordon et al. J Infect Dis. (1995) June; 171(6):1576-85). In one aspect the fusion protein comprises a sequence of “substantially all” of the C terminal portion of the CS protein. As used herein, “substantially all” of the C-terminal portion of the CS protein includes the C terminus sequence devoid of the hydrophobic anchor sequence. In one aspect the fusion protein comprises a sequence consisting of the CS protein sequence devoid of the last 12 to 14 (such as 12) amino-acids from the C terminal is envisaged. In one embodiment the fusion protein for use in the invention is a protein which comprises a contiguous amino acid sequence having at least 99%, 98%, 95%, 90% sequence similarity to amino acids 207-395 of P. falciparum 3D7 clone, derived from the strain NF54 (Caspers et al, supra) fused in frame via a linear linker to the N terminal of HBsAg. The linker may comprise part or all of the preS2 region from HBsAg. A particular fusion protein for use in the invention is the fusion protein known as RTS, as described in WO 93/10152 and WO 98/05355, incorporated herein by reference in their entirety. The RTS may be in the form of RTS,S mixed particles (wherein “S” represents an unfused monomer) or as RTS. The RTS,S particles comprise two polypeptides RTS and S that may be synthesized simultaneously and which spontaneously form composite particulate structures (RTS,S) e.g. during purification. These particles may also be referred to a Virus Uke Particles (VLP). Such particles can be prepared in a number of ways, for example by expressing the fusion protein in a suitable host such as yeast or bacteria. It is believed that the presence of the surface antigen from Hepatitis B and the formation of the RTS,S particles boosts the immunogenicity of the CS protein portion of the hybrid protein, aids stability, and/or assists reproducible manufacturing of the protein. The CS antigens may be used in conjunction with another antigen selected from any antigen which is expressed on the sporozoite or the pre-erythrocytic stage of the parasite life cycle such as the liver stage, for example liver stage antigen-1 (LSA-1), liver stage antigen-3 (LSA-3), thrombospondin related anonymous protein (TRAP), merozoite surface protein-1 (MSP1) the major merozoite surface protein, and apical merezoite antigen-1 (AMA-1). Other suitable antigens to use in conjunction with CS antigens include PfEMP-I, Pfs 16 antigen, MSP-3, LSA-3, AMA-I, TRAP, GLURP, RAPI, RAP2, Sequestrin, Pf332, STARP, SALSA, PfEXPI, Pfs25, Pfs28, PFS27/25, Pfs48/45, Pfs230. Suitable antigens from P. vivax include circumsporozoite protein (CS protein) based antigens and Duffy antigen binding protein and fragments thereof, such as PvRII (see eg WO02/12292). Suitable CS protein based antigens include a fusion protein comprising sequences derived from a CS protein of P. vivax. In one embodiment, the fusion protein is a hybrid fusion protein. The hybrid protein herein may contain protein derived from P. vivax type I and type II. In particular, the hybrid fusion protein may contain protein derived from P. vivax type I and type II fused to another protein or fragment thereof. In one aspect the hybrid fusion protein comprises a hybrid protein derived from the CS proteins of P. vivax (CSV) and a surface antigen from Hepatitis B, such as the major surface protein known as the S antigen, such as the S antigen derived from an adw serotype. Preferably, the fusion protein is an immunogenic hybrid fusion protein comprising: a. at least one repeat unit derived from the central repeat section of a type I circumsporozoite protein of P. vivax, b. at least one repeat unit derived from the central repeating section of a type II circumsporozoite protein of P. vivax, and c. surface antigen S derived from Hepatitis B virus. The CSV derived antigen component of the invention may be fused to the amino terminal end of the S protein. More specifically the C-terminus end of the CSV fragment is fused to the N-terminus of said S antigen. For example, a suitable fusion protein is CSV-S, as described in WO2008/009652. In host cells, once expressed, the hybrid fusion protein (comprising S antigen), is able to spontaneously assemble into a lipoprotein structure/particle composed of numerous monomers of said proteins (or VLPs). Such particles can be prepared by expressing the fusion protein in a suitable host such as yeast or bacteria. When the chosen recipient host cell strain also carries in its genome one or more integrated copies of a hepatitis B S expression cassette, the resulting strain synthesizes hybrid protein as a fusion proteins, and also non-fused S antigen. These may spontaneously be assembled into lipoprotein particles comprising monomers of the hybrid fusion protein and monomers of the S antigen. Suitable host cell for the expression of the fusion protein is for example yeast. Also provided, is a VLP comprising CSV-S and/or RTS units. The particle may consist essentially of CSV-S and RTS units. Alternatively, the particles produced comprise or consist essentially of CSV-S, RTS and S units. Such mixed particles are described for example in WO2008/009650. In certain embodiments the composition of the invention comprises an antigen derived from Mycobacterium spp., such as Mycobacterium bovis or Mycobacterium tuberculosis, in particular Mycobacterium tuberculosis. Antigens of interest in the field of tuberculosis include Rv1196 and Rv0125. Rv1196 (described, for example, by the name Mtb39a in Dillon et al Infection and Immunity 1999 67(6): 2941-2950) is highly conserved, with 100% sequence identity across H37Rv, C, Haarlem, CDC1551, 94-M4241A, 98-R604INH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506 strains, the F11 strain having a single point mutation Q30K (most other clinical isolates have in excess of 90% identity to H37Rv). Rv0125 (described, for example, by the name Mtb32a in Skeiky et al Infection and Immunity 1999 67(8): 3998-4007) is also highly conserved, with 100% sequence identity across many strains. Full length Rv0125 includes an N-terminal signal sequence which is cleaved to provide the mature protein. In one embodiment the antigen is derived from Rv1196, such as comprise, such as consist of, a sequence having at least 70% identity to SEQ ID No: 1, such as at least 80%, in particular at least 90%, especially at least 95%, for example at least 98%, such as at least 99%. Typical Rv1196 related antigens will comprise (such as consist of) a derivative of SEQ ID No: 1 having a small number of deletions, insertions and/or substitutions. Examples are those having deletions of up to 5 residues at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and substitution of up to 20 residues. Other derivatives of Rv1196 are those comprising (such as consisting of) a fragment of SEQ ID No: 1 which is at least 200 amino acids in length, such as at least 250 amino acids in length, in particular at least 300 amino acids in length, especially at least 350 amino acids in length. In one embodiment the antigen is derived from Rv0125, such as comprise, such as consist of, a sequence having at least 70% identity to SEQ ID No: 2, such as at least 80%, in particular at least 90%, especially at least 95%, for example at least 98%, such as at least 99%. Typical Rv0125 related antigens will comprise (such as consist of) a derivative of SEQ ID No: 2 having a small number of deletions, insertions and/or substitutions. Examples are those having deletions of up to 5 residues at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and substitution of up to 20 residues. Other derivatives of Rv0125 are those comprising (such as consisting of) a fragment of SEQ ID No: 2 which is at least 150 amino acids in length, such as at least 200 amino acids in length, in particular at least 250 amino acids in length, especially at least 300 amino acids in length. Particular derivatives of Rv0125 are those comprising (such as consisting of) the fragment of SEQ ID No: 2 corresponding to residues 1-195 of SEQ ID No: 2. Further immunogenic derivatives of Rv0125 are those comprising (such as consisting of) the fragment of SEQ ID No: 2 corresponding to residues 192-323 of SEQ ID No: 2. Particularly preferred Rv0125 related antigens are derivatives of SEQ ID No: 2 wherein at least one (for example one, two or even all three) of the catalytic triad have been substituted or deleted, such that the protease activity has been reduced and the protein more easily produced—the catalytic serine residue may be deleted or substituted (e.g. substituted with alanine) and/or the catalytic histidine residue may be deleted or substituted and/or substituted the catalytic aspartic acid residue may be deleted or substituted. Especially of interest are derivatives of SEQ ID No: 2 wherein the catalytic serine residue has been substituted (e.g. substituted with alanine). Also of interest are Rv0125 related antigens which comprise, such as consist of, a sequence having at least 70% identity to SEQ ID No: 2, such as at least 80%, in particular at least 90%, especially at least 95%, for example at least 98%, such as at least 99% and wherein at least one of the catalytic triad have been substituted or deleted or those comprising, such as consisting of, a fragment of SEQ ID No: 2 which is at least 150 amino acids in length, such as at least 200 amino acids in length, in particular at least 250 amino acids in length, especially at least 300 amino acids in length and wherein at least one of the catalytic triad have been substituted or deleted. Further immunogenic derivatives of Rv0125 are those comprising (such as consisting of) the fragment of SEQ ID No: 2 corresponding to residues 192-323 of SEQ ID No: 2 wherein at least one (for example one, two or even all three) of the catalytic triad have been substituted or deleted. Particular immunogenic derivatives of Rv0125 are those comprising (such as consisting of) the fragment of SEQ ID No: 2 corresponding to residues 1-195 of SEQ ID No: 2 wherein the catalytic serine residue (position 176 of SEQ ID No: 2) has been substituted (e.g. substituted with alanine). Suitably the antigen will comprise, such as consist of, a sequence having at least 70% identity to SEQ ID No. 3, such as at least 80%, in particular at least 90%, especially at least 95%, such as at least 98%, for example at least 99%. Typical M72 related antigens will comprise, such as consist of, a derivative of SEQ ID No: 3 having a small number of deletions, insertions and/or substitutions. Examples are those having deletions of up to 5 residues at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and substitution of up to 20 residues. Other derivatives of M72 are those comprising, such as consisting of, a fragment of SEQ ID No: 3 which is at least 450 amino acids in length, such as at least 500 amino acids in length, such as at least 550 amino acids in length, such as at least 600 amino acids in length, such as at least 650 amino acids in length or at least 700 amino acids in length. As M72 is a fusion protein derived from the two individual antigens Rv0125 and Rv1196, any fragment of at least 450 residues will comprise a plurality of epitopes from the full length sequence (Skeiky et al J. Immunol. 2004 172:7618-7628; Skeiky Infect. Immun. 1999 67(8):3998-4007; Dillon Infect. Immun. 1999 67(6):2941-2950). M72 related antigen will comprise, such as consist of, a sequence having at least 70% identity to SEQ ID No. 3, such as at least 80%, in particular at least 90%, especially at least 95%, such as at least 98%, for example at least 99%. Typical M72 related antigens will comprise, such as consist of, a derivative of SEQ ID No: 3 having a small number of deletions, insertions and/or substitutions. Examples are those having deletions of up to 5 residues at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and substitution of up to 20 residues. In particular embodiments the M72 related antigen will comprise residues 2-723 of SEQ ID No. 3, for example comprise (or consist of) SEQ ID No. 3 or comprise (or consist) of SEQ ID No. 4. A further antigen that may be employed in accordance with the present invention is the tuberculosis antigen Rv1753 and variants thereof, such as described in WO2010010180, for example a Rv1753 sequence selected from Seq ID Nos: 1 and 2-7 of WO2010010180, in particular Seq ID No: 1. Another antigen of interest in the field of tuberculosis is Rv2386 and variants thereof, such as described in WO2010010179, for example a Rv2386 sequence selected from Seq ID Nos: 1 and 2-7 of WO201000179, in particular Seq ID No: 1. Other antigens of interest in the field of tuberculosis include Rv3616 and variants thereof, such as described in WO2011092253, for example a natural Rv3616 sequence selected from Seq ID Nos: 1 and 2-7 of WO2011092253 or a modified Rv3616 sequence such as those selected from Seq ID Nos: 161 to 169, 179 and 180 of WO2011092253, in particular Seq ID No: 167. An additional antigen of interest is HBHA, such as described in WO97044463, WO03044048 and WO2010149657. Other antigens of interest are those comprising (or consisting of): Rv1174, also known as DPV, such as described in SEQ ID No 8 of WO2010010177; Rv1793, also known as MTI or Mtb9.9, such as described in SEQ ID No 10 of WO2010010177; Rv2087, also known as MSL or Mtb9.8, such as described in SEQ ID No 9 of WO2010010177; Rv3616, also known as HTCC1 or Mtb40, such as described in SEQ ID Nos 1 and 2-7 WO2010010177 or SEQ ID Nos 161-169, 179 or 180 of WO2011092253; and/or Rv3874, also known as CFP10 or Tb38.1, such as described in SEQ ID No 9 of WO2010010177; or an immunogenic portion (such as at least 20, 50, 75 or 100 residues therefrom) or variant thereof (such as having at least 70%, 80%, 90% or 95% identity thereto). (WO2010010177 and WO2011092253 are incorporated herein by reference in their entirety) Tuberculosis antigens are suitably utilised in the form of a polypeptide, but may alternatively be provided in the form of a polynucleotide encoding said polypeptide. A further antigen that may be employed in accordance with the present invention is derived from Varicella zoster virus (VZV). The VZV antigen for use in the invention may be any suitable VZV antigen or immunogenic derivative thereof, suitably being a purified VZV antigen. In one embodiment, the VZV antigen is the VZV glycoprotein gE (also known as gp1) or immunogenic derivative hereof. The wild type or full length gE protein consists of 623 amino acids comprising a signal peptide, the main part of the protein, a hydrophobic anchor region (residues 546-558) and a C-terminal tail. In one aspect, a gE C-terminal truncate (also referred to truncated gE or gE truncate) is used whereby the truncation removes 4 to 20 percent of the total amino acid residues at the carboxy terminal end. In a further aspect, the truncated gE lacks the carboxy terminal anchor region (suitably approximately amino acids 547-623 of the wild type sequence). In a further aspect gE is a truncated gE having the sequence of SEQ ID NO. 1. The gE antigen, anchorless derivatives thereof (which are also immunogenic derivatives) and production thereof is described in EP0405867 and references therein [see also Vafai A., Antibody binding sites on truncated forms of varicalla-zoster virus gpl(gE) glycoprotein, Vaccine 1994 12:1265-9]. EP192902 also describes gE and production thereof. Truncated gE is also described by Haumont et al. Virus Research (1996) vol 40, p 199-204, herein incorporated fully by reference. An adjuvanted VZV gE composition suitable for use in accordance of the present invention is described in WO2006/094756, i.e. a carboxyterminal truncated VZV gE in combination with adjuvant comprising QS21, 3D-MPL and liposomes further containing cholesterol. Leroux-Roels I. et al. (J. Infect. Dis. 2012, 206: 1280-1290) reported on a phase I/II clinical trial evaluating the adjuvanted VZV truncated gE subunit vaccine. In a further embodiment, the compositions of the present invention may comprise an immunogen or antigen that is a derivative of any of the antigens described herein. As used herein the term “derivative” refers to an antigen that is modified relative to its naturally occurring form. Derivatives of the present invention are sufficiently similar to native antigens to retain antigenic properties and remain capable of raising an immune response against the native antigen. Whether or not a given derivative raises such an immune response may be measured by a suitable immunological assay such as an ELISA or flow cytometry. Cryoprotectant A cryoprotectant suitable for use in the present invention is an amorphous sugar such as one selected from sucrose, trehalose, lactose, raffinose, and combinations thereof. In one embodiment, the cryoprotectant is sucrose or trehalose or a combination thereof. The cryoprotectant may further comprise lyocake structure enhancing sugars such as dextran. In an embodiment, the liquid mixture for drying, e.g. by lyophilisation, contains at least 3% (w/v), at least 4% (w/v), at least 5% (w/v), or at least 6% (w/v) of the cryoprotectant. In another embodiment the cryoprotectant is present in the liquid mixture in a total amount of less than 10%, less than 8%, less than 7%, less than 6% or less than 5.5% (w/v %). Alternatively stated, the cryoprotectant is present in the liquid mixture in a total amount of at least 4%, at least 4.5% or at least 5% (w/v %), but less than 10%, less than 8%, less than 7% or less than 6% (w/v %). The total concentration of cryoprotectant in the liquid mixture suitably ranges from 5 to 10% (w/v) whereby at least 5% sucrose, trehalose or a combination thereof is present. In one embodiment, 5% sucrose is used. In one embodiment, 5% trehalose is used. In specific embodiments, the liquid mixture comprises at least 5% (w/v %) or between 5 and 10% (w/v %) of sucrose, trehalose or a combination thereof. In another embodiment, the reconstituted vaccine contains at least 0.6% (w/v), at least 0.8% (w/v), at least 1% (w/v), or at least 1.2% (w/v) of the cryoprotectant. In another embodiment the cryoprotectant is present in the reconstituted vaccine in a total amount of less than 2%, less than 1.6%, less than 1.4%, less than 1.2% or less than 1.1% (w/v %). Alternatively stated, the cryoprotectant is present in the reconstituted vaccine in a total amount of at least 0.8%, at least 0.9% or at least 1% (w/v %), but less than 2%, less than 1.6%, less than 1.4% or less than 1.2% (w/v %). The total concentration of cryoprotectant in the reconstituted vaccine suitably ranges from 1 to 2% (w/v) whereby at least 1% sucrose, trehalose or a combination thereof is present. In one embodiment, 1% sucrose is used. In one embodiment, 1% trehalose is used. In specific embodiments, the reconstituted vaccine comprises at least 1% (w/v %) or between 1 and 2.5% (w/v %) of sucrose, trehalose or a combination thereof. In another embodiment, the ratio of cryoprotectant to liposome lipid concentration ranges from 10 to 20. In specific embodiments, the ratio is 10, 15 or 20. In an alternative embodiment, the amorphous sugar also contributes to or provides for tonicity in the reconstituted vaccine, thus requiring a higher concentration of the amorphous sugar, such as 8 to 10% (e.g. 9.25%) sucrose (w/v) in the reconstituted vaccine, or, 8 to 10% trehalose (w/v) (e.g. 9.25%), or a combination of sucrose and trehalose in a total amount of 8 to 10% (w/v). Further Excipients In one embodiment, the liquid mixture is a substantially aqueous mixture optionally comprising further solvents such as ethanol or isopropanol. In a further embodiment, a buffer is added to the composition. The pH of the liquid mixture is adjusted in view of the therapeutic components of the composition. Suitably, the pH of the liquid mixture is at least 4, at least 5, at least 5.5, at least 5.8, at least 6. Alternatively stated, the pH of the liquid mixture may be less than 9, less than 8, less than 7.5 or less than 7. In other embodiments, pH of the liquid mixture is between 4 and 9, between 5 and 8, between 5.5 and 7.5, or, between 5.8 and 6.4. An appropriate buffer may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS. In one embodiment, the buffer is a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4. The buffer can be present in the liquid mixture in an amount of at least 6 mM, at least 10 mM or at least 40 mM. Or, the buffer can be present in the liquid mixture in an amount of less than 100 mM, less than 60 mM or less than 40 mM. According to specific embodiments, the buffer is a phosphate buffer, present in the liquid mixture in an amount between 6 and 40 mM, such as at about 10 mM. Suitably, the buffer is selected from Na/K2PO4, K/K2PO4 and succinate. In particular, K/K2PO4 is used as buffer. The formulation of a protein antigen for lyophilisation according to the present invention may include a surfactant. Particularly suitable surfactants for use in the present invention include polysorbates, in particular polysorbate 80 (PS80), and poloxamer188. In a further embodiment, the liquid mixture or dried composition contains a limited amount of NaCl, such as less than 60 mM, less than 50 mM, less than 40 mM, less than 30 mM, less than 25 mM or less than 20 mM NaCl in the liquid mixture. According to specific embodiments the liquid mixture contains less than 10% cryoprotectant, e.g. sucrose, and less than 50 mM NaCl. Alternatively, the liquid mixture contains less than 5% cryoprotectant, e.g. sucrose, and less than 30 mM NaCl. In a further embodiment, the liquid mixture or dried composition contains a limited amount of salts, such as less than 60 mM, less than 50 mM, less than 40 mM, less than 30 mM, less than 25 mM or less than 20 mM NaCl in the liquid mixture. Tonicity of the composition upon reconstitution can be adjusted using methods know to the skilled person such as by providing sufficient isotonifying agents in the dried composition, such as by reconstituting the dried composition with an at least isotonic solvent. In particular embodiments, tonicity of the reconstituted composition can be adjusted by adding appropriate amounts of NaCl upon reconstitution, e.g. reconstituting the dried composition with saline, or, by increasing the initial amount of cryoprotectant to levels yielding isotonicity upon reconstitution with water for injection. Alternatively the dried composition is reconstituted with an isotonic aqueous solution of a non-ionic isotonifier, e.g. sorbitol. In a preferred embodiment, the lyophilised composition is reconstituted with saline. It is well known that for parenteral administration solutions should have a pharmaceutically acceptable osmolality to avoid cell distortion or lysis. A pharmaceutically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the compositions of the present invention when reconstituted will have an osmolality in the range of 250 to 750 mOsm/kg, for example, the osmolality may be in the range of 250 to 550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg. Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA). The present invention further provides a composition as described herein for use in the treatment or prevention of disease, wherein the composition is an immunogenic composition or a vaccine composition. In a specific example of this embodiment, the invention provides an immunogenic composition such as a vaccine composition for use in the treatment or prevention of a disease associated with one or more antigen described above. In one embodiment the invention provides an immunogenic composition as described herein for use in the treatment or prevention of a disease selected from malaria, tuberculosis, COPD, HIV and herpes. The present invention further provides methods of therapy or prophylaxis of malaria, tuberculosis, COPD, HIV or herpes in an individual in need thereof, comprising the step of providing to said individual an effective amount of an immunogenic or vaccine composition as described herein. The invention also provides a method for producing a dried composition as described herein, comprising the steps of: i. Admixing a plurality of components to provide a liquid mixture, said components comprising: a. a liquid liposomal preparation comprising liposomes, said liposomes comprising a neutral lipid and a sterol; b. a saponin; and c. a cryoprotectant; and ii. drying the mixture under reduced pressure. In one embodiment, the liquid liposomal preparation of step i(a) further comprises a lipopolysaccharide. Stated alternatively, the liquid liposomal preparation optionally comprises a lipopolysaccharide. The lipopolysaccharide is as defined herein above. In a further embodiment, the admixed liquid composition of step (i) further (or optionally) comprises one or more components selected from antigens, immunogens, buffers, and surfactants. Thus, the method for producing a dried composition as described herein may comprise the steps of: i. Admixing a plurality of components to provide a liquid mixture, said components comprising: a. a liquid liposomal preparation comprising liposomes, said liposomes comprising a neutral lipid and a sterol; b. a saponin; c. a cryoprotectant; and d. one or more ingredient selected from an antigen, a buffer, and a surfactant; and ii. drying the mixture under reduced pressure. As used herein, an admixed liquid composition is a composition comprising multiple components, where an isolated component need not be a liquid, but the resulting admixed composition (the mixture) is in liquid form, i.e., the admixed composition is amorphous, flows freely, and is of constant volume under a given pressure. In one embodiment, the admixed liquid composition of step (i) comprises any two, or all three, of: an antigen, a buffer, and a surfactant. Stated alternatively, the admixed liquid composition optionally comprises any two, or all three of: an antigen, a buffer, and a surfactant. In one embodiment of the present invention, the liposomes in the liquid liposomal preparation do not contain any cryoprotectant, e.g., were not formed in the presence of a cryoprotectant. In one embodiment of the present invention, the liquid liposomal formulation does not contain any cryoprotectant, e.g., does not contain an amorphous sugar such as one selected from sucrose, trehalose, lactose, raffinose, and combinations thereof. In an alternative embodiment, the components of the liquid composition are admixed in a specific order. First, a solution of the cryoprotectant in water is provided, to which (if present) the buffer solution is added. Second the liquid liposomal preparation is added. Third, the saponin component is added. Fourth, (if present) the surfactant is added, and, fifth, the antigen (if present) is added. In between certain steps of the process, the mixture may be stirred for some time, e.g. 10 minutes or longer, 15 minutes or longer, 30 minutes or longer, 45 minutes or longer, or, between 15 and 45 minutes. In one embodiments the mixture is stirred after addition of the saponin. In another embodiment, the mixture is stirred for at least 15 minutes after the addition of the surfactant. In yet another embodiment, the mixture is stirred for at least 15 minutes after the addition of the antigen. In a further embodiment, the mixture is stirred for at least 15 minutes after each of the steps of adding the saponin, the surfactant and/or the antigen. In one embodiment, some or all of the activities in step i. are performed at room temperature. Drying under reduced pressure of a liquid mixture as provided under step ii. can be achieved using different methodologies known in the art. In one embodiment, the drying in step ii. is done by lyophilisation. The terms “freeze-drying” and “lyophilising” or “lyophilisation”, and, “freeze-dried” and “lyophilised” are used interchangeably and refer to the same process of rapidly freezing a wet substance, followed by dehydration under reduced pressure. Lyophilisation or freeze-drying cycle usually consists of three process phases. In the first phase of the process, a mostly aqueous solution or mixture is frozen, i.e. “freezing of the admixed liquid composition of step i.”. Subsequently, water is removed, i.e. “drying of the frozen composition”, first by sublimation during primary drying. In the third phase, non-frozen water is removed by diffusion and desorption during secondary drying. For the purpose of defining the method described the following terms are used as they are known in the art. The term “glass transition temperature” or “Tg” is the temperature at which an amorphous solid becomes soft upon heating or brittle upon cooling. The term “Tg′” refers to the glass transition temperature in the frozen state. The term “collapse temperature” or “Tc” refers to the temperature applied during the primary drying and at which an amorphous material softens to the extent that it can no longer support its own structure. In the lyophilisation of step ii. the admixed liquid composition of step i. is frozen prior to the drying by bringing the product temperature below Tg′ of the composition. In an embodiment, freezing is achieved by exposing the sample or aqueous mixture to a constant shelf temperature at a freezing temperature which is below Tg′. In an alternative embodiment, the product may be frozen by applying shelf-ramp freezing, i.e. gradually reducing the shelf temperature to a freezing temperature below Tg′. According to embodiments, the freezing temperature is a temperature below Tg′ minus 5° C., below Tg′ minus 7.5° C., or below Tg′ minus 10° C., such as at or below −50° C. Drying of the frozen composition under reduced pressure as contemplated in the lyophilisation of step ii. described herein will typically be done in two phases, i.e. primary drying and secondary drying. In an embodiment, drying will include: primary drying at a temperature below Tc of the product, and, secondary drying at a temperature above Tc of the product and below the Tg of the product. In one embodiment, the drying step ii. of the method described herein is completed within 48 hours, within 36 hours, within 30 hours, within 28 hours. In a specific embodiment, step ii. is completed within 28 hours. In one embodiment, the antigen is RTS,S and primary drying is done at a pressure lower than 90 μbar and/or above 45 μbar. Within the same embodiment, primary drying conditions may be applied for up to 19 hours and should be applied for at least 15 hours. In an alternative embodiment, e.g. when the antigen is a VZV gE derivative, a more conservative freeze-drying cycle is used such as illustrated by FIG. 1-B. The dried composition obtained by the method described is capable of eliciting an immune response in a subject. The said immune response is in correspondance with the adjuvant, and with any antigen present in the composition. In one embodiment, the cryoprotectant (which may be in a liquid form or other form) is mixed with the liposomal preparation prior to mixing with the saponin. In a further embodiment, the surfactant is admixed prior to the antigen. According to another embodiment, the order of mixture is first mixing the cryoprotectant and buffer, followed by the addition of liquid liposomal preparation, saponin, surfactant, and antigen in respective order. In the description of the method, each of the terms has the same meaning as set forth for the compositions herein. Methods for obtaining or preparing the liposome preparation are described in WO 2013/041572, which is incorporated herein by reference in its entirety. A suitable method described therein comprises: (a) producing a lipidic film by (i) dissolving a lipid mix in isopropanol to form a homogeneous mix, and (ii) removing the solvent from the homogeneous mix to form a lipidic film, wherein the lipid mix comprises the lipid and sterol; (b) hydrating the lipidic film with a hydrating solution to form a coarse liposome suspension; (c) reducing size of the coarse liposome suspension produced in step (b) with high shear and high pressure homogenizer to form liposomes; and optionally (d) sterilising the liposomes. Suitably, step (c) comprises steps: (c′) pre-homogenising the coarse liposome suspension solution with a high shear mixer, and (c″) homogenising the solution produced in step (c′) with a high pressure homogeniser. Surprisingly, for the dried composition to retain its immune potentiating capacity or immunogenicity, the liposomes of the adjuvant were not required to be formed and/or formulated, e.g. during the manufacture of the liposome preparation, in the presence of a cryoprotectant. The following examples illustrate the invention. EXAMPLES 1. Example 1—Co-Lyophilisation of RTS,S/AS01 Vaccine (Quadridose) RTS,S/AS01 Vaccine The RTS,S antigen consists of two polypeptide chains, RTS and S. The RTS polypeptide contains a portion (aa 207-395) of the P. falciparum CS protein fused to the surface antigen (S) of the hepatitis B virus. The RTS fusion protein and the S polypeptide are coexpressed in Saccharomyces cerevisiae and spontaneously assemble into virus-like particles referred to as RTS,S. These purified particles constitute the RTS,S antigen as used in the formulation of the vaccine. Full details for obtaining the RTS,S antigen are available in WO93/10152, incorporated herein by reference in its entirety. AS01 refers to a vaccine adjuvant comprising QS21, 3D-MPL in a cholesterol containing liposomal formulation. Concentrated Liposome Bulk The concentrated liposome bulk was prepared as described in example 3 of WO2013/041572 (incorporated herein by reference in its entirety). Briefly, the concentrated liposome bulk has been prepared in 2 steps. The first step was the lipidic film preparation. DOPC (Dioleoyl phosphatidylcholine), 3D-MPL and cholesterol were dissolved sequentially in isopropanol. Then isopropanol was stripped off under stirring and reduced pressure gradient in a warming bath at 55° C. to obtain a film residue. The pressure was then gradually reduced and a final drying was applied to obtain a lipidic film. The second step was the preparation of the concentrated liposomes bulk. To that end, the lipidic film was rehydrated in PBS to form a coarse suspension of liposomes. The liposome suspension was then homogenized with a high-shear mixer in-line with a high-pressure homogenizer to produce the desired nano-sized liposomes. The resulting concentrated liposome bulk is filtered through a 0.22 μm PES membrane. The concentrated liposome bulk for use in the example contained 40 mg/ml of DOPC, 10 mg/ml Cholesterol, 2 mg/ml MPL in 10 mM phosphate buffer (pH 6.1) and 150 mM NaCl. Vaccine Formulation Antigen, i.e. RTS,S, and adjuvant, i.e. AS01, were co-formulated for lyophilisation in water for injection adding 1) 30% sucrose (ad 5%), 2) 100 mM buffer, either PO4 (K/K2) or succinate, pH 6.1 (ad 10 mM), 3) 40 mg/ml liposome bulk (ad 5 mg/ml), 4) 5 mg/ml QS21 (ad 0.25 mg/ml), followed by stirring of the thus obtained adjuvant preparation during 15 to 45 minutes at room temperature. Subsequently 3% (w/v) Polysorbate 80 (ad 0.0312%) was added and the mixture stirred during 15 to 45 minutes at room temperature. The antigen RTS,S was added ad 0.25 mg/ml and the obtained solution stirred for 15-45 minutes at room temperature. pH was measured and adjusted to 6.1 if needed. Control formulations containing either adjuvant or antigen were also prepared. The samples tested are as follows: 1. RTS,S (lot A) PO4 (Na/Na2) pH 6.8 2. RTS,S (lot B) PO4 (Na/Na2) pH 6.8 3. AS01E3 PO4 (K/K2) pH 6.1 4. colyo RTS,S (lot A)/AS succinate 10 mM pH 6.1 5. colyo RTS,S (lot A)/AS PO4 (K/K2) 10 mM pH 6.1 6. colyo RTS,S (lot B)/AS succinate 10 mM pH 6.1 7. colyo RTS,S (lot B)/AS PO4 (K/K2) 10 mM pH 6.1 8. RTS,S (lot A) succinate pH 6.1 9. RTS,S (lot A) PO4 (K/K2) pH 6.1 10. RTS,S (lot B) succinate pH 6.1 11. RTS,S (lot B) PO4 (K/K2) pH 6.1 12. AS01 succinate pH 6.1 13. AS01 PO4 (K/K2) pH 6.1 14. Sucrose 5% Freeze-Drying The formulations thus obtained were filled into glass vials (0.5 ml fill volume) and lyophilized by applying a 28 hour lyophilisation cycle as presented in FIG. 1-A. Evaluation Several aspects were assessed to evaluate the thermal stability of the samples for up to 1 year at 4 and 30° C., 6 months at 37° C. and 3 months at 45° C. 1. Visual Aspect of the Cakes The cakes had an elegant pharmaceutical appearance (similar to Mosquirix bidose formulation) for all formulation groups. Intringuingly, the formulations containing RTS,S but not the adjuvant displayed slight retraction. The cake appearance proved to be stable up to 12 months at 30° C. and 6 months at 37° C. A slight shrinkage was observed after 6 months at 45° C., which is probably due to a decrease in Tg because of an increased moist content of the cake. 2. Morphology of the Liposomes by Electron Microscopy The structure of liposome was also analysed by transmission electron microscopy under a Zeiss Libra120. Negative staining analysis was performed according to standard two-step negative staining method using sodium phosphotungstate as contrasting agent (Hayat M A. & Miller S. E., 1990, Negative Staining, Mc Graw-Hill ed.), using glow discharged carbon-formvar coated nickel grids (200 mesh) and analyzed at 100 kV. The samples were also analyzed by cryo-microscopy at 80 kV, without any contrasting agent, following vitrification at 107° K in the holes of a carbon-coated plastic mesh (Dubochet et al., 1987, in Cryotechniques in Biological EM; R. A. Steinbrecht and K. Zierold, ed; Springer Verlag). The analysis revealed that in the phosphate-buffered solutions, the liposome morphology was preserved after RTS,S/AS01 co-lyophilization and stable up to 6 months at 45° C. 3. Antigen-Adjuvant Interactions The interaction between the RTS,S antigen and the AS01 components DOPC and Cholesterol are studied by ultracentrifugation in a sucrose gradient, followed by the quantification of RTS,S, DOPC and cholesterol in the collected fractions. The tested samples were reconstituted in 150 mM NaCl are compared to Mosquirix™ (freeze-dried RTS,S reconstituted with liquid AS01). Similar to Mosquirix™, no interaction was observed between RTS,S and the adjuvant components. 4. Liposome Particle Size Colloidal stability was evaluated by nephelometry, indicating a lightly higher stability of phosphate-buffered formulation compared to succinate-buffered formulations. The size of AS01 liposomes in colyophilized samples was measured by DLS, indicating a hydrodynamic radius of ca 95 nm (the hydrodynamic radius in the control liquid formulation is 110 nm). This is most probably due to the presence of PS80 in the formulation (and not due to the freeze-dying step, neither to the presence of RTS,S). The AS01 liposome size remained stable over time at higher temperature. Results of the nephelometry after incubation at different temperatures are represented in FIG. 3. 5. RTS,S Particle Size The size of RTS,S particles was measured by SEC-HPLC on a TSKgel G5000PWXL with fluorescence detection (λEx: 280 nm/λEm: 320 nm) in order to avoid interference with adjuvant components when using UV detection. The retention time of RTS,S particles in samples where the adjuvant and antigen were colyophilized was identical to the RTS,S control purified bulk and remained stable up to 1 year at 4 and 30° C., 6 months at 37° C. and 3 months at 45° C. 6. RTS and S Proteins The integrity of RTS and S proteins was demonstrated by SDS-PAGE and ELISA. SDS-PAGE profiles were similar for up to 1 year at 4 and 30° C., 6 months at 37° C. At 45° C., some slight smears were visible after 3 months of storage. However, antigenicity by ELISA remained stable for up to 1 year at 4 and 30° C., 6 months at 37° C. and 3 months at 45° C. The antigenicity of RTS,S was measured by a mix CS-S sandwich capture ELISA (coating with monoclonal anti-CSP and revelation with a polyclonal anti-S). 7. Chemical Integrity of the Adjuvant Components The chemical integrity of AS01 components (QS21 and MPL) was evaluated, since both components are known to be sensitive to hydrolysis. Hydrolysed QS21 (QS21H) and MPL congeners are quantified by HPLC methods. QS21 concentration and hydrolysis (QS21H) were determined by reverse phase HPLC on a Symetry RP18 column, with UV detection at 214 nm. MPL congeners were determined following derivatization with DNBA and RP-HPLC on a Waters Symmetry C18 column and fluorescence detection (excitation at 345 nm and emission at 515 nm). QS21H remained below 3% in all freeze-dried samples. On the contrary, the QS21H content in the liquid reference adjuvant formulation (1 mg/ml DOPC, 0.25 mg/ml Cholesterol, 50 μg/ml QS21, 50 μg/ml MPL in 10 mM phosphatebuffer (pH 6.1), 150 mM NaCl) rapidly increased at high temperature (value above 3% after 1 month at 37° C. and after 3 months at 30° C.). MPL congeners also remained stable up to 12 months at 30° C. and up to 6 months at 45° C. in all lyophilized formulations. On the contrary, the liquid reference adjuvant formulation is not stable at high temperature, as indicated by MPL deacylation (decrease of the proportion of penta and hexa congeners, together with an increase of the proportion of tetra congeners). The proportion is higher than 35% after 1 month at 45° C., after 3 months at 37° C. or after 6 months at 30° C. 8. Preclinical Immunogenicity Immunogenicity of the co-lyophilized samples was compared to the immunogenicity of Mosquirix™ in a mouse model. The antibody responses and CD8 T-cell responses against both S and CS antigen were evaluated, as well as CD4 responses against S antigen were assessed in CB6F1 mice. Fresh pools of leukocytes collected at different time points, were stimulated for 6 hours with pools of 15-mer peptides covering the CSP or HBs sequence. The CSP and HBs-specific cellular responses were evaluated by ICS measuring the amount of CD4+ or CD8+T cells expressing IFN-γ and/or IL-2 and/or TNFα. All ICS analysis were performed using FlowJo software. The study results showed that the co-lyophilization of RTS,S and AS01 had no impact on the immunogenicity (same cellular and humoral responses for both RTS and S at T0, compared to the current Mosquirix™. Also, the co-lyophilization RTS,S/AS01 proved to be stable up to 1 year at 30° C. (and 1 year at 30° C. plus 1 month at 45° C.), 6 months at 37° C. and 3 months at 45° C. (except a slight increase of HBs-specific CD8+ T cell responses observed after 3 months at 37° C. but not at 45° C.). Upon reconstitution of lyophilized RTS,S in the liquid reference adjuvant formulation pre-incubated for 3 months at 37° C., there was a slight decrease of CSP-specific CD4+ T cell responses). The liquid reference adjuvant formulation incubated for 3 months at 45° C. could not be injected because it was proven to be haemolytic. Immune response is illustrated by FIG. 2. 2. Example 2—Colyophilisation of VZV gE/AS01 Vaccine (Unidose) The VZV gE antigen (also referred to herein as gE) is a truncated form of the Varicella Zoster Virus glycoprotein E, has the sequence as disclosed in FIG. 4, and is obtained as disclosed in Example 2 of WO2006/094756. AS01 refers to the vaccine adjuvant comprising QS21, 3D-MPL in a cholesterol containing liposomal formulation. The concentrated liposome bulk used is the same as described for example 1. The antigen, i.e. VZV gE, and adjuvant, i.e. AS01, were co-formulated for lyophilisation in water for injection mixing: 1) 30% sucrose (ad 5%), 2) 100 mM buffer, either PO4 (K/K2), pH 6.1 (ad 10 mM), 3) 40 mg/ml liposome bulk (ad 2.5 mg/ml), and, 4) 5 mg/ml QS21 (ad 0.125 mg/ml), followed by stirring of the thus obtained adjuvant preparation during 15 to 45 minutes at room temperature. Subsequently 3% (w/v) Polysorbate 80 (ad 0.02%) was added and the mixture stirred during 15 to 45 minutes at room temperature. The antigen VZV gE was added ad 0.125 mg/ml and the obtained solution was stirred for 15-45 minutes at room temperature. pH was measured and adjusted to 6.1 if needed. Control formulations containing either adjuvant or antigen were also prepared. The samples tested were as follows: 1) VZV gE/AS01 2) VZV gE 3) AS01 Freeze-Drying The formulations thus obtained were filled into glass vials (0.5 ml fill volume) and lyophilized by applying a 40-hour lyophilisation cycle as represented in FIG. 1-B. Evaluation The purpose of this experiment was to evaluate the feasibility of co-lyophilisation of another antigen (VZV gE) with the adjuvant AS01. The integrity of both antigen and adjuvant were evaluated directly after co-lyophilisation. The lyophilised material was analysed following reconstitution with 150 mM NaCl and compared to control VZV gE vaccine (lyophilized VZV gE reconstituted in liquid AS01 or in buffered saline (10 mM phosphate, 150 mM NaCl, pH 6.1). 1. Injectability The pH measured in the different groups are slightly lower (by ca. 0.4 units) that in the control Shingrix vaccine, although the pH was fixed at 6.1 in the corresponding final bulks (before lyophilisation). The osmolality determined in the 3 groups is similar to the control. Group pH Osmolality (mOsm/kg) 1 (colyo gE/AS) Before lyophilisation 6.1 206 After lyophilisation 5.8 436 2 (gE) Before lyophilisation 6.1 208 After lyophilisation 5.8 432 3 (AS01) Before lyophilisation 6.1 209 After lyophilisation 5.8 430 Control 6.2 436 2. Morphology of the Liposomes by Electron Microscopy The structure of liposomes was also analysed by transmission electron microscopy with negative staining, using the same protocol as in example 1. The adjuvant displayed the characteristic morphology of the AS01 structural pattern at the EM level, i.e. liposomes of various size and shape, with membrane perforations clearly visible. The putative gE antigens were observed as very small amorphous material spread between liposomes. The same pattern was observed in the gE/AS01 sample before and after lyophilisation, and in the to control gE cake reconstituted in AS01 adjuvant, indicating that the liposome morphology was preserved after gE/AS01 co-lyophilization. 3. Liposome Particle Size The size of AS01 liposomes in samples was measured by DLS (in the groups containing AS01), indicating a hydrodynamic radius of ca 90 nm similar to the hydrodynamic radius in the control liquid formulation. The value obtained was close to the expected value for AS01 liposome. Group DLS_ZAD (nm) VZV gE/AS01 Before lyophilisation 93 After lyophilisation 91 AS01 Before lyophilisation 91 After lyophilisation 91 4. Size of gE in Solution The size of gE antigen was measured by SEC-HPLC on a TSKgel G4000PWXL with fluorescence detection (λAEx: 280 nm/λEm: 320 nm) in order to avoid interference with adjuvant components when using UV detection. The retention time of VZV gE in samples where the adjuvant and antigen were colyophilized was identical to the gE control purified bulk, indicating that the colyophilization process had no impact on the size of gE in solution. 5. Integrity of gE Protein The integrity of VZV gE protein was demonstrated by SDS-PAGE analysis of samples before and after lyophilisation. SDS-PAGE profiles (see FIG. 5) were similar for co-lyophilized samples and VZV gE control (purified bulk and drug product), in both reducing (R) and non-reducing (NR) conditions. A slight band of higher molecular weight was observed in co-lyophilized sample, corresponding presumably to aggregation, but not representing a significant amount of protein. Legend for FIG. 5 1 MW marker 2 Sample buffer 3 Control VZV gE (purified bulk) 4 Control VZV gE (reconstituted in AS01 buffer) 5 Sample buffer 6 VZV gE/AS01 before colyo (group 1) 7 VZV gE before lyo (group 2) 8 AS01 before lyo (group 3) 9 Colyo VZV gE/AS01 (group 1) 10 Lyo VZV gE (group 2) 11 Lyo AS01 (group 3) 12 Sample buffer 6. In Vitro Potency (Antigenic Activity by ELISA) The in vitro potency was measured by ELISA in the samples containing VZV gE. The test is an inhibition ELISA assay based on a human polyclonal antibodies directed against Varicella Zoster antigens (VARITECT®). Briefly, serial dilutions of samples containing VZV gE antigen are incubated with a fixed amount of VARITECT®. After incubation, human anti-gE antibodies, which do not react with VZV gE antigen samples are detected by incubation on gE antigen coated microplate. The antigen-antibody complex is revealed by addition of a rabbit anti-human IgG antibody labelled with peroxidase, followed by addition of tetra methyl benzidine. The VZV gE antigenic activity is obtained by dividing the VZV gE content by the protein content (measured by Lowry). The potency, which can only be applied to final containers, is obtained by dividing the VZV gE content by the titer of the standard that was used for the validation of the method. The ratio of in vivo potency to VZV gE content was close to 1 in all tested groups, as in the control. These results further confirmed the integrity of VZV gE antigen following lyophilisation in the presence of AS01. Theoretical gE Protein content by gE content by Antigenic content (μg/ml) Lowry (μg/ml) ELISA (μg/ml) activity (gE/prot) Potency gE/AS01 no lyo (group 1) 125 128 123 0.96 N/A Colyo gE/AS01 (group 1) 100 104 103 0.99 1.02 gE no lyo (group 2) 125 125 132 1.05 N/A Lyo gE (group 2) 100 100 101 1.01 0.98 Control 100 94 108 1.15 0.92 7. Chemical Integrity of the Adjuvant Components As in Example 1, the chemical integrity of AS01 components (QS21 and MPL) was evaluated in AS01-containing samples. Hydrolysed QS21 (QS21H) and MPL congeners were quantified by HPLC methods. QS21H remained below 3% in all freeze-dried samples. There was no impact of the lyophilisation process on the chemical integrity of MPL, as indicated by the similar proportions of tetra-, penta- and hexa-congeners before and after lyophilisation in both groups. Acceptance Group 1 (gE/AS01) Group 3 (AS01) Test criteria for AS01B3 liquid Colyo liquid Lyo QS21H LIMIT TEST BY HPLC Not more than 3%. <3% <3% <3% <3% MPL CONGENER Between 15 and 35%. 23.9 24.7 24.5 24.3 DISTRIBUTION BY HPLC-FLUO (Tetra-acyl component) MPL CONGENER Between 35 and 60%. 44.8 44.2 44.8 45 DISTRIBUTION BY HPLC-FLUO (Penta-acyl component) MPL CONGENER Between 20 and 40%. 31.3 31.1 30.7 30.7 DISTRIBUTION BY HPLC-FLUO (Hexa-acyl component) QS21 elicits a haemolytic activity when not quenched with cholesterol within the liposomal membrane. The hemolytic activity was therefore evaluated in the AS01 containing formulations, before and after lyophilisation. Whatever the conditions tested, the hemolytic rate remained under the acceptable baseline fixed at 1% (see FIG. 6). None of them were responsible for a QS21 dequenching. 16881236 glaxosmithkline biologicals sa USA B2 Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001. Open Apr 20th, 2022 03:04PM Apr 20th, 2022 03:04PM GlaxoSmithKline Health Care Pharmaceuticals & Biotechnology
nyse:gsk GlaxoSmithKline Apr 12th, 2022 12:00AM May 12th, 2020 12:00AM https://www.uspto.gov?id=US11299457-20220412 Chemical compounds This invention relates to non-steroidal compounds that are modulators of androgen receptor, and also to the methods for the making and use of such compounds. 11299457 1. A method of treating sarcopenia, wherein said method comprises administering the compound: or a pharmaceutically acceptable salt thereof, to a human subject in need thereof. 2. The method according to claim 1, wherein 0.1-50 mgs of the compound is administered. 3. A method of treating sarcopenia, wherein said method comprises administering the compound: to a human subject in need thereof. 4. The method according to claim 3, wherein 0.1-50 mgs of the compound is administered. 4 This application is a continuation of U.S. application Ser. No. 16/226,763 filed on Dec. 20, 2018, which is a continuation of U.S. application Ser. No. 15/358,458 filed on Nov. 22, 2016, (which issued as U.S. Pat. No. 10,196,353 on Feb. 5, 2019), which is a continuation of U.S. application Ser. No. 14/549,034 filed on 20 Nov. 2014 (which issued as U.S. Pat. No. 9,533,948 on 3 Jan. 2017), which is a continuation of U.S. application Ser. No. 13/941,911 filed on 15 Jul. 2013 (which issued as U.S. Pat. No. 8,957,104 on 17 Feb. 2015), which claims the benefit of U.S. Provisional Application 61/748,874, filed on 4 Jan. 2013, and U.S. Provisional Application 61/672,455 filed on 17 Jul. 2012, which are herein incorporated by reference. FIELD OF THE INVENTION This invention relates to non-steroidal compounds that are modulators of the androgen receptor and methods for their use in treatment. BACKGROUND OF THE INVENTION Steroidal nuclear receptor (NR) ligands are known to play important roles in the health of both men and women. Testosterone (T) and dihydrotestosterone (DHT) are endogenous steroidal ligands for the androgen receptor (AR) that appear to play a role in every tissue type found in the mammalian body. During the development of the fetus, androgens play a role in sexual differentiation and development of male sexual organs. Further sexual development is mediated by androgens during puberty. Androgens play diverse roles in the adult, including stimulation and maintenance of male sexual accessory organs and maintenance of the musculoskeletal system. Cognitive function, sexuality, aggression, and mood are some of the behavioral aspects mediated by androgens. Androgens have a physiologic effect on the skin, bone, and skeletal muscle, as well as blood, lipids, and blood cells (Chang, C. and Whipple, G. Androgens and Androgen Receptors. Kluwer Academic Publishers: Boston, Mass., 2002) Many clinical studies with testosterone have demonstrated significant gains in muscle mass and function along with decreases in visceral fat. See, for example, Bhasin (2003) S. J. Gerontol. A Biol. Sci. Med. Sci. 58:1002-8, and Ferrando, A. A. et al. (2002) Am. J. Phys. Endo. Met. 282: E601-E607. Androgen replacement therapy (ART) in men improves body composition parameters such as muscle mass, strength, and bone mineral density (see, for example, Asthana, S. et al. (2004) J. Ger., Series A: Biol. Sci. Med. Sci. 59: 461-465). There is also evidence of improvement in less tangible parameters such as libido and mood. Andrologists and other specialists are increasingly using androgens for the treatment of the symptoms of androgen deficiency. ART, using T and its congeners, is available in transdermal, injectable, and oral dosage forms. All current treatment options have contraindications (e.g., prostate cancer) and side-effects, such as increased hematocrit, liver toxicity, and sleep apnoea. Side-effects from androgen therapy in women include: acne, hirsutism, and lowering of high-density lipoprotein (HDL) cholesterol levels, a notable side-effect also seen in men. Agents that could selectively afford the benefits of androgens and greatly reduce the side-effect profile would be of great therapeutic value. Interestingly, certain NR ligands are known to exert their action in a tissue selective manner (see, for example, Smith et al. (2004) Endoc. Rev. 2545-71). This selectivity stems from the particular ability of these ligands to function as agonists in some tissues, while having no effect or even an antagonist effect in other tissues. The term “selective receptor modulator” (SRM) has been given to these molecules. A synthetic compound that binds to an intracellular receptor and mimics the effects of the native hormone is referred to as an agonist. A compound that inhibits the effect of the native hormone is called an antagonist. The term “modulators” refers to compounds that have a spectrum of activities ranging from full agonism to partial agonism to full antagonism. SARMs (selective androgen receptor modulators) represent an emerging class of small molecule pharmacotherapeutics that have the potential to afford the important benefits of androgen therapy without the undesired side-effects. Many SARMs with demonstrated tissue-selective effects are currently in the early stages of development See, for example, Mohler, M. L. et al. (2009) J. Med. Chem. 52(12): 3597-617. One notable SARM molecule, Ostarine™, has recently completed phase I and II clinical studies. See, for example, Zilbermint, M. F. and Dobs, A. S. (2009) Future Oncology 5(8):1211-20. Ostarine™ appears to increase total lean body mass and enhance functional performance. Because of their highly-selective anabolic properties and demonstrated androgenic-sparing activities, SARMs should be useful for the prevention and/or treatment of many diseases in both men and women, including, but not limited to sarcopenia, cachexias (including those associated with cancer, heart failure, chronic obstructive pulmonary disease (COPD), and end stage renal disease (ESRD), urinary incontinence, osteoporosis, frailty, dry eye and other conditions associated with aging or androgen deficiency. See, for example, Ho et al. (2004) Curr Opin Obstet Gynecol. 16:405-9; Albaaj et al. (2006) Postgrad Med J 82:693-6; Caminti et al. (2009) J Am Coil Cardiol. 54(10):919-27; lellamo et al. (2010) J Am Coll Cardiol. 56(16):1310-6; Svartberg (2010) Curr Opin Endocrinol Diabetes Obes. 17(3):257-61, and Mammadov et al. (2011) Int Urol Nephrol 43:1003-8. SARMS also show promise for use in promoting muscle regeneration and repair (see, for example, Serra et al. (Epub 2012 Apr. 12) doi:10.1093/Gerona/g1s083), in the areas of hormonal male contraception and benign prostatic hyperplasia (BPH), and in wound healing (see, for example, Demling (2009) ePiasty 9:e9). Preclinical studies and emerging clinical data demonstrate the therapeutic potential of SARMs to address the unmet medical needs of many patients. The demonstrated advantages of this class of compounds in comparison with steroidal androgens (e.g., tissue-selective activity, oral administration, AR selectivity, and lack of androgenic effect) position SARMs for a bright future of therapeutic applications. Accordingly, there remains a need in the art for new SARMs for therapeutic use. BRIEF SUMMARY OF INVENTION The present invention relates to non-steroidal compounds that are modulators of androgen receptor, and also to the use of these compounds in therapy. Briefly, in one aspect, the present invention provides compounds of formula (I): or a salt thereof wherein: indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is H or C1-3 alkyl; R4 is —C(O)OCH3, —C(CH3)2OH, —CH2OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3, or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo; and R5 is H or methyl. In another aspect of the invention, R1, R2, R3, and R5 are as defined above and R4 is —C(O)OCH3, —C(CH3)2OH, —C(CH3)(CF3)OH, —CH2OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3, or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo. In a particular embodiment of the invention, indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is H; R4 is —C(CH3)(CF3)OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3 or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo, and R5 is methyl. In an alternate embodiment of the invention, indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is C1-3 alkyl; R4 is —C(CH3)(CF3)OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3 or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo, and R5 is H or methyl. Another aspect of the present invention provides a pharmaceutical composition comprising a compound of the present invention and one or more pharmaceutically acceptable excipients. Another aspect of the present invention provides a compound of the present invention for use as an active therapeutic substance. Another aspect of the present invention provides a compound of the present invention for use in the acceleration of wound healing and burn healing, and in the treatment of hypogonadism, sarcopenia, osteoporosis, muscle wasting, wasting diseases, cachexia (including cachexias associated with cancer, chronic obstructive pulmonary disease (COPD), end stage renal disease (ESRD), heart failure, HIV illness, HIV treatment, and diabetes mellitus type 1 and type 2), frailty, dry eye, prostatic hyperplasia, prostate cancer, breast cancer, menopausal and andropausal vasomotor conditions, sexual dysfunction, erectile dysfunction, depression, uterine fibroid disease, endometriosis, urinary incontinence (including urinary incontinence associated with muscle and/or tissue wasting of the pelvic floor), acne, hirsutism, male contraception, impotence, and in the use as male and female hormone replacement therapy, as a stimulant of hematopoiesis, and as an anabolic agent. Another aspect of the present invention provides the use of a compound of the present invention in the manufacture of a medicament for use in the acceleration of wound healing and the treatment of hypogonadism, sarcopenia, osteoporosis, muscle wasting, wasting diseases, muscle wasting and cachexia (including muscle wasting and cachexias associated with cancer, chronic obstructive pulmonary disease (COPD), end stage renal disease (ESRD), heart failure, HIV illness, HIV treatment, and diabetes mellitus type 1 and type 2), frailty, dry eye, prostatic hyperplasia, prostate cancer, breast cancer, menopausal and andropausal vasomotor conditions, urinary incontinence (including urinary incontinence associated with muscle and/or tissue wasting of the pelvic floor), sexual dysfunction, erectile dysfunction, depression, uterine fibroid disease, endometriosis, acne, hirsutism, male contraception, impotence, and in the use as male and female hormone replacement therapy, as a stimulant of hematopoiesis, and as an anabolic agent. Another aspect of the present invention provides a method for the treatment of hypogonadism, sarcopenia, osteoporosis, muscle wasting, wasting diseases, cachexia and muscle wasting (including muscle wasting and cachexias associated with cancer, chronic obstructive pulmonary disease (COPD), end stage renal disease (ESRD), heart failure, HIV illness, HIV treatment, and diabetes mellitus type 1 and type 2), frailty, prostatic hyperplasia, prostate cancer, breast cancer, menopausal and andropausal vasomotor conditions, chronic obstructive pulmonary disease (COPD), urinary incontinence (including urinary incontinence associated with muscle and/or tissue wasting of the pelvic floor), sexual dysfunction, erectile dysfunction, depression, uterine fibroid disease, endometriosis, acne, hirsutism, male contraception, impotence, and a method of male and female hormone replacement therapy, stimulation of hematopoiesis, and anabolism, comprising the administration of a compound of the present invention. In another aspect, the present invention provides a method for the treatment of a muscle injury, and for accelerating muscle repair comprising the administration of a compound of the present invention. Also provided is the use of a compound of the present invention in the treatment of a muscle injury, or in the acceleration of muscle repair. Additionally included is the use of a compound of the present invention in the manufacture of a medicament for the treatment of muscle injury or the acceleration of muscle repair. DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention provides compounds of formula (I): or a salt thereof wherein: indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is H or C1-3 alkyl; R4 is —C(O)OCH3, —C(CH3)2OH, —CH2OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3, or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo; and R5 is H or methyl. In another aspect of the invention, R1, R2, R3, and R5 are as defined above and R4 is —C(O)OCH3, —C(CH3)2OH, —C(CH3)(CF3)OH, —CH2OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3, or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo. In one embodiment, indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is H or C1-3 alkyl; R4 is, —CH2S(O)2CH3, and R5 is H or methyl. In a particular embodiment of the invention, indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is H; R4 is —C(CH3)(CF3)OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3 or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo, and R5 is methyl. In an alternate embodiment of the invention, indicates a single or double bond; R1 is —CF3, —C≡N, or halo; R2 is H, C1-3 alkyl, or —CHF2; R3 is C1-3 alkyl; R4 is —C(CH3)(CF3)OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3 or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo, and R5 is H or methyl. In some embodiments, R1 is —CF3, —C≡N, or halo. In certain embodiments, R1 is —CF3 or —C≡N. In certain embodiments, R1 is halo. In particular embodiments, R1 is Cl. In some preferred embodiments, R1 is —CF3. In some embodiments, R2 is H, methyl, ethyl, propyl, or —CHF2. In particular embodiments, R2 is H, methyl, or —CHF2. In certain preferred embodiments, R2 is H. In other preferred embodiments, R2 is methyl. In certain embodiments, R3 is H. In other embodiments, R3 is C1-3 alkyl. In particular embodiments, R3 is methyl or ethyl. In certain preferred embodiments, R3 is methyl. In some embodiments, R4 is —C(O)OCH3, —C(CH3)2OH, —CH2OH, —CH2SCH3, —CH2S(O)2CH3, or —C(O)CH3. In other embodiments, R4 is —C(CH3)(CF3)OH. In preferred embodiments R4 is —C(CH3)2OH or —CH2S(O)2CH3. In particularly preferred embodiments, R4 is —CH2S(O)2CH3. In other embodiments, R4 is phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo. In certain embodiments, R4 is: In one embodiment, R5 is H. In another embodiment, R5 is methyl. In some embodiments, R3 is H; R4 is —C(CH3)2OH, —CH2SCH3, or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo, and R5 is H. In alternate embodiments, R3 is methyl, ethyl, or propyl, R4 is —C(O)OCH3, —C(CH3)2OH, —CH2OH, —CH2SCH3, —CH2S(O)2CH3, —C(O)CH3, or phenyl or pyridinyl, wherein said phenyl or pyridinyl is optionally substituted with one or two groups selected from —C≡N and halo, and R5 is H. In an additional embodiment, R3 is methyl, ethyl, or propyl; R4 is —C(CH3)2OH; and R5 is H. In an another embodiment, R3 is methyl, ethyl, or propyl; R4 is —C(CH3) (CF53)OH; and R5 is H. In one preferred embodiment, the compound is a compound of Formula I′: where R1, R2, R3, R4, and R5, are as defined above. In an alternate embodiment, the compound is a compound of Formula I″: where R1, R2, R3, R4, and R5, are as defined above. As used herein the term “halo” or “halogen” refers to fluoro, chloro, bromo, or iodo groups. As used herein the term “alkyl” refers to a straight or branched chain hydrocarbon, preferably having the specified number of carbon atoms. Examples of “alkyl” as used herein include, but are not limited to, methyl, ethyl, propyl, isopropyl. As used throughout this specification, the preferred number of atoms, such as carbon atoms, will be represented by, for example, the phrase “Cx-Cy alkyl,” which refers to an alkyl group, as herein defined, containing the specified number of carbon atoms. While the embodiments and preferred groups for each variable have generally been listed above separately for each variable, compounds of this invention include those in which several of each variable in formula (I), (I′), or (I″) are selected from the aspects or embodiments, and preferred, more preferred, or most preferred groups for each variable. Therefore, this invention is intended to include all combinations of all aspects, embodiments, and preferred, more preferred, and most preferred groups. The invention also provides compounds selected from the list consisting of: Methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]propanoate; Methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]butanoate; 2-Methyl-1-(1-methyl-2-oxopropyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(2-Hydroxy-1,2-dimethylpropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-Ethyl-2-oxopropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-Ethyl-2-hydroxy-2-methylpropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-Hydroxypropan-2-yl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 2-Methyl-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 2-Methyl-1-(1-(methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 2-Methyl-1-(1-(methylthio)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 2-Methyl-1-(1-(methylsulfonyl)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(2-Hydroxy-2-methylpropyl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(3-Hydroxy-3-methylbutan-2-yl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(2-(Methylthio)ethyl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-(Methylthio)propan-2-yl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 2-(Difluoromethyl)-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile; 2-(Difluoromethyl)-1-(1-(methylthio)propan-2-yl)-1H-indole-4,5-dicarbonitrile; 2-(Difluoromethyl)-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-4,5-dicarbonitrile; 1-(3-Oxobutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (S)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (R)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (R)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)indoline-5-carbonitrile; 1-(2-Hydroxy-2-methylpentan-3-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-(Methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (R)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (R)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)indoline-5-carbonitrile; 1-(1-(Methylthio)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-(Methylsulfonyl)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 4-Chloro-1-(3-oxobutan-2-yl)-1H-indole-5-carbonitrile; (S)-4-Chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile; (R)-4-Chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile; 4-Chloro-1-(2-hydroxy-2-methylpentan-3-yl)-1H-indole-5-carbonitrile; 4-Chloro-1-(3-hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-5-carbonitrile; (S)-4-Chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile; (S)-4-Chloro-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-5-carbonitrile; (R)-4-Chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile; (R)-4-Chloro-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-5-carbonitrile; (S)-1-(3-Hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile; (R)-1-(3-Hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile; 1-(2-Hydroxy-2-methylpentan-3-yl)-1H-indole-4,5-dicarbonitrile; 1-(3-Hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-4,5-dicarbonitrile; (R)-1-(1-(Methylsulfonyl)propan-2-yl)-1H-indole-4,5-dicarbonitrile; (R)-1-(1-(3-Cyanophenyl)ethyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; 1-(1-(3-Cyanophenyl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (R)-1-(1-(5-Cyanopyridin-3-yl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (R)-4-Chloro-1-(1-(5-cyanopyridin-3-yl)propyl)-1H-indole-5-carbonitrile; (R)-1-(1-Phenylethyl)-1H-indole-4,5-dicarbonitrile; (R)-1-(1-(3-Cyanophenyl)ethyl)-1H-indole-4,5-dicarbonitrile; (R)-1-(1-(5-Cyanopyridin-3-yl)propyl)-1H-indole-4,5-dicarbonitrile; and salts thereof. The invention also encompasses the compound 4-Chloro-1-((2R,3S)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile and salts thereof. The invention also provides compounds selected from the list consisting of: 1-((2R,3S)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (S)-1-(1-(Methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; (S)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile; and salts thereof. In a preferred embodiment, the compound is (R)-1-(1-(methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile. The compounds of the present invention are believed to modulate the function of one or more nuclear hormone receptor(s). Particularly, the compounds of the present invention modulate the androgen receptor (“AR”). The present invention includes compounds that are selective agonists, partial agonists, antagonists, or partial antagonists of the AR. Compounds of the present invention are useful in the treatment of AR-associated diseases and conditions, for example, a disease or condition that is prevented, alleviated, or cured through the modulation of the function or activity of AR. Such modulation may be isolated within certain tissues or widespread throughout the body of the subject being treated. As used herein, the term “treatment” refers to alleviating the specified condition, eliminating or reducing the symptoms of the condition, slowing or eliminating the progression of the condition. The compounds of the present may invention may also be useful in preventing or delaying the initial occurrence of the condition in a subject, or reoccurrence of the condition in a previously afflicted subject. One embodiment of the present invention provides compounds of the present invention for use in medical therapy. Particularly, the present invention provides for the treatment of disorders mediated by androgenic activity. More particularly, the present invention provides treatment of disorders responsive to tissue-selective anabolic and or androgenic activity. A further embodiment of the invention provides a method of treatment of a mammal suffering from a disorder mediated by androgenic activity, which includes administering to said subject an effective amount of a compound of the present invention. One embodiment of the present invention is the use of the compounds of the present invention for the treatment of a variety of disorders including, but not limited to, osteoporosis and/or the prevention of reduced bone mass, density, or growth, osteoarthritis, acceleration of bone fracture repair and healing, acceleration of healing in joint replacement, periodontal disease, acceleration of tooth repair or growth, Paget's disease, osteochondrodysplasias, muscle wasting, the maintenance and enhancement of muscle strength and function, frailty or age-related functional decline (ARFD), dry eye, sarcopenia, end-stage renal disease (ESRD), chronic fatigue syndrome, chronic myalgia, acute fatigue syndrome, sepsis, acceleration of wound healing, maintenance of sensory function, chronic liver disease, AIDS, weightlessness, burn and trauma recovery, thrombocytopenia, short bowel syndrome, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease and ulcerative colitis, obesity, eating disorders including anorexia associated with cachexia or aging, hypercortisolism and Cushing's syndrome, cardiovascular disease or cardiac dysfunction, congestive heart failure, high blood pressure, malignant tumor cells containing the androgen receptor including breast, brain, skin, ovary, bladder, lymphatic, liver, kidney, uterine, pancreas, endometrium, lung, colon, and prostate, prostatic hyperplasia, hirsutism, acne, seborrhea, androgenic alopecia, anemia, hyperpilosity, adenomas and neoplasis of the prostate, hyperinsulinemia, insulin resistance, diabetes, syndrome X, dyslipidemia, menopausal vasomotor conditions, urinary incontinence, atherosclerosis, libido enhancement, sexual dysfunction, depression, nervousness, irritability, stress, reduced mental energy and low self-esteem, improvement of cognitive function, endometriosis, polycystic ovary syndrome, counteracting preeclampsia, premenstrual syndrome, contraception, uterine fibroid disease, aortic smooth muscle cell proliferation, male hormone replacement, or ADAM. A further embodiment of the invention provides a method of treatment of a mammal requiring the treatment of a variety of disorders including, but not limited to, osteoporosis and/or the prevention of reduced bone mass, density, or growth, osteoarthritis, acceleration of bone fracture repair and healing, acceleration of healing in joint replacement, periodontal disease, acceleration of tooth repair or growth, Paget's disease, osteochondrodysplasias, muscle wasting, the maintenance and enhancement of muscle strength and function, frailty or age-related functional decline (ARFD), dry eye, sarcopenia, end-stage renal disease (ESRD), chronic fatigue syndrome, chronic myalgia, acute fatigue syndrome, acceleration of wound healing, maintenance of sensory function, chronic liver disease, AIDS, weightlessness, burn and trauma recovery, thrombocytopenia, short bowel syndrome, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease and ulcerative colitis, obesity, eating disorders including anorexia associated with cachexia or aging, hypercortisolism and Cushing's syndrome, cardiovascular disease or cardiac dysfunction, congestive heart failure, high blood pressure, malignant tumor cells containing the androgen receptor including breast, brain, skin, ovary, bladder, lymphatic, liver, kidney, uterine, pancreas, endometrium, lung, colon, and prostate, prostatic hyperplasia, hirsutism, acne, seborrhea, androgenic alopecia, anemia, hyperpilosity, adenomas and neoplasis of the prostate, hyperinsulinemia, insulin resistance, diabetes, syndrome X, dyslipidemia, menopausal vasomotor conditions, urinary incontinence (including urinary incontinence associated with muscle and/or tissue wasting of the pelvic floor), atherosclerosis, libido enhancement, sexual dysfunction, depression, nervousness, irritability, stress, reduced mental energy and low self-esteem, improvement of cognitive function, endometriosis, polycystic ovary syndrome, counteracting preeclampsia, premenstrual syndrome, contraception, uterine fibroid disease, aortic smooth muscle cell proliferation, male hormone replacement, or ADAM. Preferably the compounds of the present invention are used as male and female hormone replacement therapy or for the treatment or prevention of hypogonadism, osteoporosis, muscle wasting, wasting diseases, cancer cachexia, frailty, prostatic hyperplasia, prostate cancer, breast cancer, menopausal and andropausal vasomotor conditions, urinary incontinence, sexual dysfunction, erectile dysfunction, depression, uterine fibroid disease, and/or endometriosis, treatment of acne, hirsutism, stimulation of hematopoiesis, male contraception, impotence, and as anabolic agents, which use includes administering to a subject an effective amount of a compound of the present invention. In some embodiments, the invention encompasses the use of a compound of the invention in the treatment of muscle injury. In particular embodiments, the muscle injury is a surgery-related muscle injury, a traumatic muscle injury, a work-related skeletal muscle injury, or an overtraining-related muscle injury. Non-limiting examples of surgery-related muscle injuries include muscle damage due to knee replacement, anterior cruciate ligament (ACL) repair, plastic surgery, hip replacement surgery, joint replacement surgery, tendon repair surgery, surgical repair of rotator cuff disease and injury, and amputation. Non-limiting examples of traumatic muscle injuries include battlefield muscle injuries, auto accident-related muscle injuries, and sports-related muscle injuries. Traumatic injury to the muscle can include lacerations, blunt force contusions, shrapnel wounds, muscle pulls or tears, burns, acute strains, chronic strains, weight or force stress injuries, repetitive stress injuries, avulsion muscle injury, and compartment syndrome. In one embodiment, the muscle injury is a traumatic muscle injury and the treatment method provides for administration of at least one high dose of a compound of the invention immediately after the traumatic injury (for example, within one day of the injury) followed by periodic administration of a low dose of a compound of the invention during the recovery period. Non-limiting examples of work-related muscle injuries include injuries caused by highly repetitive motions, forceful motions, awkward postures, prolonged and forceful mechanical coupling between the body and an object, and vibration. Overtraining-related muscle injuries include unrepaired or under-repaired muscle damage coincident with a lack of recovery or lack of an increase of physical work capacity. In an additional embodiment, the muscle injury is exercise or sports-induced muscle damage resulting including exercise-induced delayed onset muscle soreness (DOMS). In another aspect, the invention provides a method of treating a muscle degenerative disorder comprising administering to a human a compound of the invention. In particular embodiments, the muscle degenerative disorder is muscular dystrophy, myotonic dystrophy, polymyositis, or dermatomyositis. For example, the methods may be used to treat a muscular dystrophy disorder selected from Duchenne MD, Becker MD, Congenital MD (Fukuyama), Emery Dreifuss MD, Limb girdle MD, and Fascioscapulohumeral MD. The methods of the invention may also be used to treat myotonic dystrophy type I (DM1 or Steinert's), myotonic dystrophy type II (DM2 or proximal myotonic myopathy), or congenital myotonia. In some embodiments, the invention encompasses a therapeutic combination in which the compound of the invention is administered in a subject in combination with the implantation of a biologic scaffold (e.g. a scaffold comprising extracellular matrix) that promotes muscle regeneration. Such scaffolds are known in the art. See, for example, Turner and Badylack (2012) Cell Tissue Res. 347(3):759-74 and U.S. Pat. No. 6,576,265. Scaffolds comprising non-crosslinked extracellular matrix material are preferred. In another aspect, the invention provides a method of treating tendon damage where the method comprises administering a compound of the invention to a subject in need thereof. In a particular embodiment, the invention includes a method of enhancing the formation of a stable tendon-bone interface. In a related embodiment, the invention provides a method of increasing the stress to failure of tendons, for example surgically-repaired tendons. In an additional embodiment, the invention provides a method of reducing fibrosis at the repair site for surgically-repaired tendons. In a particular embodiment, the invention provides a method of treating tendon damage associated with rotator cuff injury, or tendon damage associated with surgical repair of rotator cuff injury. The mammal requiring treatment with a compound of the present invention is typically a human being. In one preferred embodiment, the disorder to be treated is muscle wasting associated with chronic obstructive pulmonary disease (COPD). In another preferred embodiment, the disorder to be treated is muscle wasting associated with chronic kidney disease (CKD) or end stage renal disease (ESRD). In an alternate preferred embodiment, the disorder to be treated is muscle wasting associated with chronic heart failure (CHF). In an additional preferred embodiment, the compound is used to accelerate bone fracture repair and healing, for example to accelerate the repair and healing of a hip fracture. In yet another preferred embodiment, the compound is used to treat urinary incontinence (including urinary incontinence associated with muscle and/or tissue wasting of the pelvic floor). The compounds of the present invention may crystallize in more than one form, a characteristic known as polymorphism, and such polymorphic forms (“polymorphs”) are within the scope of formula (I), (I′), or (I″). Polymorphism generally may occur as a response to changes in temperature, pressure, or both. Polymorphism may also result from variations in the crystallization process. Polymorphs may be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point. Certain of the compounds described herein contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. The scope of the present invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds represented by formula (I), (I′), or (I″)., as well as any wholly or partially equilibrated mixtures thereof. The present invention also includes the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted. Typically, but not absolutely, the salts of the present invention are pharmaceutically acceptable salts. Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention. Salts of the compounds of the present invention may comprise acid addition salts. Representative salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamine, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium, and valerate salts. Other salts, which are not pharmaceutically acceptable, may be useful in the preparation of compounds of this invention and these should be considered to form a further aspect of the invention. As used herein, the term “solvate” refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of formula (I), (I′), or (I″)) and a solvent. Such solvents, for the purpose of the invention, should not interfere with the biological activity of the solute. Non-limiting examples of suitable solvents include, but are not limited to water, methanol, ethanol, and acetic acid. Preferably the solvent used is a pharmaceutically acceptable solvent. Non-limiting examples of suitable pharmaceutically acceptable solvents include water, ethanol, and acetic acid. Most preferably the solvent used is water. As used herein, the term “effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. The biological or medical response may be considered a prophylactic response or a treatment response. The term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts of a compound of formula (I) (I′), or (I″) may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition. Accordingly, the invention further provides pharmaceutical compositions that include effective amounts of compounds of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compounds of the present invention are as herein described. The carrier(s), diluent(s) or excipient(s) must be acceptable, in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient of the pharmaceutical composition. In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing a compound of the present invention with one or more pharmaceutically acceptable carriers, diluents or excipients. A therapeutically effective amount of a compound of the present invention will depend upon a number of factors. For example, the species, age, and weight of the recipient, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration are all factors to be considered. The therapeutically effective amount ultimately should be at the discretion of the attendant physician or veterinarian. An effective amount of a compound of the present invention for the treatment of humans suffering from disorders such as frailty, generally, should be in the range of 0.01 to 100 mg/kg body weight of recipient (mammal) per day. More usually the effective amount should be in the range of 0.001 to 1 mg/kg body weight per day. Thus, for a 70 kg adult mammal the actual amount per day would usually be from 0.07 to 70 mg, such as 0.1-20 mg, for example 1-10 mg. This amount may be given in a single dose per day or in a number (such as two, three, four, five, or more) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt, solvate may be determined as a proportion of the effective amount of the compound of formula (I), (I′), or (I″) per se. Similar dosages should be appropriate for treatment of the other conditions referred to herein. Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Such a unit may contain, as a non-limiting example, 0.1 mg to 100 mg of a compound of the present invention, such as 0.1-50 mg, for example 0.5-15 mg depending on the condition being treated, the route of administration, and the age, weight, and condition of the patient. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Such pharmaceutical formulations may be prepared by any of the methods well known in the pharmacy art. Pharmaceutical formulations may be adapted for administration by any appropriate route, for example by an oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions, each with aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. For instance, for oral administration in the form of a tablet or capsule, the active drug component may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Generally, powders are prepared by comminuting the compound to a suitable fine size and mixing with an appropriate pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavorings, preservatives, dispersing agents, and coloring agents may also be present. Capsules can be made by preparing a powder, liquid, or suspension mixture and encapsulating with gelatin or some other appropriate shell material. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol may be added to the mixture before the encapsulation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate may also be added to improve the availability of the medicament when the capsule is ingested. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may also be incorporated into the mixture. Examples of suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants useful in these dosage forms include, for example, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Tablets can be formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture may be prepared by mixing the compound, suitably comminuted, with a diluent or base as described above. Optional ingredients include binders such as carboxymethylcellulose, aliginates, gelatins, or polyvinyl pyrrolidone, solution retardants such as paraffin, resorption accelerators such as a quaternary salt, and/or absorption agents such as bentonite, kaolin, or dicalcium phosphate. The powder mixture may be wet-granulated with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials, and forcing through a screen. As an alternative to granulating, the powder mixture may be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules may be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention may also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax may be provided. Dyestuffs may be added to these coatings to distinguish different unit dosages. Oral fluids such as solutions, syrups, and elixirs may be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups may be prepared, for example, by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions may be formulated generally by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers may be added. Solubilizers that may be used according to the present invention include Cremophor EL, vitamin E, PEG, and Solutol. Preservatives and/or flavor additives such as peppermint oil, or natural sweeteners, saccharin, or other artificial sweeteners; and the like may also be added. Where appropriate, dosage unit formulations for oral administration may be microencapsulated. The formulation may also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like. The compounds of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes may be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers may include polyvinylpyrrolidone (PVP), pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethyl-aspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug; for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels. Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from a patch by chemical enhancers, iontophoresis, noncavitational ultrasound, microneedles, thermal ablation, microdermabrasion, and electroporation as generally described in Nature Biotechnology, 26(11), 1261-1268 (2008), incorporated herein by reference as related to such delivery systems. Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. For treatments of the eye or other external tissues, for example mouth and skin, the formulations may be applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles, and mouthwashes. Pharmaceutical formulations adapted for nasal administration, where the carrier is a solid, include a coarse powder having a particle size for example in the range 20 to 500 microns. The powder is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators. Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas. Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. In addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question. For example, formulations suitable for oral administration may include flavoring or coloring agents. The compounds of the present invention and their salts, and solvates thereof, may be employed alone or in combination with other therapeutic agents for the treatment of the above-mentioned conditions. For example, in frailty therapy, combination may be had with other anabolic or osteoporosis therapeutic agents. As one example, osteoporosis combination therapies according to the present invention would thus comprise the administration of at least one compound of the present invention and the use of at least one other osteoporosis therapy such as, for example, Boniva® (ibandronate sodium), Fosamax® (alendronate), Actonel® (risedronate sodium), or Prolia™ (denosumab) The compound(s) of the present invention and the other pharmaceutically active agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order. The amounts of the compound(s) of the present invention and the other pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect. The administration in combination of a compound of the present invention with other treatment agents may be in combination by administration concomitantly in: (1) a unitary pharmaceutical composition including both compounds; or (2) separate pharmaceutical compositions each including one of the compounds. Alternatively, the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time. Other potential therapeutic combinations include the compounds of the present invention combined with other compounds of the present invention, growth promoting agents, growth hormone secretagogues (e.g., ghrelin), growth hormone releasing factor and its analogs, human growth hormone and its analogs (e.g., Genotropin®, Humatrope®, Norditropin®, Nutropin®, Saizen®, Serostim®), somatomedins, alpha-adrenergic agonists, serotonin 5-HTD agonists, agents that inhibit somatostatin or its release, 5-α-reductase inhibitors, aromatase inhibitors, GnRH agonists or antagonists, parathyroid hormone, estrogen, testosterone, SERMs, progesterone receptor agonists or antagonists, and/or with other modulators of nuclear hormone receptors. The compounds of the present invention may be used in the treatment of a variety of disorders and conditions and, as such, the compounds of the present invention may be used in combination with a variety of other suitable therapeutic agents useful in the treatment of those disorders or conditions. Non-limiting examples include combinations of the present invention with anti-diabetic agents, anti-osteoporosis agents, anti-obesity agents, anti-inflammatory agents, anti-anxiety agents, anti-depressants, anti-hypertensive agents, anti-platelet agents, anti-thrombotic and thrombolytic agents, cardiac glycosides, cholesterol or lipid lowering agents, mineralocorticoid receptor antagonists, phosphodiesterase inhibitors, kinase inhibitors, thyroid mimetics, anabolic agents, viral therapies, cognitive disorder therapies, sleeping disorder therapies, sexual dysfunction therapies, contraceptives, cytotoxic agents, radiation therapy, anti-proliferative agents, and anti-tumor agents. Additionally, the compounds of the present invention may be combined with nutritional supplements such as amino acids, triglycerides, vitamins (including vitamin D; see, for example Hedström et al. (2002) J Bone Joint Surg Br. 84(4):497-503), minerals, creatine, piloic acid, carnitine, or coenzyme Q10. In particular, the compounds of the present invention are believed useful, either alone or in combination with other agents in the acceleration of wound healing and burn healing and the treatment of hypogonadism, sarcopenia, osteoporosis, muscle wasting, wasting diseases, cachexia (including cachexias associated with cancer, chronic obstructive pulmonary disease (COPD), end stage renal disease (ESRD), heart failure, HIV illness, HIV treatment, and diabetes mellitus type 1 and type 2), frailty, dry eye, prostatic hyperplasia, prostate cancer, breast cancer, menopausal and andropausal vasomotor conditions, urinary incontinence, sexual dysfunction, erectile dysfunction, depression, uterine fibroid disease, endometriosis, acne, hirsutism, male contraception, impotence, and in the use as male and female hormone replacement therapy, as a stimulant of hematopoiesis, and as an anabolic agent. The compounds of this invention may be made by a variety of methods, including well-known standard synthetic methods. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the working Examples. In all of the schemes described below, protecting groups for sensitive or reactive groups are employed where necessary in accordance with general principles of synthetic chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T. W. Green and P. G. M. Wuts (1991) Protecting Groups in Organic Synthesis, John Wiley & Sons, incorporated by reference with regard to protecting groups). These groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art. The selection of processes as well as the reaction conditions and order of their execution shall be consistent with the preparation of compounds of formula (I), (I′), or (I″). Those skilled in the art will recognize if a stereocenter exists in compounds of formula (I), (I′), or (I″). Accordingly, the present invention includes all possible stereoisomers and includes not only racemic compounds but the individual enantiomers as well. When a compound is desired as a single enantiomer, such may be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, an intermediate, or a starting material may be effected by any suitable method known in the art. See, for example, Stereochemistry of Organic Compounds by E. L. Eliel, S. H. Wlen, and L. N. Mander (Wiley-Interscience, 1994), incorporated by reference with regard to stereochemistry. Compounds of formula (I) can be synthesized by alkylation of highly substituted indoles with alpha haloesters (Scheme 1). The starting indoles can be made according to published procedures (see, for example, US2008139631A1). The respective esters are then subjected to addition of Grignard reagents such as methylmagnesium iodide to afford mixtures of methylketones and tertiary alcohols. Further structural diversification to afford compounds of formula (I) comes from reduction of the same ester bearing indoles of Scheme 1 (Scheme 2). The resulting primary alcohols are then treated with mesyl chloride followed by sodium thiomethoxide to provide thioethers. Oxidation with Oxone provides the corresponding methyl sulfones. Another method affords compounds of formula (I) stems from highly substituted aryl fluorides made by simple aryl lithiation of commercially available 4-fluorobenzonitriles followed by quenching with iodine (Scheme 3). The corresponding iodoarenes are then coupled to TMS-acetylene through standard palladium mediated synthetic methods. The resulting alkynylarenes are then treated with amines to afford secondary aniline intermediates which cyclize to the corresponding indoles upon treatment with a base. Non-commercially available amine partners for the nucleophilic substitution step are synthesized by standard methods. ABBREVIATIONS As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, the following abbreviations may be used in the examples and throughout the specification: g (grams); L (liters); μL (microliters); M (molar); Hz (Hertz); mol (moles); rt (room temperature); h (hour); MS (mass spec); GCMS (gas chromatography mass spec; HPLC (high performance liquid chromatography); psi (pounds per square inch); Pd(C) palladium on carbon; NH4Cl (ammonium chloride); MeCN (acetonitrile); Pd(PPh3)4 (palladium tetrakistriphenyl phosphine); NaOH (sodium hydroxide); CDCl3 (deuterated chloroform); SiO2 (silica); DMSO (dimethylsulfoxide); EtOAc (ethyl acetate); HCl (hydrochloric acid); DMF (N,N-dimethylformamide); Cs2CO3 (cesium carbonate); Et (ethyl); MeOH (methanol); Et2O (diethyl ether); sat'd (saturated); K2CO3 (potassium carbonate); NMP (N-methyl-2-pyrrolidone); LiBH4 (lithium borohydride); Oxone (potassium peroxomonosulfate); Na2S2O3 (sodium thiosulphate); PTFE (polytetrafluoroethylene); hex (hexanes); NaCNBH3 (sodium cyanoborohydride); Pd(PPh3)2Cl2 (bis(triphenylphosphine)palladiumchloride); anhyd (anhydrous); dppf (1,1′-bis(diphenylphosphino)ferrocene); Pd2(dba)3 (tris(dibenzylideneacetone)dipalladium(0); PMHS (polymethylhydrosiloxane); Aq (aqueous); n-BuLi (n-butyllithium); MTBE (methyl t-butyl ether); mg (milligrams); mL (milliliters); N (normal); mM (millimolar); MHz (megahertz); mmol (millimoles); min (minute); d (day); LCMS (liquid chromatography mass spec); ESI (electrospray ionization); H2 (hydrogen gas) ee (enantiomeric excess); THF (tetrahydrofuran); CH2Cl2 (methylene chloride); TFA (trifluoroacetic acid); CD3OD (deuterated methanol); Na2SO4 (sodium sulfate); CHCl3 (chloroform); PhMe (toluene); Me (methyl); EtOH (ethanol); t-Bu (tert-butyl); N2 (nitrogen); NaHCO3 (sodium bicarbonate); Zn(CN)2 (zinc cyanide); DIEA (diisopropylethyl amine); Et3N (triethylamine); LDA (lithium diisopropylamide); DTPA (diisopropylamine); KOtBu (potassium t-butoxide); semiprep (semipreparative); CuI (copper iodode); DMAC (dimethyacemide); MSCl (mesyl chloride); TBAF (tetra-n-butylammonium fluoride) TsOH (tosic acid); Boc2O (di-t-butyl dicarbonate). Unless otherwise indicated, all temperatures are expressed in ° C. (degrees Centigrade). All reactions conducted under an inert atmosphere at room temperature unless otherwise noted. Reagents employed without synthetic details are commercially available or made according to literature procedures. UPLC-MS analysis was conducted on a Waters Acquity UPLC system using a Waters BEH C18 column with dimensions 2.1×50 mm at 40° C. A 0.5 uL partial loop with needle overfill injection was made, and UV detection was performed from 210 to 350 nm scanning at 40 Hz on a Waters Acquity PDA detector. A water+0.2% formic acid v/v (solvent A)/acetonitrile+0.15% formic acid v/v (solvent B) gradient was implemented with initial conditions 95/5% (A/B) to 1/99% over 1.10 min, and held until 1.5 min. A flow rate of 1 mL/min was used. Mass spectral analysis was performed on a Waters Acquity SQD with alternating positive/negative electrospray ionization scanning from 125-1000 amu, with a scan time of 105 msec, and an interscan delay of 20 msec. 1H NMR spectra were acquired on a Varian Inova 400 MHz NMR spectrometer. The samples were dissolved in 99.9% Deuterated Chloroform-D, DMSO-d6, or d4-Methanol, as indicated for each sample. Chemical shifts are expressed in parts per million (ppm, 8 units). Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or b (broad). EXAMPLES For the purposes of the following examples, when it is recited that a compound was “synthesized as described” in another example, it indicates that the compound was synthesized essentially as described in the other example with such modifications as are within the purview of the art. Example 1 Methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]propanoate A mixture of 2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (see, for example, US2008139631A1) (0.300 g, 1.338 mmol), cesium carbonate (0.654 g, 2.007 mmol) and methyl 2-bromopropanoate (0.223 mL, 2.007 mmol) in DMF (3 mL) was heated at 90° C. for 1 h. Upon cooling, the reaction mixture was partitioned between Et2O (30 mL) and water (25 mL). The organic phase was washed with water (20 mL) and brine (10 mL). The combined aqueous phases were washed with Et2O (2×25 ml). The organic phases were combined, washed with brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel eluting with 5-40% EtOAc-hexane gradient to give methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]propanoate (0.419 g, 94% yield): MS (ESI): m/z 311 (MH+). Example 2 Methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]butanoate Synthesized in a manner similar to Example 1 using 2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile and methyl 2-bromobutanoate: MS (ESI): m/z 325 (MH+). Examples 3 and 4 2-Methyl-1-(1-methyl-2-oxopropyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 3) and 1-(2-Hydroxy-1,2-dimethylpropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 4) To an ice-cold solution of methyl magnesium iodide (3M in Et2O) (0.322 ml, 0.967 mmol) in Et2O (1 mL) was added a solution of methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]propanoate (Example 1) (0.100 g, 0.322 mmol) in Et2O (1 mL). The heterogeneous mixture was stirred in an ice bath for 5 min, at rt for 10 min, and then at 38° C. for ˜1 h. Upon cooling, the reaction mixture was diluted with EtOAc (5 mL) and treated with aq. saturated NH4Cl (5 mL). The mixture was partitioned between EtOAc (25 mL) and water (15 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel eluting sequentially with 50%, 75% and 100% CH2Cl2-hexanes to give 2-methyl-1-(1-methyl-2-oxopropyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.008 g, 8% yield, less polar product) (MS (ESI): m/z 295 (MH+)) and 1-(2-hydroxy-1,2-dimethylpropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.069 g, 60% yield, more polar product) (MS (ESI): m/z 311 (MH+). Examples 5 and 6 1-(1-Ethyl-2-oxopropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 5) and 1-(1-Ethyl-2-hydroxy-2-methylpropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 6) Synthesized in a manner similar to Examples 3 and 4 using methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]butanoate. Example 5 (8% yield): 1-(1-Ethyl-2-oxopropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile: MS (ESI): m/z 309 (MH+). Example 6 (53% yield): 1-(1-Ethyl-2-hydroxy-2-methylpropyl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile: MS (ESI): m/z 325 (MH+). Example 7 1-(1-Hydroxypropan-2-yl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile To an ice-cold solution of methyl 2-(5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl)propanoate (Example 1) (0.263 g, 0.848 mmol) in THF (5 mL) was added dropwise LiBH4 (2M in THF) (1.695 mL, 3.39 mmol). After complete addition of the reducing agent, the cold bath was removed and the mixture was stirred at rt. After 2 h, the reaction mixture was cooled in an ice bath and a saturated aqueous NH4Cl solution (15 mL) was added slowly. The mixture was then diluted with EtOAc (40 mL) and treated slowly with 1N HCl (10 mL). The phases were separated and the aqueous phase was washed with EtOAc (20 mL). The combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 20-60% EtOAc-hexane gradient to give 1-(1-hydroxypropan-2-yl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.212 g, 83% yield) as a white solid: MS (ESI): m/z 283 (MH+). Example 8 2-Methyl-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile A. 2-(5-Cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl)propyl methanesulfonate To a solution of 1-(1-hydroxypropan-2-yl)-2-methyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 7) (0.110 g, 0.390 mmol) and Et3N (0.068 mL, 0.487 mmol) in CH2Cl2 (4 mL) was added methanesulfonyl chloride (0.038 mL, 0.487 mmol) dropwise. After stirring at rt for 2 h, the reaction mixture was concentrated to dryness. The residue was partitioned between EtOAc (30 mL) and 0.2N HCl (15 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 25-60% EtOAc-hexane gradient to give 2-(5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl)propyl methanesulfonate (0.145 g, 97% yield) as a colorless oil: MS (ESI): m/z 361 (MH+). B. 2-Methyl-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile To a solution of 2-(5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl)propyl methanesulfonate (0.145 g, 0.402 mmol) in DMF (3 mL) was added sodium thiomethoxide (0.056 g, 0.805 mmol) in one portion. After 90 min, additional sodium thiomethoxide (2 eq) was added, and the mixture stirred for another 1 h. The reaction mixture was diluted with water (25 mL) and extracted with EtOAc (30 mL). The organic phase was washed with 0.1N HCl (1×20 mL) and brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 0-30% EtOAc-hexane gradient to give 2-methyl-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.094 g, 71% yield) as a colorless oil: MS (ESI): m/z 313 (MH+). Example 9 2-Methyl-1-(1-(methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile To an ice-cold solution of 2-methyl-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 8) (0.045 g, 0.144 mmol) in MeOH (4 mL) was added a solution of Oxone (0.133 g, 0.216 mmol) in water (2 mL). After 1 h, additional Oxone (0.100 g, 0.163 mmol) was added, and the mixture was stirred at rt. After 30 min, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (20 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by preparative HPLC (Phenomenex Luna column; gradient: 10-100% MeCN-water with 0.1% TFA). The fractions with product were basified with aq. saturated K2CO3 solution, and then concentrated down to the aqueous phase, which was extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated to give 2-methyl-1-(1-(methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile: MS (ESI): m/z 345 (MH+). Example 10 2-Methyl-1-(1-(methylthio)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in 3 steps, starting with methyl 2-[5-cyano-2-methyl-4-(trifluoromethyl)-1H-indol-1-yl]butanoate (Example 2) and using procedures similar to those described for Examples 7 and 8: MS (ESI): m/z 327 (MH+). Example 11 2-Methyl-1-(1-(methylsulfonyl)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 9 using 2-methyl-1-(1-(methylthio)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 10): MS (ESI): m/z 359 (MH+). Example 12 1-(2-Hydroxy-2-methylpropyl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile A mixture of 2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.025 g, 0.099 mmol) (see, for example, US2008139631A1), Cs2CO3 (0.129 g, 0.396 mmol), potassium iodide (0.0165 g, 0.099 mmol) and commercially available 1-chloro-2-methylpropan-2-ol (0.041 mL, 0.396 mmol) in DMF (2 mL) was heated at 80° C. for 90 min and then at 120° C. for 1 h. Additional 1-chloro-2-methylpropan-2-ol (0.041 mL, 0.396 mmol), Cs2CO3 (0.129 g, 0.396 mmol) and potassium iodide (0.0165 g, 0.099 mmol) were added, and heating continued at 120° C. for another 6 h. Upon cooling, the mixture was partitioned between EtOAc (25 mL) and water (20 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC (Phenomenex Luna column; gradient: 10-90% MeCN-water with 0.1% TFA). The fractions with product were concentrated down to the aqueous phase, which is then partitioned between EtOAc (25 mL) and saturated aqueous NaHCO3 solution (20 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. This chromatography did not separate product from unreacted starting indole, so the material was chromatographed over silica gel using a 50%-100% CH2Cl2-hexanes gradient to give 1-(2-(methylthio)ethyl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.014 g, 42% yield) as a white solid: MS (ESI): m/z 325 (M+H). Example 13 1-(3-Hydroxy-3-methylbutan-2-yl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile A. Methyl 2-(5-cyano-2-propyl-4-(trifluoromethyl)-1H-indol-1-yl)propanoate Synthesized in a manner similar to Example 1 using 2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile and methyl 2-bromopropanoate: MS (ESI): m/z 339 (MH+). B. 1-(3-Hydroxy-3-methylbutan-2-yl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 4 using methyl 2-(5-cyano-2-propyl-4-(trifluoromethyl)-1H-indol-1-yl)propanoate: MS (ESI): m/z 339 (MH+). Example 14 1-(2-(Methylthio)ethyl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile A mixture of 2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.025 g, 0.099 mmol), Cs2CO3 (0.129 g, 0.396 mmol), (2-chloroethyl)(methyl)sulfane (0.039 mL, 0.396 mmol) and potassium iodide (0.0165 g, 0.099 mmol) in DMF (2 mL) was heated at 80° C. After ˜1 h, additional Cs2CO3 (0.129 g, 0.396 mmol), (2-chloroethyl)(methyl)sulfane (0.039 mL, 0.396 mmol) and potassium iodide (0.0165 g, 0.099 mmol) were added, and heating was continued for 1 h. Upon cooling, the mixture was partitioned between EtOAc (25 mL) and water (20 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by preparative HPLC (Phenomenex Luna column; gradient: 10-90% MeCN-water with 0.1% TFA). The fractions with product were concentrated down to the aqueous phase and then partitioned between EtOAc (25 mL) and saturated aqueous NaHCO3 solution (20 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated to give 1-(2-(methylthio)ethyl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile: MS (ESI): m/z 327 (M+H). Example 15 1-(1-(Methylthio)propan-2-yl)-2-propyl-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in 3 steps, starting with methyl 2-(5-cyano-2-propyl-4-(trifluoromethyl)-1H-indol-1-yl)propanoate (Example 13A) using procedures similar to those described for Examples 7 and 8: MS (ESI): m/z 341 (M+H). Example 16 2-(Difluoromethyl)-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile A. Methyl 2-(4,5-dicyano-2-(difluoromethyl)-1H-indol-1-yl)propanoate Synthesized in a manner similar to Example 1 using 2-(difluoromethyl)-1H-indole-4,5-dicarbonitrile (see, for example, US2008139631A1) and methyl 2-bromopropanoate: MS (ESI): m/z 304 (M+H). B. 2-(Difluoromethyl)-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile Synthesized in a manner similar to Example 4 using methyl 2-(4,5-dicyano-2-(difluoromethyl)-1H-indol-1-yl)propanoate: MS (ESI): m/z 304 (M+H). Example 17 2-(Difluoromethyl)-1-(1-(methylthio)propan-2-yl)-1H-indole-4,5-dicarbonitrile Synthesized in 3 steps, starting with methyl 2-(4,5-dicyano-2-(difluoromethyl)-1H-indol-1-yl)propanoate (Example 16A) using procedures similar to those described for Examples 7 and 8: MS (ESI): m/z 306 (M+H). Example 18 2-(Difluoromethyl)-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-4,5-dicarbonitrile Synthesized in a manner similar to Example 9 using 2-(difluoromethyl)-1-(1-(methylthio)propan-2-yl)-1H-indole-4,5-dicarbonitrile (Example 17): MS (ESI): m/z 338 (M+H). Examples 19 and 20 1-(3-Oxobutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 19) and 1-(3-Hydroxy-3-methyl butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 20) A. Methyl 2-(5-cyano-4-(trifluoromethyl)-1H-indol-1-yl)propanoate Synthesized in a manner similar to Example 1 using 4-(trifluoromethyl)-1H-indole-5-carbonitrile (see, for example, US2008139631A1) and methyl 2-bromopropanoate: MS (ESI): m/z 297 (MH+). B. 1-(3-Oxobutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 19) and 1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Ex. 20) Synthesized in a manner similar to Examples 3 and 4 using methyl 2-(5-cyano-4-(trifluoromethyl)-1H-indol-1-yl)propanoate. Example 19 (8% yield): 1-(3-oxobutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile: MS (ESI): m/z 281 (MH+). Example 20 (53% yield): 1-(3-hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile: MS (ESI): m/z 297 (MH+). Example 21 (S)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile A. 2-(4-Fluoro-2-(trifluoromethyl)phenyl)-1,3-dioxolane To a solution of commercially available 4-fluoro-2-(trifluoromethyl)benzaldehyde (15 g, 78 mmol) in toluene (90 mL) was added ethylene glycol (21.77 mL, 390 mmol) and TsOH (0.743 g, 3.90 mmol). The mixture was then heated (under a Dean-Stark trap attached to a reflux condenser) in an oil bath at 140° C. for 4 h, about 1.4-1.5 mL of water was collected, which was close to the expected volume. TLC (20% EtOAc-hexane) showed a major, new more polar spot. The mixture is diluted with EtOAc (100 mL) and washed with water (50 mL). The organic phase is washed with water (1×50 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (330 g ISCO column) eluting with 0-10% EtOAc-hexane gradient. The cleanest fractions with product afforded 9.83 g (51% yield): MS (ESI): m/z 237 (M+H). B. 2-(4-Fluoro-3-iodo-2-(trifluoromethyl)phenyl)-1,3-dioxolane To a solution of 2-(4-fluoro-2-(trifluoromethyl)phenyl)-1,3-dioxolane (2.52 g, 10.65 mmol) and DIPA (0.150 mL, 1.067 mmol) in anhyd THF (30 mL) at −78° C. was added a solution of n-BuLi in hexanes (4.26 mL, 10.65 mmol), dropwise at such a rate that the internal temperature remained <−70° C. The resulting pale yellow solution was stirred 3 h at −78° C. during which time a blue color developed. Iodine (2.97 g, 11.71 mmol) was added in one portion (internal temp −78° C.→−66° C.). The mixture was stirred 30 min, removed from the cooling bath and quenched by addition of 10% Na2S2O3. Upon warming, the mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 2.49 g of a mixture of desired product and unreacted starting material (ca. 9:1 by 1H NMR). The mixture was resolved by reversed phase low pressure liquid chromatography (C18 column, MeOH/water gradient) affording 2-(4-fluoro-3-iodo-2-(trifluoromethyl)phenyl)-1,3-dioxolane (2.13 g, 5.88 mmol, 55.2% yield) as a pale yellow oil: 1H NMR (400 MHz, CDCl3) δ ppm 7.87 (dd, J=8.8, 5.7 Hz, 1H) 7.23 (m, J=8.2, 7.5, 0.6, 0.6 Hz, 1H), 6.23 (q, J=2.1 Hz, 1H), 4.10-4.03 (m, 4H). C. 4-Fluoro-3-iodo-2-(trifluoromethyl)benzonitrile Step 1 To a solution of 2-(4-fluoro-3-iodo-2-(trifluoromethyl)phenyl)-1,3-dioxolane (9.43 g, 26.0 mmol) in acetone (60 mL) was added aqueous hydrochloric acid (52.1 mL, 52.1 mmol) and the mixture was heated under reflux for 15 h (complete conversion by 1H NMR). The mixture was cooled, slowly poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo affording 8.09 g pale yellow syrup which crystallized on standing (assume 25.4 mmol benzaldehyde). Step 2 To a solution of benzaldehyde from step 1 and Et3N (7.08 mL, 50.8 mmol) in chloroform (75 mL) was added hydroxylamine hydrochloride (1.864 g, 26.8 mmol) in one portion and the mixture was stirred at rt. An additional portion of hydroxylamine hydrochloride (0.441 g; 6.35 mmol) was added after 3 h and stirring was continued overnight. 1H NMR after 18 h indicated complete conversion to the oxime. Step 3 To the solution from step 2 was added Et3N (7.08 mL, 50.8 mmol) and the mixture was cooled in an ice bath. A solution of triphosgene (8.27 g, 27.9 mmol) in chloroform (20 mL) was added dropwise over 15 min. 1H NMR after 1 h, indicated complete conversion. The mixture was washed (water×2, NaHCO3, brine), dried over Na2SO4 and concentrated in vacuo. The crude solid obtained was recrystallized from heptane affording 4-fluoro-3-iodo-2-(trifluoromethyl)benzonitrile (5.88 g, 18.67 mmol, 71.7% yield) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) δ 7.85 (ddd, J=8.6, 5.1, 0.5 Hz, 1H), 7.36 (ddd, J=8.6, 6.6, 0.5 Hz, 1H); MS (GCMS El) m/z 315 ([M]+, 100%). Alternative Route to Example 21C 4-Fluoro-3-iodo-2-(trifluoromethyl)benzonitrile To a freshly prepared solution of LDA (119 mmol) in anhyd THF (250 mL) at −45° C. was added a solution of commercially available 4-fluoro-2-(trifluoromethyl)benzonitrile (21.5 g, 114 mmol) in THF (30 mL), dropwise at a rate such that the internal temperature remained <−40° C. (became dark brown during addition). The mixture was stirred 30 min at −45° C., cooled to −70° C. and iodine (31.7 g, 125 mmol) was added in one portion (−70° C.→−52° C.). The mixture was stirred for 1 h, removed from the cooling bath and quenched by addition of 10% Na2S2O3 (ca. 250 mL) and 1N HCl (ca. 125 mL). The mixture was extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) followed by recrystallization from heptane (30 mL), twice, affording 4-fluoro-3-iodo-2-(trifluoromethyl)benzonitrile (15.79 g, 50.1 mmol, 44.1% yield) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) δ 7.85 (ddd, J=8.6, 5.1, 0.5 Hz, 1H), 7.36 (ddd, J=8.6, 6.6, 0.5 Hz, 1H); MS (GCMS El) m/z 315 ([M]+, 100%). D. 4-Fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile A 20 mL vial was charged with 4-fluoro-3-iodo-2-(trifluoromethyl)benzonitrile, (0.315 g, 1.00 mmol), Pd(PPh3)2Cl2 (0.014 g, 0.020 mmol) and CuI (0.0076 g, 0.040 mmol), and sealed with a rubber septum. Anhyd PhMe (5 mL) and DIPA (0.210 mL, 1.500 mmol) were added via syringe and the mixture was degassed 10 min by sparging with N2 while immersed in an ultrasonic bath. Ethynyltrimethylsilane (0.155 mL, 1.100 mmol) was added dropwise via syringe and the septum was replaced by a PTFE-faced crimp top. The mixture was stirred in a heating block at 60° C. Upon cooling the mixture was diluted with EtOAc and filtered through Celite. The filtrate was washed (satd NH4Cl, water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 4-fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile (0.231 g, 81% yield) as a light orange oil: 1H NMR (400 MHz, CDCl3) δ 7.75 (ddd, J=8.7, 5.0, 0.6 Hz, 1H), 7.39 (ddd, J=8.6, 7.8, 0.5 Hz, 1H), 0.28 (s, 9H); MS (GCMS El) m/z 285 ([M]+, 15%), 270 ([M−CH3]+, 100%). E. (S)-Methyl 2-(dibenzylamino)propanoate Commercially available (S)-methyl 2-aminopropanoate, hydrochloride (10.0 g, 71.6 mmol) was suspended in DMF (35 mL) and then K2CO3 (31.7 g. 229 mmol) was added followed by benzyl bromide (18.21 mL, 158 mmol). The mixture was left to stir for 38 h at rt. LCMS showed good conversion to the desired product at this time. The reaction was filtered and the solid components were rinsed with EtOAc. The filtrate was then diluted with water and EtOAc and the layers were partitioned. The aqueous portion was extracted with small portions of EtOAc. The combined organic portions were dried over Na2SO4, filtered, and concentrated to a pale yellow, viscous oil. This oil was then chromatographed (ISCO, silica 120 g column, 254 collection, general gradient; hexanes/EtOAc) to afford the desired product (15.76 g, 75%): MS (ESI) m/z 284 (M+H). F. (S)-3-(Dibenzylamino)-2-methylbutan-2-ol (S)-Methyl 2-(dibenzylamino)propanoate (15.76 g, 55.6 mmol) was dissolved in Et2O (400 mL) and then cooled to ca. 0° C. Methylmagnesium iodide (27.7 mL, 3 M) was added next. The mixture turned heterogeneous white with addition of the latter. The mixture was allowed to warm to ambient temperature. LCMS the next day (17 h) indicated conversion to the desired product. The reaction was slowly quenched with sat. aqueous NH4Cl and then diluted with water and EtOAc. The layers were separated and the aqueous portion was further extracted with EtOAc. The combined organic portions were dried over Na2SO4, filtered and concentrated to a pale yellow oil. LCMS after thorough drying showed the desired product. This material was used directly for the next step: MS (ESI) m/z 284 (M+1). G. (S)-3-Amino-2-methylbutan-2-ol S)-3-(Dibenzylamino)-2-methylbutan-2-ol (15.76 g, 55.6 mmol) was dissolved in MeOH (250 mL) and then treated with Pd(C) (2.0 g, 10% dry weight, 50% water). The reaction vessel was then purged with N2 and vacuum cycles (7×) and then charged with H2 (two vacuum and charge cycles) to 65 psi on a Fischer Porter apparatus. The vessel pressure was held at 65 psi for the first 2 h with charging as needed. The pressure held after 2 h. The reaction was left to stir at ambient temperature overnight. The reaction vessel was purged with alternating cycles of vacuum and N2. The catalyst was filter away with Celite and the cake was rinsed with MeOH. Water was added to the spent cake to minimize fire potential. The filtrate was carefully concentrated to a pale yellow, thick liquid (5.60 g, 98%) via rotavap (40 torr 45° C.) followed by high vac. 1HNMR confirmed the absence of methanol. Excessive exposure to high vacuum will result in loss of product: 1H NMR (400 MHz, DMSO-d6) δ 4.12 (bs, 1H), 2.57 (q, J=6.5 Hz, 1H), 1.40 (bs, 2H), 1.03 (s, 3H), 1.00 (s, 3H), 0.90 (d, J=6.7 Hz, 3H). H. (S)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile 4-Fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile (0.063 g, 0.221 mmol), (S)-3-amino-2-methylbutan-2-ol (0.060 g, 0.582 mmol) and DIEA (0.077 mL, 0.442 mmol) were combined in NMP (0.5 mL) and heated to 90° C. LCMS after heating for 9 h showed good conversion to the aniline intermediate and some desired indole formation. The mixture was cooled to rt and then treated with KOtBu (1.98 mL, 1 M in THF). The base did not afford conversion to the desired indole despite heating. The mixture was quenched with sat. aqueous NH4Cl, and then extracted with EtOAc. The combined organic fractions were concentrated to a yellow oil and then diluted with NMP (1 mL). Addition of more KOtBu (1.98 mL, 1 M in THF) afforded a dark brown solution that was heated to 50° C. LCMS after 0.5 h showed conversion to the desired indole. The reaction was again quenched with sat. aqueous NH4Cl and then extracted with EtOAc. The combined organic portions were concentrated to a yellow oil and then chromatographed (ISCO, std grad, hex/EtOAc, 24 g silica) to afford the desired product. The mixture was next subjected to reverse phase semiprep (Agilent, 230 nm detection) to afford the desired product as a colorless gum: MS (ESI): m/z 297 (MH+). Example 22 (R)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 21 staring with commercially available (R)-methyl 2-aminopropanoate, hydrochloride: MS (ESI): m/z 297 (MH+). Example 23 (R)-1-(3-Hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)indoline-5-carbonitrile To a solution of (R)-1-(3-hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 22) (0.017 g, 0.057 mmol) in TFA (1.5 mL), in an ice bath, was added NaCNBH3 (0.0721 g, 1.148 mmol) in portions. After stirring in the cold bath for 1 h, the reaction mixture was partially concentrated. The residue was dissolved in CH2Cl2 (20 mL) and washed with 0.5 N NaOH (10 mL). The organic phase was washed with 0.5 N NaOH (1×10 mL) and brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 10-40% EtOAc-hexane gradient to give (R)-1-(3-hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)indoline-5-carbonitrile: MS (ESI): m/z 299 (M+H). Example 24 1-(2-Hydroxy-2-methylpentan-3-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile A. Methyl 2-(5-cyano-4-(trifluoromethyl)-1H-indol-1-yl)butanoate Synthesized in a manner similar to Example 1 using 4-(trifluoromethyl)-1H-indole-5-carbonitrile and methyl 2-bromobutanoate: MS (ESI): m/z 311 (MH+). B. 1-(2-Hydroxy-2-methyl pentan-3-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 4 using methyl 2-(5-cyano-4-(trifluoromethyl)-1H-indol-1-yl)butanoate: MS (ESI): m/z 311 (MH+). Example 25 1-(1-(Methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in 3 steps, starting with methyl 2-(5-cyano-4-(trifluoromethyl)-1H-indol-1-yl)propanoate (Example 19A) using procedures similar to those described for Examples 7 and 8: MS (ESI): m/z 299 (MH+). Example 26 1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 9 using 1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 25): MS (ESI): m/z 331 (MH+). Example 27 (R)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile A. (R)-1-(Methylthio)propan-2-amine Step 1 To a solution of commercially available (R)-2-aminopropan-1-ol (5 g, 66.6 mmol) in MeCN (20 mL), in an ice bath, was added very slowly, dropwise, chlorosulfonic acid (4.46 mL, 66.6 mmol) (very exothermic). A gummy beige precipitate formed. The reaction mixture was kept in the cold bath for ˜10 min, and then at rt for ˜30 min. The reaction mixture was scratched with a spatula to try to solidify the gummy precipitate. After a few minutes, a beige solid formed. After stirring for another ˜10 minutes, the solids were collected by filtration, washed sequentially with MeCN (40 mL) and hexanes (100 mL), and dried by air suction for ˜40 min. The intermediate ((R)-2-aminopropyl hydrogen sulfate, weighed 0.46 g (˜96% yield). Step 2 To a solution of sodium thiomethoxide (5.60 g, 80 mmol) in water (20 mL) was added solid NaOH (2.66 g, 66.6 mmol) in portions over ˜10 min. Then the intermediate from step 1 was added as a solid over ˜5 min. The mixture was then heated at 90° C. for −10 h. The reaction mixture was biphasic. Upon cooling, MTBE (20 mL) was added, and the organic phase (brownish color) was separated. The aqueous phase was extracted with MTBE (2×20 mL). The original organic phase is washed with 1N NaOH (15 mL) (this removes most of the color). The basic aqueous phase was re-extracted with MTBE (2×20 mL). All the ether phases are combined, dried over Na2SO4, filtered, and concentrated (carefully, since the product is volatile) to afford the crude product as a light yellow oil: 1H NMR (400 MHz, DMSO-d6) δ 2.91-2.87 (m, 1H), 2.43-2.31 (m, 2H), 2.04 (s, 3H), 1.50 (bs, 2H), 1.01 (d, J=6.3 Hz, 3H). Alternative Synthesis of Example 27A (R)-1-(Methylthio)propan-2-amine Hydrochloride A. (R)-2-((tert-Butoxycarbonyl)amino)propyl methanesulfonate Step 1 Commercially available (R)-2-aminopropan-1-ol (135 g, 1797 mmol) was dissolved in MeOH 1350 mL). The solution was cooled to 5° C. with an icebath, then Boc2O (392 g, 1797 mmol) was added as a solution in MeOH (1000 mL). The reaction temperature was kept below 10° C. After the addition, the cooling bath was removed, and the mixture was stirred for 3 h. The MeOH was removed under vacuum (rotavap bath: 50° C.). The resulting residue was a colorless oil that solidified overnight to a white solid. This material was used as is for the next step. Step 2 The residue was dissolved in CH2Cl2 (1200 mL) and NEt3 (378 mL, 2717 mmol) was added, then the mixture was cooled on an ice bath. Next, MsCl (166.5 mL, 2152 mmol) was added over ˜2 h, while keeping the reaction temperature below 15° C. The mixture was stirred in an icebath for 1 h then the bath was removed. The mixture was stirred for 3 d, then washed with a 10% NaOH solution (500 mL 3×), then with water. The organic phase was dried with MgSO4, filtered, then stripped off (rota, 50° C. waterbath. The impure residue was dissolved in a mix of 500 mL EtOAc (500 mL) and MTBE (500 mL) and then. extracted with water to remove all water-soluble salts. The organic phase was dried with MgSO4, filtered, then stripped off to afford a white solid residue: 1H NMR (400 MHz, DMSO-d6) δ 6.94-6.92 (m, 1H), 4.02 (d, J=5.8 Hz, 2H), 3.78-3.71 (m, 1H), 3.16 (s, 3H), 1.38 (s, 9H), 1.06 (d, J=6.8 Hz, 3H). B. (R)-tert-Butyl (1-(methylthio)propan-2-yl)carbamate NaSMe (30 g, 428 mmol) was stirred with DMF (200 mL) to afford a suspension. Next, (R)-2-((tertbutoxycarbonyl)amino)propyl methanesulfonate (97 g, 383 mmol) was added portionwise while the temperature was kept below 45° C. (exothermic). After the addition, the mixture was stirred for 2 h, then toluene (100 mL) was added. The mixture was washed with water (500 mL, 4×), then dried with MgSO4, and filtered. The filtrate was stripped off (rotavap) to a pale yellow oil: 1H NMR (400 MHz, DMSO-d6) δ 6.77-6.75 (m, 1H), 3.60-3.54 (m, 1H), 2.54-2.50 (m, 1H), 2.43-2.38 (m, 1H), 2.05 (s, 3H), 1.38 (s, 9H), 1.08 (d, J=7.8 Hz, 3H). C. (R)-1-(Methylthio)propan-2-amine hydrochloride Acetyl chloride (150 mL,) was added to a stirred solution of MeOH (600 mL) cooled with an icebath. The mixture was stirred for 30 min in an icebath, then added to (R)-tert-butyl (1-(methylthio)propan-2-yl)carbamate (78 g, 380 mmol). The mixture was stirred at rt for 2 h, (CO2, (CH3)2C═CH2 evolution) and then stripped off to a white solid: 1H NMR (400 MHz, DMSO-d6) δ 8.22 (bs, 3H), 3.36-3.29 (m, 1H), 2.80-2.75 (m, 1H), 2.64-2.59 (m, 1H), 2.10 (s, 3H), 1.27 (d, J=6.6 Hz, 3H). D. (R)-1-(1-(Methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile A mixture of 4-fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile (Example 21D, 1.16 g, 4.07 mmol), (R)-1-(methylthio)propan-2-amine (0.599 g, 5.69 mmol) and DIEA (1.42 mL, 8.13 mmol) in DMSO (7 mL) was heated (sealed tube) at 100° C. for 50 min. Upon cooling, the reaction mixture was diluted with EtOAc (50 mL) and washed with water (30 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated to give the intermediate aniline. This intermediate was dissolved in NMP (7 mL), treated with KOtBu (1 M in THF) (5.69 mL, 5.60 mmol) and heated at 50° C. The reaction was monitored by LCMS, and deemed complete after 40 min. Upon cooling, the reaction mixture was diluted with EtOAc (40 mL) and washed with water (30 mL). The organic phase was washed with more water and brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 5-40% EtOAc-hexane gradient to give the thioether intermediate: MS (ESI): m/z 299 (MH+). E. (R)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile To an ice-cold solution of (R)-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.560 g, 1.88 mmol) in MeOH (10 mL) was added a solution of Oxone (4.04 g, 6.57 mmol) in water (10 mL). After 50 min, the reaction mixture was diluted with water (30 mL) and extracted with EtOAc (50 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using 100% CH2Cl2 to give (R)-1-(1-(methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile as a white foam that was crystallized from CH2Cl2/hexanes to afford a white solid (0.508 g, 79% yield): 1H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J=8.6 Hz, 1H), 8.12 (d, J=3.5 Hz, 1H), 7.81 (d, J=8.5 Hz, 1H), 6.87-6.84 (m, 1H), 5.43-5.35 (m, 1H), 4.01 (dd, J=14.8, 8.6 Hz, 1H), 3.83 (dd, J=14.8, 4.9 Hz, 1H), 2.77 (s, 3H), 1.59 (d, J=6.8 Hz, 3H); MS (ESI): m/z 331 (M+H). Example 28 (R)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)indoline-5-carbonitrile Synthesized in a manner similar to Example 23 using (R)-1-(1-(methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 27): MS (ESI): m/z 333 (M+H). Example 29 1-(1-(Methylthio)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in 3 steps, starting with methyl 2-(5-cyano-4-(trifluoromethyl)-1H-indol-1-yl)butanoate (Example 24A) using procedures similar to those described for Examples 7 and 8: MS (ESI): m/z 313 (MH+). Example 30 1-(1-(Methylsulfonyl)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 9 using 1-(1-(methylthio)butan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (Example 29): MS (ESI): m/z 345 (MH+). Example 31 4-Chloro-1-(3-oxobutan-2-yl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 1 using 4-chloro-1H-indole-5-carbonitrile (see, for example, US2008139631A1) and 3-bromobutan-2-one: MS (ESI): m/z 247 (MH+). Example 32 (S)-4-Chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile A. 2-Chloro-4-fluoro-3-iodobenzonitrile To a freshly-prepared solution of LDA (33.7 mmol) in anhydrous THF (30 mL) at −78° C. was added a solution of commercially available 2-chloro-4-fluorobenzonitrile (5.00 g, 32.1 mmol) in THF (10 mL), dropwise at such a rate that the internal temperature remained <−70° C. The mixture was stirred for 2 h and a solution of iodine (8.97 g, 35.4 mmol) in THF (20 mL) was added dropwise (temp<−70° C.). The mixture was stirred 30 min, removed from the cooling bath and quenched by addition of 10% Na2S2O3. The mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was dissolved in a small amount of CH2Cl2 and filtered through a pad of silica (25% EtOAc/hexanes eluent). Fractions containing the major product were concentrated in vacuo and the residue was recrystallized from heptane affording 3.24 g tan solid. The mother liquor was concentrated and the residue was purified by flash chromatography (EtOAc/hexanes, gradient elution) affording 2.85 g of a pale yellow solid. Solids were combined to give 2-chloro-4-fluoro-3-iodobenzonitrile (6.09 g, 67% yield): 1H NMR (400 MHz, CDCl3) δ 7.70 (dd, J=8.6, 5.5 Hz, 1H), 7.08 (dd, J=8.6, 6.8 Hz, 1H); MS (GCMS El) m/z 281 ([M]+, 100%). B. 2-Chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile A thick-walled glass pressure vessel was charged with 2-chloro-4-fluoro-3-iodobenzonitrile (2.815 g, 10.00 mmol), Pd(PPh3)2Cl2 (0.351 g, 0.500 mmol), and CuI (0.095 g, 0.500 mmol) and sealed with a rubber septum. Anhydrous THF (25 mL) and DIPA (4.22 mL, 30.0 mmol) were added via syringe and the mixture was degassed 10 min by sparging with N2 while immersed in an ultrasonic cleaning bath. To the degassed mixture was added ethynyltrimethylsilane (4.24 mL, 30.0 mmol), the vessel was resealed with a PTFE bushing, and the mixture was stirred in a heating block at 50° C. After 41 h, the mixture was cooled and poured into half-satd NH4Cl. The whole was filtered through a pad of Celite and the filtrate was extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (2.29 g, 91% yield) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) δ 7.60 (dd, J=8.7, 5.4 Hz, 1H), 7.12 (dd, J=8.7, 7.9 Hz, 1H), 0.30 (s, 9H); MS (GCMS El) m/z 251 ([M]+, 14%), 236 ([M−CH3]+, 100%). C. (S)-4-Chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile A mixture of 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile, (0.229 g, 0.91 mmol), (S)-3-amino-2-methylbutan-2-ol (Example 21E) (0.113 g, 1.092 mmol), and K2CO3 (0.252 g, 1.820 mmol) in anhyd NMP (3 mL) was stirred in a heating block at 60° C. under N2 for 2 h. CuI (0.017 g; 0.091 mmol) was added and the mixture was subjected to microwave heating (140° C.) for 30 min. The reaction mixture was poured into EtOAc/water and the whole was filtered through a pad of Celite. Layers of the filtrate were separated and the aqueous layer was extracted with EtOAc (×2). Combined organics were filtered (Whatman #2), washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (S)-4-chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile (0.148 g, 62% yield) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) δ 7.50 (d, 1H), 7.42-7.39 (m, 1H), 7.39-7.34 (m, 1H), 6.74 (d, J=3.3 Hz, 1H), 4.41 (q, J=7.0 Hz, 1H), 1.60 (d, J=7.1 Hz, 3H), 1.41 (s, 1H), 1.33 (s, 3H), 1.09 (s, 3H); MS (LCMS ES+) m/z 263 ([M+H]+, 88%), 304 ({[M+H]+MeCN}+, 100%). Example 33 (R)-4-Chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile To a solution of (R)-3-amino-2-methylbutan-2-ol (made in a manner similar to Example 21G using commercially available (S)-methyl 2-aminopropanoate, hydrochloride) (0.1084 g, 1.051 mmol) and 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) (0.212 g, 0.841 mmol) in anhyd NMP (4 mL) at rt was added DBU (0.475 mL, 3.15 mmol), dropwise via syringe. The reaction vial was sealed with a crimp top and subjected to microwave heating (140° C.) for 40 min. Upon cooling the mixture was poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative HPLC (C18 stationary phase, MeCN/water gradient with 0.1% TFA additive) affording (R)-4-chloro-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile (0.0188 g, 7% yield) as a tan film: 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J=3.4 Hz, 1H), 7.41 (d, J=8.6 Hz, 1H), 7.37 (d, J=8.6 Hz, 1H), 6.74 (d, J=3.3 Hz, 1H), 4.41 (q, J=7.0 Hz, 1H), 1.60 (d, J=7.0 Hz, 3H), 1.33 (s, 3H), 1.10 (s, 3H); MS (LCMS ES+) m/z 263 ([M+H]+, 52%), 304 ({[M+H]+MeCN}+f, 100%). Example 34 4-Chloro-1-(2-hydroxy-2-methylpentan-3-yl)-1H-indole-5-carbonitrile A. 3-Amino-2-methylpentan-2-ol Synthesized in a manner similar to Example 21G starting with commercially available methyl 2-aminobutanoate hydrochloride: 1H NMR (400 MHz, CDCl3) δ 2.37-2.35 (m, 1H), 1.69-1.64 (m, 2H), 1.19 (s, 3H), 1.05 (s, 3H), 1.01-0.98 (m, 3H). B. 4-Chloro-1-(2-hydroxy-2-methylpentan-3-yl)-1H-indole-5-carbonitrile A mixture of 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) (0.163 g, 0.647 mmol), 3-amino-2-methylpentan-2-ol (0.091 g, 0.776 mmol), and K2CO3 (0.179 g, 1.294 mmol) in anhyd NMP (3 mL) was stirred in a heating block at 60° C. under N2. After 18 h, the mixture was subjected to microwave heating (140° C.) for 15 min. Upon cooling the mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 4-chloro-1-(2-hydroxy-2-methylpentan-3-yl)-1H-indole-5-carbonitrile (0.0761 g, 43% yield) as a yellow gum.: 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J=2.2 Hz, 1H), 7.42 (d, J=8.6 Hz, 1H), 7.35 (d, J=8.7 Hz, 1H), 6.77 (d, J=3.3 Hz, 1H), 4.07 (dd, J=11.6, 3.6 Hz, 1H), 2.09 (m, J=3.6 Hz, 2H), 1.44 (s, 1H), 1.35 (s, 3H), 1.08 (s, 3H), 0.65 (t, J=7.3 Hz, 3H); MS (LCMS ES+) m/z 277 ([M+H]+, 70%), 318 ({[M+H]+MeCN}+, 100%). Example 35 4-Chloro-1-(3-hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-5-carbonitrile A. 3-Amino-2,3-dimethylbutan-2-ol Synthesized in a manner similar to Example 21G starting with commercially available methyl 2-amino-2-methylpropanoate: 1H NMR (400 MHz, CDCl3) δ 1.18 (s, 6H), 1.16 (s, 6H). B. 4-Chloro-1-(3-hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-5-carbonitrile An oven-dried vial was charged with 3-amino-2,3-dimethylbutan-2-ol (0.063 g, 0.539 mmol), 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) (0.113 g, 0.449 mmol), and K2CO3 (0.137 g, 0.988 mmol) and sealed with a rubber septum. Anhyd NMP (3 mL) was added via syringe and the mixture was stirred in a heating block at 60° C. under N2. After 1 h, the vial was sealed with a PTFE-faced crimp top and subjected to microwave heating; 1 h at 140° C. followed by 45 min at 160° C. (with air cooling). The mixture was poured into water/EtOAc and the whole was filtered through a pad of Celite. Layers of the filtrate were separated and the aqueous layer was extracted with EtOAc (×2). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by preparative HPLC (C18 stationary phase, MeCN/water gradient with 0.1% TFA additive) affording 4-chloro-1-(3-hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-5-carbonitrile (0.0066 g, 5% yield) as a tan solid (ca. 85% purity): 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J=9.0 Hz, 1H), 7.49 (d, J=3.5 Hz, 1H), 7.32 (d, J=8.9 Hz, 1H), 6.71 (d, J=3.4 Hz, 1H), 1.87 (s, 6H), 1.20 (s, 6H); MS (LCMS ES+) m/z 277 ([M+H]+, 65%), 318 ({[M+H]+MeCN}+, 100%). Example 36 (S)-4-Chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile A mixture of 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) (0.120 g, 0.477 mmol), (S)-1-(methylthio)propan-2-amine (0.075 g, 0.715 mmol) (prepared essentially as described in US2005182275A1) and DIEA (0.166 mL, 0.953 mmol) in DMSO (2 mL) was heated (sealed tube) at 100° C. for 45 min. Upon cooling, the reaction mixture was diluted with EtOAc (25 mL) and washed with water (20 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated to give the intermediate (S)-2-chloro-3-ethynyl-4-((1-(methylthio)propan-2-yl)amino)benzonitrile. This intermediate was dissolved in NMP (2 mL), treated with KOtBu (1 M in THF) (1.430 mL, 1.430 mmol) and heated at 60° C. The reaction was monitored by LCMS, and after 45 min, additional KOtBu (1 M in THF) (1.430 mL, 1.430 mmol) was added and heating continued for another 1 h. Upon cooling, the reaction mixture was diluted with EtOAc (25 mL) and washed with water (20 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 5-30% EtOAc-hexane gradient to give (S)-4-chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile (0.056 g, 42% yield): MS (ESI): m/z 265 (M+H). Example 37 (S)-4-Chloro-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 9 using (S)-4-chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile (Example 36): MS (ESI): m/z 297 (MH+). Example 38 (R)-4-Chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 36 using 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) and (R)-1-(methylthio)propan-2-amine (Example 27C): MS (ESI): m/z 265 (M+H). Example 39 (R)-4-Chloro-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 9 using (R)-4-chloro-1-(1-(methylthio)propan-2-yl)-1H-indole-5-carbonitrile (Example 38): MS (ESI): m/z 297 (MH+). Example 40 (S)-1-(3-Hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile A. 1,2-Dibromo-4-fluoro-3-iodobenzene To a solution of freshly-prepared LDA (33.9 mmol) in anhyd THF (100 mL) at −78° C. was added a solution of 1,2-dibromo-4-fluorobenzene (4 mL, 32.3 mmol) in THF (8 mL), dropwise at such a rate that the internal temperature remained <−70° C. The mixture was stirred 30 min and iodine (9.02 g, 35.5 mmol) was added in one portion. The mixture was stirred 30 min, quenched by addition of 10% Na2S2O3, and removed from the cooling bath. Upon warming the mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was eluted from a pad of silica (hexanes→2% EtOAc/hexanes) and recrystallized from MeOH-water affording 1,2-dibromo-4-fluoro-3-iodobenzene (8.59 g, 70% yield) as a white solid: 1H NMR (400 MHz, CDCl3) δ 7.64 (dd, J=8.8, 5.5 Hz, 1H), 6.93 (dd, J=8.8, 7.0 Hz, 1H); MS (GCMS El) m/z 378 ([M]+, Br isotopes, 56%), 380 ([M]+, Br isotopes, 100%), 382 ([M]+, Br isotopes, 51%). B. ((2,3-Dibromo-6-fluorophenyl)ethynyl)trimethylsilane A thick-walled glass vessel was charged with 1,2-dibromo-4-fluoro-3-iodobenzene (8.31 g, 21.88 mmol), Pd(PPh3)2Cl2 (0.768 g, 1.094 mmol), and CuI (0.292 g, 1.532 mmol) and sealed with a rubber septum. Anhyd THF (30 mL) and DIPA (30.8 mL, 219 mmol) were added via syringe and the mixture was degassed 10 min by sparging with N2 while immersed in an ultrasonic bath. Ethynyltrimethylsilane (3.40 mL, 24.07 mmol) was added via syringe and the septum was replaced with a PTFE bushing. The mixture was stirred in an oil bath at 40° C. After 40 h, the mixture was cooled, diluted with EtOAc and filtered through a pad of Celite. The filtrate was washed (satd NH4Cl×2, water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording ((2,3-dibromo-6-fluorophenyl)ethynyl)trimethylsilane (6.08 g, 17.37 mmol, 79% yield) as a yellow oil: 1H NMR (400 MHz, CDCl3) δ 7.53 (dd, J=8.9, 5.4 Hz, 1H), 6.95 (dd, J=8.9, 8.1 Hz, 1H), 0.29 (s, 9H); MS (GCMS El) m/z 348 ([M]+, Br isotopes, 18%), 350 ([M]+, Br isotopes, 34%), 352 ([M]+, Br isotopes, 18%), 333 ([M-CH3]+, Br isotopes, 56%), 335 ([M-CH3]+, Br isotopes, 100%), 337 ([M-CH3]+, Br isotopes, 54%). C. 4-Fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile An oven-dried flask was charged with ((2,3-dibromo-6-fluorophenyl)ethynyl)trimethylsilane, (6.08 g, 17.37 mmol), Zn(CN)2 (2.039 g, 17.37 mmol), Pd2(dba)3 (0.477 g, 0.521 mmol), and dppf (0.481 g, 0.868 mmol) and sealed with a rubber septum. Anhyd DMAC (25 mL) and PMHS (0.344 mL, 17.37 mmol) were added via syringe and the mixture was degassed 10 min by sparging with N2 while immersed in an ultrasonic cleaning bath. The mixture was stirred in an oil bath at 100° C. under nitrogen. After 26 h the mixture was cooled, poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (2.98 g, 71% yield) as a tan solid: 1H NMR (400 MHz, CDCl3) δ 7.72 (dd, J=8.7, 4.7 Hz, 1H), 7.43 (dd, J=8.6, 8.0 Hz, 1H), 0.32 (s, 9H); MS (GCMS El) m/z 242 ([M]+, 7%), 227 ([M-CH3]+, 100%). D. (S)-1-(3-Hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile An oven-dried vial was charged with (S)-3-amino-2-methylbutan-2-ol (Example 21G) (0.064 g, 0.622 mmol), 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (0.126 g, 0.518 mmol), and K2CO3 (0.143 g, 1.036 mmol) and sealed with a rubber septum. Anhyd NMP (3 mL) was added via syringe and the mixture was stirred in a heating block at 60° C. under N2. After 30 min, the vial was sealed with a PTFE-faced crimp top and the mixture was subjected to microwave heating (140° C.) for 15 min. The mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (S)-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile (0.0659 g, 50% yield) as a tan solid: 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J=8.7, Hz, 1H), 7.69 (d, J=3.3 Hz, 1H), 7.51 (d, J=8.7 Hz, 1H), 6.84 (d, J=3.2 Hz, 1H), 4.47 (q, J=7.0 Hz, 1H), 1.63 (d, J=7.0 Hz, 3H), 1.53 (s, 1H), 1.34 (s, 3H), 1.11 (s, 3H). Example 41 (R)-1-(3-Hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile An oven-dried vial was charged with (R)-3-amino-2-methylbutan-2-ol (made in a manner similar to Example 21G using commercially available (S)-methyl 2-aminopropanoate, hydrochloride) (0.072 g, 0.696 mmol), 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (Example 40C) (0.141 g, 0.58 mmol), anhyd NMP (3.5 mL) and DIEA (0.304 mL, 1.740 mmol), and the vial was sealed with a crimp top. The mixture was subjected to microwave heating (140° C.) for 20 min. Upon cooling the mixture was poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 0.0572 g (0.226 mmol) of the aniline intermediate. The aniline was dissolved in anhyd NMP (2 mL) and a solution of KOtBu in THF (0.250 mL, 0.25 mmol) was added via syringe. The mixture was stirred overnight at rt under N2. After ca. 24 h, the mixture was poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-1-(3-hydroxy-3-methylbutan-2-yl)-1H-indole-4,5-dicarbonitrile (0.0285 g, 19% yield) as a colorless film: 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J=8.7, Hz, 1H), 7.69 (d, J=3.4 Hz, 1H), 7.51 (d, J=8.6 Hz, 1H), 6.84 (d, J=3.4 Hz, 1H), 4.46 (q, J=7.1 Hz, 1H), 1.63 (d, J=7.0 Hz, 3H), 1.48 (s, 1H), 1.34 (s, 3H), 1.11 (s, 3H). Example 42 1-(2-Hydroxy-2-methylpentan-3-yl)-1H-indole-4,5-dicarbonitrile An oven-dried 20 mL microwave vial was charged with 3-amino-2-methylpentan-2-ol (Example 34A) (0.0705 g, 0.602 mmol), 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (Example 40C) (0.146 g, 0.602 mmol), and K2CO3 (0.100 g, 0.722 mmol). Anhyd NMP (3 mL) was added via syringe and the vial was sealed with a PTFE-faced crimp top. The mixture was subjected to microwave heating (140° C.) for 35 min. The mixture was cooled, poured into satd NaHCO3, layered with EtOAc and the whole was filtered (Whatman #2). Layers were separated and the aqueous layer was extracted with EtOAc (×2). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution). Fractions containing the desired product were decolorized with activated carbon affording 1-(2-hydroxy-2-methylpentan-3-yl)-1H-indole-4,5-dicarbonitrile (0.0285 g, 18% yield) as an amber film: 1H NMR (400 MHz, CDCl3) δ 7.75-7.61 (m, 2H), 7.53 (d, J=8.6 Hz, 1H), 6.88 (d, J=3.3 Hz, 1H), 4.16-4.07 (m, 1H), 2.20-2.01 (m, 2H), 1.37 (s, 3H), 1.08 (s, 3H), 0.65 (t, J=7.3 Hz, 3H); MS (LCMS ES+) m/z 268 ([M+H]+, 26%), 285 (100%), 309 ({[M+H]+MeCN}+, 78%). Example 43 1-(3-Hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-4,5-dicarbonitrile An oven-dried vial was charged with 3-amino-2,3-dimethylbutan-2-ol (Example 35A) (0.063 g, 0.540 mmol), and 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (Example 40C) (0.109 g, 0.45 mmol) and sealed with a rubber septum. DIEA (0.157 mL, 0.900 mmol) and anhyd DMSO (2 mL) were added via syringe and the mixture was stirred at rt under N2. After 18 h, the temperature was increased to 60° C. and stirring continued an additional 30 h. Upon cooling the mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo to a dark, oily residue. An oven-dried vial was charged with the residue, followed by CuI (0.043 g, 0.225 mmol) and sealed with a rubber septum. Anhyd DMF (3 mL) was added via syringe and the septum was replaced with a PTFE-faced crimp top. The mixture was subjected to microwave heating (140° C.) for 20 min. The mixture was poured into satd NH4Cl and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) followed by preparative HPLC (C18 stationary phase, MeCN/water gradient with 0.1% TFA additive) affording 1-(3-hydroxy-2,3-dimethylbutan-2-yl)-1H-indole-4,5-dicarbonitrile (0.0093 g, 8% yield) as a colorless solid: 1H NMR (400 MHz, CDCl3) δ 8.21 (dd, J=9.0, 0.8 Hz, 1H), 7.65 (d, J=3.5 Hz, 1H), 7.42 (d, J=9.0 Hz, 1H), 6.81 (dd, J=3.5, 0.8 Hz, 1H), 1.88 (s, 6H), 1.21 (s, 6H); MS (LCMS ES+) m/z 268 ([M+H]+, 29%), 309 ({[M+H]+MeCN}+, 100%). Example 44 (R)-1-(1-(Methylsulfonyl)propan-2-yl)-1H-indole-4,5-dicarbonitrile A mixture of (R)-4-chloro-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-5-carbonitrile (Example 39) (0.043 g, 0.145 mmol), Zn(CN)2 (0.034 g, 0.290 mmol) and Pd(PPh3)4 (0.0335 g, 0.029 mmol) in DMF (3 mL) was sparged with N2 for 5 minutes, and then heated at 120° C. in a sealed tube for 4 h. The reaction was monitored by LCMS, and additional zinc cyanide and tetrakis(triphenylphosphine)palladium(0) was added accordingly. After about 50% conversion, the reaction mixture was diluted with EtOAc (20 mL) and washed with water (15 mL). The organic phase was washed with brine. The combined aqueous phases were extracted with EtOAc (1×20 mL). The organic phases were combined, dried over Na2SO4, filtered and concentrated. The residue was purified by preparative HPLC (Phenomenex Luna column; gradient: 10-100% MeCN-water with 0.1% TFA). The fractions with product were combined and concentrated down to the aqueous phase, which was partitioned between EtOAc (20 ml) and aq. saturated Na2CO3 solution (20 mL). The organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated. The product was subsequently crystallized from CH2Cl2-hexanes to give (R)-4-chloro-1-(1-(methylsulfonyl)propan-2-yl)-1H-indole-5-carbonitrile as a white solid (0.015 g, 33% yield): MS (ESI): m/z 288 (M+H). Example 45 (R)-1-(1-(3-Cyanophenyl)ethyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile An oven-dried vial was charged with 4-fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile (Example 21D) (0.173 g, 0.606 mmol), commercially available (R)-3-(1-aminoethyl)benzonitrile (0.098 g, 0.667 mmol) and K2CO3 (0.092 g, 0.667 mmol) and sealed with a rubber septum. Anhyd NMP (3 mL) was added via syringe and the mixture was stirred in a heating block at 60° C. under N2 for 17 h. The mixture was cooled, poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-1-(1-(3-cyanophenyl)ethyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.0845 g, 41% yield) as a pale yellow gum which solidified upon trituration with Et2O/hexanes: 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J=7.6 Hz, 1H), 7.57 (d, J=4.6 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.47 (t, J=7.8 Hz, 1H), 7.42 (s, 1H), 7.39 (d, J=8.6 Hz, 1H), 7.29 (d, J=6.7 Hz, 1H, overlapping with solvent), 6.96 (m, 1H), 5.76 (q, J=7.0 Hz, 1H), 2.01 (d, J=7.1 Hz, 3H); MS (LCMS ES+) m/z 340 ([M+H]+, 86%), 381 ({[M+H]+MeCN}+, 100%). Example 46 1-(1-(3-Cyanophenyl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile A. 1-(3-Cyanophenyl)propyl methanesulfonate A mixture of 3-(1-hydroxypropyl)benzonitrile (0.273 g, 1.694 mmol; ref. Synlett (2002), (11), 1922-1924), Et3N (0.354 mL, 2.54 mmol) and MsCl (0.198 mL, 2.54 mmol) in CH2Cl2 (5 mL) was stirred at rt. After 90 min, an additional 0.75 eq each of Et3N and MsCl were added. After 1 h, the reaction mixture was concentrated to dryness, and the residue was partitioned between EtOAc (20 mL) and 0.1N HCl (20 mL). The organic phase was washed with water and brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using a 5-30% EtOAc-hexane gradient to give 1-(3-cyanophenyl)propyl methanesulfonate (0.289 g, 68% yield) (the product is somewhat unstable, and it needs to be used shortly thereafter or stored at low temperatures). B. 1-(1-(3-Cyanophenyl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile To a suspension of 4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.030 g, 0.143 mmol) in THF (5 mL) was added KOtBu (1M in THF) (0.157 mL, 0.157 mmol). After stirring at rt for a couple of min, a solution of 1-(3-cyanophenyl)propyl methanesulfonate (0.0512 g, 0.214 mmol) in THF (1 mL) was added, and the mixture was heated at 80° C. in a sealed tube. The reaction was monitored by LCMS. After ˜30 min, additional 1-(3-cyanophenyl)propyl methanesulfonate (0.0342 g, 0.143 mmol) in THF (1 mL) was added, and the mixture was heated at 80° C. for another 30 min. Upon cooling, the reaction mixture was diluted with EtOAc (20 mL) and washed with water (20 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was chromatographed over silica gel using 0-25% EtOAc-hexane gradient. The material was further purified by preparative HPLC (Phenomenex Luna column; gradient: 10-100% MeCN-water with 0.1% TFA). The fractions with product were combined and concentrated down to the aqueous phase, which was then partitioned between EtOAc (25 mL) and saturated aq. NaHCO3 solution (20 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated to give 1-(1-(3-cyanophenyl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.014 g, 26% yield): MS (ESI): m/z 354 (M+H). Example 47 (R)-1-(1-(5-Cyanopyridin-3-yl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile An oven-dried vial was charged with 4-fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile (Example 21D), (0.171 g, 0.598 mmol), commercially available (R)-5-(1-aminopropyl)nicotinonitrile (0.106 g, 0.658 mmol) and K2CO3 (0.091 g, 0.658 mmol) and sealed with a rubber septum. Anhydrous NMP (3 mL) was added via syringe and the mixture was stirred in a heating block at 60° C. under N2. After 22 h the mixture was cooled, poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-1-(1-(5-cyanopyridin-3-yl)propyl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile (0.0758 g, 36% yield) as a tan gum: 1H NMR (400 MHz, CDCl3) δ 8.82 (s, 1H), 8.69 (br. s., 1H), 7.64 (br. s., 1H), 7.60-7.43 (m, 3H), 7.00 (br. s., 1H), 5.49 (t, J=7.5 Hz, 1H), 2.42 (sxt, J=7.1 Hz, 2H), 1.04 (t, J=7.1 Hz, 3H); MS (LCMS ES+) m/z 355 ([M+H]+, 62%), 396 ({[M+H]+MeCN}+, 100%). Example 48 (R)-4-Chloro-1-(1-(5-cyanopyridin-3-yl)propyl)-1H-indole-5-carbonitrile An oven-dried vial was charged with commercially available (R)-5-(1-aminopropyl)nicotinonitrile (0.165 g, 1.023 mmol), 2-chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) (0.234 g, 0.93 mmol), and K2CO3 (0.141 g, 1.023 mmol) and sealed with a rubber septum. Anhyd NMP (3 mL) was added via syringe and the mixture was stirred in a heating block at 60° C. under N2. After 18 h, the septum was replaced with a PTFE-faced crimp top and the mixture was subjected to microwave heating (140° C.) for 15 min. Upon cooling the mixture was poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), the combined washes were filtered (Whatman #2) and re-extracted with EtOAc (×1). Combined organics were dried over Na2SO4, filtered through a short pad of silica and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-4-chloro-1-(1-(5-cyanopyridin-3-yl)propyl)-1H-indole-5-carbonitrile (0.1024 g, 34% yield) as a gum which formed a tan solid upon trituration with Et2O/hexanes: 1H NMR (400 MHz, CDCl3) δ 8.81 (d, J=1.1 Hz, 1H), 8.68 (d, J=1.8 Hz, 1H), 7.66-7.58 (m, 1H), 7.44 (d, J=3.4 Hz, 1H), 7.42 (d, J=8.8 Hz, 1H), 7.18 (d, J=8.5 Hz, 1H), 6.88 (d, J=3.1 Hz, 1H), 5.43 (dd, J=8.5, 6.9 Hz, 1H), 2.50-2.30 (m, 2H), 1.03 (t, J=7.3 Hz, 3H); MS (LCMS ES+) m/z 321 ([M+H]+, 55%), 362 ({[M+H]+MeCN}+, 100%). Example 49 (R)-1-(1-Phenylethyl)-1H-indole-4,5-dicarbonitrile An oven-dried vial was charged with 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (Example 40C) (0.100 g, 0.413 mmol) and K2CO3 (0.057 g, 0.413 mmol) and sealed with a rubber septum. Anhyd NMP (2 mL) and (R)-1-phenylethanamine (0.053 mL, 0.413 mmol) were added via syringe and the mixture was stirred in a heating block at 60° C. under N2. After 15 h, the mixture was cooled, quenched by addition of satd NH4Cl, poured into water and extracted with EtOAc (×3). Combined organics were washed (water×2, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-1-(1-phenylethyl)-1H-indole-4,5-dicarbonitrile (0.0574 g, 51% yield) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J=3.3 Hz, 1H), 7.48 (dd, J=8.6, 0.7 Hz, 1H), 7.43 (d, J=8.7 Hz, 1H), 7.37-7.27 (m, 3H), 7.12-7.06 (m, 2H), 6.88 (dd, J=3.3, 0.7 Hz, 1H), 5.72 (q, J=7.1 Hz, 1H), 1.98 (d, J=7.0 Hz, 3H); MS (LCMS ES+) m/z 272 ([M+H]+, 5%), 289 (100%), 313 ({[M+H]+MeCN}, 23%), 335 ({[M+Na]+MeCN}, 22%). Example 50 (R)-1-(1-(3-Cyanophenyl)ethyl)-1H-indole-4,5-dicarbonitrile An oven-dried vial was charged with 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (Example 40C) (0.128 g, 0.528 mmol), (R)-3-(1-aminoethyl)benzonitrile (0.085 g, 0.581 mmol), and K2CO3 (0.080 g, 0.581 mmol) and sealed with a rubber septum. Anhyd NMP (3 mL) was added via syringe and the mixture was stirred in a heating block at 60° C. under N2. After 3.5 h the septum was replaced with a PTFE-faced crimp top and the mixture was subjected to microwave heating (140° C.) for 20 min. Upon cooling, the mixture was poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were filtered (Whatman #2), washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-1-(1-(3-cyanophenyl)ethyl)-1H-indole-4,5-dicarbonitrile (0.0705 g, 0.238 mmol, 45.0% yield) as a tan foam: 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J=3.4 Hz, 1H), 7.61 (dt, J=7.8, 1.3 Hz, 1H), 7.50-7.41 (m, 3H), 7.40 (t, J=1.8 Hz, 1H), 7.30-7.27 (m, 1H), 6.93 (dd, J=3.4, 0.7 Hz, 1H), 5.76 (q, J=7.1 Hz, 1H), 2.01 (d, J=7.1 Hz, 3H); MS (LCMS ES+) m/z 297 ([M+H]+, 24%), 338 ({[M+H]+MeCN}+, 100%). Example 51 (R)-1-(1-(5-Cyanopyridin-3-yl)propyl)-1H-indole-4,5-dicarbonitrile A. 3-Ethynyl-4-fluorophthalonitrile To a solution of 4-fluoro-3-((trimethylsilyl)ethynyl)phthalonitrile (Example 40C) (0.302 g, 1.246 mmol) in anhyd THF (5 mL) was added a solution of TBAF in THF (1.246 mL, 1.246 mmol), dropwise. The resulting black mixture was stirred at rt under N2 for 5 min. The mixture was poured into water and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording 3-ethynyl-4-fluorophthalonitrile (0.0635 g, 30% yield) as a tan solid: 1H NMR (400 MHz, CDCl3) δ 7.79 (dd, J=8.7, 4.7 Hz, 1H), 7.49 (dd, J=8.7, 8.0 Hz, 1H), 3.86 (s, 1H); MS (GCMS El) m/z 170 ([M]+, 100%). B. (R)-1-(1-(5-Cyanopyridin-3-yl)propyl)-1H-indole-4,5-dicarbonitrile To a solution of (R)-5-(1-aminopropyl)nicotinonitrile hydrochloride (0.081 g, 0.411 mmol) in anhyd NMP (2.0 mL) was added DIEA (0.215 mL, 1.232 mmol) via syringe. The mixture was stirred 15 min and 3-ethynyl-4-fluorophthalonitrile (0.0635 g, 0.373 mmol) was added in one portion. The mixture was stirred at rt under N2 for 36 h, poured into satd NaHCO3 and extracted with EtOAc (×3). Combined organics were washed (water, brine), dried over Na2SO4 and concentrated in vacuo. The residue was dissolved in anhyd DMF (3 mL), CuI (0.036 g, 0.187 mmol) was added and the mixture was subjected to microwave heating (140° C.) for 30 min. The mixture was diluted with EtOAc and filtered through a pad of Celite. The filtrate was diluted 1:1 with heptane and concentrated in vacuo (3× heptane chase). The residue was purified by low pressure liquid chromatography (silica gel, EtOAc/hexanes, gradient elution) affording (R)-1-(1-(5-cyanopyridin-3-yl)propyl)-1H-indole-4,5-dicarbonitrile (0.0407 g, 0.131 mmol, 35.0% yield) as a yellow gum: 1H NMR (400 MHz, CDCl3) δ 8.83 (d, J=8.7 Hz, 1H), 8.69 (d, J=2.2 Hz, 1H), 7.66 (t, J=2.1 Hz, 1H), 7.63 (d, J=3.4 Hz, 1H), 7.53 (s, 2H), 7.00 (d, J=3.4 Hz, 1H), 5.50 (dd, J=8.7, 6.8 Hz, 1H), 2.53-2.34 (m, 2H), 1.03 (t, J=7.3 Hz, 3H); MS (LCMS ES+) m/z 312 ([M+H]+, 6%), 353 ({[M+H]+MeCN}+, 100%). Example 52 4-Chloro-1-((2R,3S)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile A. (R)-3-(Bibenzylamino)-1,1,1-trifluorobutan-2-one (R)-Methyl 2-(dibenzylamino)propanoate (made in a manner similar to Example 21E using commercially available (R)-methyl 2-aminopropanoate, hydrochloride (8.36 g, 29.5 mmol) was dissolved in toluene (15 mL) and treated with trimethyl(trifluoromethyl)silane (6.53 mL, 44.3 mmol). The mixture was cooled on an ice bath and tetrabutylammonium acetate (0.445 g, 1.48 mmol) was added. The reaction was left on the ice bath. TLC and LCMS after 1.5 h showed excellent conversion to a less polar (TLC) product. The mixture was quenched with sat. aq. NH4Cl and extracted with EtOAc. The organic portions were dried over Na2SO4, filtered, and concentrated to a brown oil that was diluted with THF (40 mL) and then treated with 1N aqueous HCl (10 mL). The mixture was allowed to stir overnight. LCMS the next day showed the desired product along with a trace of the diaddition product. The mixture was neutralized with NaHCO3 (saturated aqueous to pH ca. 9) and extracted with EtOAc. The combined organic portions were washed with sat NaHCO3 followed by brine. The organic portion was then dried over Na2SO4, filtered, and concentrated to a dark amber oil that was chromatographed (ISCO, 220 g silica, hex/EtOAC; 0-30%; 230 and 254 nm) to afford the desired product as a bright yellow oil (6.83 g, 72%): MS (ESI): m/z 340 (M+H as hydrate). B. (2S,3R)-3-(Dibenzylamino)-1,1,1-trifluoro-2-methylbutan-2-ol (R)-3-(Dibenzylamino)-1,1,1-trifluorobutan-2-one (Example 52A) (3.41 g, 10.61 mmol) was dissolved in Et2O (80 mL) and then cooled to ca. 0° C. (ice external temp) prior to the addition of MeMgl (7.07 mL, 3 M). Addition of the Grignard reagent caused the reaction to become heterogeneous. After stirring for 10 min, TLC indicated good conversion to a slightly less polar than sm product (a trace of what appeared to be sm remained), the mixture was quenched with sat. aq. NH4Cl and extracted with EtOAc. The combined organic portions were dried over Na2SO4 and concentrated. The resulting bright yellow residue was purified by flash chromatography (ISCO, 80 g silica, 0% to 40% over 27 min. ca. 10 min ret time; hex/EtOAc) to afford the desired product as a bright yellow oil. TLC, LCMS, and NMR showed ca. 15-20% of the bis CF3 alcohol contaminating the desired product. A trace of the other diastereomer also existed. The material was concentrated and rechromatographed (straight CH2Cl2, 80 g SiO2, 254/230 nm) to afford separation of the bis-CF3 alcohol and desired product (1.94 g, 54%). This material was used in its entirety for the debenzylation step: 1H NMR (400 MHz, DMSO-d6) δ 7.62-7.48 (m, 10H), 6.11 (s, 1H), 4.16 (d, J=13.6 Hz, 2H), 3.61 (d, J=13.7 Hz, 2H), 3.15 (q, J=6.8 Hz, 1H), 1.42-1.40 (m, 6H). C. (2S,3R)-3-Amino-1,1,1-trifluoro-2-methylbutan-2-ol (2S,3R)-3-(Dibenzylamino)-1,1,1-trifluoro-2-methylbutan-2-ol (Example 52B) (1.94 g, 5.75 mmol) was dissolved in MeOH (50 mL) and an then treated with the catalyst (0.612 g, 10% dry weight, 50% water). The reaction vessel was then purged with alternating vacuum and N2 (7×). H2 was introduced and then the vessel was purged again with vacuum alternated with H2 (3×). The reaction vessel was then finally charged with H2 (90 psi). The pressure was allowed to drop to ca. 80 psi and allowed to stay there overnight (H2 uptake appeared to stop). After 15 h, the reaction was purged with N2/vacuum cycles and the catalyst/carbon was removed by filtration through celite. The celite cake was rinsed with MeOH and the resulting filtrate was carefully concentrated to a white solid by rotavap followed by finishing the last part of the liquid volume with an N2 blow down. The resulting grey solid/film was dissolved in CH2Cl2 and then filtered through a microfilter to remove remaining Pd/C. The resulting pale yellow filtrate was then blown down and exposed to light vacuum to afford a pale yellow solid (0.726 g, 80%) PMR of this material showed excellent purity and no remaining sm: 1H NMR (400 MHz, DMSO-d6) δ 2.95 (q, J=6.7 Hz, 1H), 1.60 (bs, 2H), 1.13-1.11 (m, 3H), 0.96-0.94 (m, 3H). D. 4-Chloro-1-((2R,3S)-4,4,4-trifluoro-3-hydroxy-3-methylbutan-2-yl)-1H-indole-5-carbonitrile 2-Chloro-4-fluoro-3-((trimethylsilyl)ethynyl)benzonitrile (Example 32B) (0.08 g, 0.318 mmol), (2S,3R)-3-amino-1,1,1-trifluoro-2-methylbutan-2-ol (Example 52C) (0.079 mg, 0.503 mmol), and Hunig's base (0.094 mL, 0.540 mmol) were combined in DMSO (1.0 mL) in a sealed tube and then heated to 100° C. Formation of the aniline intermediate was monitored by LCMS. Excellent conversion to this intermediate was realized after ca. 3 h of heating. The mixture was diluted with water and extracted with EtOAc. The combined organic portions were dried over Na2SO4 and concentrated to a brown oil. Further traces of DMSO were removed by high vac. The brown residue was diluted with NMP (ca. 1.0 mL) and then treated with KOtBu (0.095 mL, 1.0 M in THF). The resulting solution was then heated to 60° C. for 45 min at which time LCMS indicated formation of a very slightly less polar product. The UV trace of this product was far different from that of the aniline intermediate. The crude mixture was diluted with water and extracted with EtOAc. The combined organic portions were washed with water and brine and then dried over Na2SO4. Concentration afforded a thick brown oil that was purified by ISCO (24 g silica, hex/EtOAc up to 70%, detection at 254 and 230 nm) to afford the desired product as a pale yellow solid (0.078 g, 78%) in excellent purity: 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J=3.5 Hz, 1H), 7.71 (d, J=8.6 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 6.68 (d, J=3.3 Hz, 1H), 6.50 (s, 1H), 5.03 (q, J=7.0 Hz, 1H), 1.51 (d, J=6.9 Hz, 3H), 1.37 (s, 3H); MS (ESI): m/z 317 (M+H). Example 53 1-((2R,3S)-4,4,4-Trifluoro-3-hydroxy-3-methylbutan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 52 using 4-fluoro-2-(trifluoromethyl)-3-((trimethylsilyl)ethynyl)benzonitrile (Example 21D): 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J=8.8 Hz, 1H), 7.94 (d, J=3.5 Hz, 1H), 7.76 (d, J=8.6 Hz, 1H), 6.78-6.76 (m, 1H), 6.54 (s, 1H), 5.15 (q, J=7.0 Hz, 1H), 1.54 (d, J=7.0 Hz, 3H), 1.39 (s, 3H); MS (ESI): m/z 351 (M+H). Example 54 (S)-1-(1-(Methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 27D using (S)-1-(methylthio)propan-2-amine which was made in a manner similar to Example 27C: MS (ESI): m/z 299 (M+H). Example 55 (S)-1-(1-(Methylsulfonyl)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile Synthesized in a manner similar to Example 27 using (S)-1-(1-(methylthio)propan-2-yl)-4-(trifluoromethyl)-1H-indole-5-carbonitrile: 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J=8.7 Hz, 1H), 8.12 (d, J=3.5 Hz, 1H), 7.81 (d, J=8.6 Hz, 1H), 6.85-6.84 (m, 1H), 5.40-5.35 (m, 1H), 4.01 (dd, J=14.6, 8.2 Hz, 1H), 3.83 (dd, J=14.9, 5.1 Hz, 1H), 2.76 (s, 3H), 1.59 (d, J=6.6 Hz, 3H); MS (ESI): m/z 331 (M+H). Biological Section Compounds of the current invention are modulators of the androgen receptor. Additionally, the compounds of the present invention may also prove useful as modulators of the glucocorticoid receptor, the mineralocorticoid receptor, and/or the progesterone receptor. Activity mediated through oxosteroid nuclear receptors was determined using the following in vitro and in vivo assays. In Vitro Assays: The following abbreviations and sources of materials are used Fluormone PL Red—a commercially available PR fluoroprobe (Invitrogen, P2964) Fluormone GS Red—a commercially available GR fluoroprobe (PanVera Corp, Product No P2894) Fluormone AL Red—a commercially available AR fluoroprobe (Invitrogen, PV4294,) MBP-hPR-LBD—maltose binding protein Purified human progesterone ligand binding domain (made in house) GR—purified human glucocorticoid receptor (PanVera Corp, Product No P2812) MBP-hAR-LBD—maltose binding protein Purified rat androgen ligand binding domain (made in house) PR Screening Buffer—100 mM potassium phosphate (pH 7.4), 100 μG/ml bovine gamma globulin, 15% ethylene glycol, 10% glycerol with 2 mM CHAPS, 1 mM DTT added fresh and 4% DMSO added fresh (final of 5% DMSO in assay with 1% concentration coming from compound dispense) AR Screening Buffer—50 mM Tris pH 7.5, 100 mM Ammonium Sulfate, 20% glycerol, 3% xyliltol with 5 mM Chaps, 2 mM DTT added fresh and 4% DMSO added fresh (final of 5% DMSO in assay with 1% concentration coming from compound dispense) GR Screening Buffer—100 mM potassium phosphate (pH 7.4), 200 mM Na2MoO2, 1 mM EDTA, 20% DMSO (PanVera Corp Product No P2814) with GR stabilizing peptide (100 μM) (PanVera Corp Product No P2815) DTT—dithiothreitol (PanVera Corp Product No P2325) Discovery Analyst—is an FP reader DMSO—dimethylsulphoxide Progesterone Receptor Fluorescence Polarization Assay: The progesterone receptor fluorescence polarization assay is used to investigate the interaction of the compounds with the progesterone receptor. Compounds are added to the 384 well black low-volume plates to a final volume of 0.1 μL. DTT and DMSO are added to the chilled assay buffer just before beginning assay. Sufficient Fluormone PL Red and PR-LBD are defrosted on ice and added to the chilled buffer in a glass tube to give a final concentration of 2 nM and 8 nM, respectively. A volume of 10 μL of the assay mix is added to compound plates with a multidrop. The assay is allowed to incubate at 20-22° C. (room temp) for 2-3 hours. The plates are counted in a Discovery Analyst with suitable 535 nM excitation and 590 nM emission interference filters (Dichroic 561 nM). Compounds that interact with the PR result in a lower fluorescence polarization reading. Test compounds are dissolved and diluted in DMSO. Compounds are assayed in singlicate, a four parameter curve fit of the following form being applied y = a - d 1 + ( x / c ) b + d where a is the minimum, b is the Hill slope, c is the IC50 and d is the maximum. Maximum and minimum values are compared to adhesion in the absence of compound and in the presence of 10−5M progesterone. Data is presented as the mean pIC50 with the standard error of the mean of n experiments. Androgen Receptor Fluorescence Polarization Assay: The androgen receptor fluorescence polarization assay is used to investigate the interaction of the compounds with the androgen receptor. Compounds are added to the 384 well black low-volume plates to a final volume of 0.1 μL. DTT and DMSO are added to the chilled assay buffer just before beginning assay. Sufficient Fluormone AL Red and AR-LBD are defrosted on ice and added to the chilled buffer in a glass tube to give a final concentration of 1 nM and 100 nM, (for current batch) respectively. A volume of 10 μL of the assay mix is added to compound plates with a multidrop. The assay is allowed to incubate at 20° C. for 2-3 hours. The plates are counted in a Discovery Analyst with suitable 535 nM excitation and 590 nM emission interference filters (Dichroic 561 nM). Compounds that interact with the AR result in a lower fluorescence polarization reading. Test compounds are dissolved and diluted in DMSO. Compounds are assayed in singlicate, a four parameter curve fit of the following form being applied y = a - d 1 + ( x / c ) b + d where a is the minimum, b is the Hill slope, c is the IC50 and d is the maximum. Maximum and minimum values are compared to adhesion in the absence of compound and in the presence of 10−5M control compound, 2-((4-cyano-3-(trifluoromethyl)phenyl)(2,2,2-trifluoroethyl)amino)acetamide. Data is presented as the mean pIC50 with the standard error of the mean of n experiments. Results from selected examples are shown in Table 1. TABLE 1 Example Binding pIC50 % Max Std. error 2 7.0 72 0.18 7 6.2 100 n/a (n = 1) 12 6.6 90 0.4 17 7.1 99 0.32 21 6.5 100 0.23 22 6.8 100 0.3 27 7.1 91 0.21 32 7.5 78 0.04 43 6.2 105 0.34 52 7.8 97 0.11 Glucocorticoid Receptor Fluorescence Polarization Assay The glucocorticoid receptor fluorescence polarization assay is used to investigate the interaction of the compounds with the glucocorticoid receptor. Compounds are added to the 384 well black plates to a final volume of 0.5 μL. Sufficient Fluormone GS Red and GR are defrosted on ice to give a final concentration of 1 nM and 4 nM, respectively. GR screening buffer is chilled to 4° C. prior to addition of DTT to give a final concentration of 1 mM. The Fluormone GS Red, and GR in GR Screening Buffer are added to compound plates to give a final volume of 10 μL. The assay is allowed to incubate at 4° C. for 12 hours. The plates are counted in a Discovery Analyst with suitable 535 nM excitation and 590 nM emission interference filters. Compounds that interact with the GR result in a lower fluorescence polarization reading. Test compounds are dissolved and diluted in DMSO. Compounds are assayed in singlicate, a four parameter curve fit of the following form being applied y = a - d 1 + ( x / c ) b + d where a is the minimum, b is the Hill slope, c is the EC50 and d is the maximum. Maximum and minimum values are compared to adhesion in the absence of compound and in the presence of 10−5M dexamethasone. Data is presented as the mean pIC50 with the standard error of the mean of n experiments. AR Functional Assay: AR DNA Preparation A plasmid containing an N-terminal truncation of the human AR gene was obtained from ATCC which was missing 154 residues from the N-terminus of the protein. The N-terminal region of the AR gene from a human liver cDNA library generated in-house, was cloned using PCR technique. The N-terminus and C-terminus pieces were PCR-ed together and subcloned in to the pSG5 vector at the BamHI site along with a Kozak sequence. The sequence differs from the published sequence in two regions of high variability within the receptor amongst published sequences. This clone has 1 additional glutamine residue (residue 79) and 3 additional glycine residues (position 475). MMTV DNA Preparation pGL3-Basic Vector was digested with SmaI and XhoI. pMSG was digested with HindIII blunt ended and then digested with XhoI to excise the pMMTV-LTR. The pMMTV-LTR fragment was then ligated to the SmaI and XhoI sites of pGL3-Basic Vector. The resulting plasmid contains the MMTV promoter from position 26 to the XhoI site, followed by luciferase which is contained between the NcoI and SalI (position 3482) sites. Assay Protocol Monkey kidney CV-1 cells (ECACC No. 87032605) were transiently transfected with Fugene-6 reagent according to the manufacturer's protocol. Briefly, a T175 flask of CV-1 cells at a density of 80% confluency was transfected with 25 g of mix DNA and 751 of Fugene-6. The DNA mix (1.25 microg pAR, 2.5 microg pMMTV Luciferase and 18.75 microg pBluescript (Stratagene)) was incubated with Fugene in 5 ml OptiMEM-1 for 30 min and then diluted up to 20 ml in transfection media (DMEM containing 1% Hyclone, 2 mM L-Glutamine and 1% Pen/Strep) prior to addition to the cells. After 24 h, cells were washed with PBS, detached from the flask using 0.25% trypsin and counted using a Sysmex KX-21N. Transfected cells were diluted in assay media (DMEM containing 1% Hyclone, 2 mM L-Glutamine and 1% Pen/Strep) at 70 cells/microlitre I. 70 microlitres of suspension cells were dispensed to each well of white Nunc 384-well plates, containing compounds at the required concentration. After 24 h, 10 microlitres of Steady Glo were added to each well of the plates. Plates were incubated in the dark for 10 min before reading them on a Viewlux reader. Analysis All data was normalized to the mean of 16 high and 16 low control wells on each plate. A four parameter curve fit of the following form was then applied y = a - d 1 + ( x / c ) b + d Where a is the minimum, b is the Hill slope, c is the XC50 and d is the maximum. Data is presented as the mean pXC50 with the standard deviation of the mean of n experiments. The compounds shown in Examples 1 through 55 were tested in the AR functional assay and all had a pIC50≥5.01 in the agonist mode of this assay. Those of skill in the art will recognize that in vitro binding assays and cell-based assays for functional activity are subject to variability. Accordingly, it is to be understood that the values for the pIC50's recited above are exemplary only. Castrated Male Rat Model (ORX Rat) The activity of the compounds of the present invention as modulators of the androgen receptor was investigated using a castrated male rat model (ORX) as described in C. D. Kockakian, Pharmac. Therap. B 1(2), 149-177 (1975); C. Tobin and Y. Joubert, Developmental Biology 146, 131-138 (1991); J. Antonio, J. D. Wilson and F. W. George, J Appl. Physiol. 87(6) 2016-2019 (1999)) the disclosures of which herein are included by reference. Androgens have been identified as playing important roles in the maintenance and growth of many tissues in both animals and humans. Muscles, like the levator ani and bulbocavernosus, and sexual accessory organs, such as the prostate glands and seminal vesicles have high expression levels of the androgen receptor and are known to respond quickly to exogenous androgen addition or androgen deprivation through testicular ablation. Castration produces dramatic atrophy of muscle and sexual accessory organs; whereas the administration of exogenous androgens to the castrated animal results in effective hypertrophy of these muscles and sexual accessory organs. Although the levator ani muscle, also known as the dorsal bulbocavernosus, is not ‘true skeletal muscle’ and definitely sex-linked, it is reasonable to use this muscle to screen muscle anabolic activities of test compounds because of its androgen responsiveness and simplicity of removal. Male Sprague-Dawley rats weighing 160-180 grams were used in the assay. The rats were singly caged upon receiving and throughout the study. Bilateral orchidectomies were performed in sterilized surgical conditions under isoflurane anesthesia. An anteroposterior incision was made in the scrotum. The testicles were exteriorized and the spermatic artery and vas deferens were ligated with 4.0 silk 0.5 cm proximal to the ligation site. The testicles then were removed by a surgical scissors distal to the ligation sites. The tissue stumps were returned to the scrotum, the scrotum and overlying skin were closed by a surgical stapler. The Sham-ORX rats underwent all procedures except ligation and scissors cutting. The rats were assigned randomly into study groups 7-10 days post surgery based on the body weight. Dihydrotestosterone (DHT) and the standard SARM, S-22, (J. Pharma. Exper. Thera. Vol 315, p. 230) were used as a positive control (1-10 mg/kg s.c. for DHT and 0.1 to 3 mg/kg p.o. for S-22). Compounds of the current invention were administered subcutaneously or orally for 4-28 days. Alternatively, some compounds of the current invention were administered subcutaneously or orally for 7-49 days. The rats were weighed daily and doses were adjusted accordingly. The general well being of the animal was monitored throughout the course of the study. At the end of the study, the rats were euthanized in a CO2 chamber. The ventral prostate glands (VP), seminal vesicles (SV), levator ani muscle (LA) and bulbocavernosus (BC) were carefully dissected. The tissues were blotted dry; the weights were recorded, and then saved for histological and molecular analysis. The VP and SV weights serve as androgenic indicators and LA and BC as anabolic indicators. The ratio of anabolic to androgenic activities was used to evaluate the test compounds. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and other potential serum markers of anabolic activities were also analyzed. In general, preferred compounds show levator ani hypertrophy and very little prostate stimulation. The compounds shown in Examples 9, 20, 26, 27, 33, 51, 52, and 53 were tested in the castrated male rate model essentially as described above. Test compounds were employed in free or salt form. The compounds shown in Examples 9, 26, 27, 51, 52, and 53 showed favorable levator ani hypertrophy and spared the prostate. Compounds having favorable levator ani hypertrophy were defined as those that show a 30% or greater increase in levator ani weight when compared to vehicle-treated castrates and dosed orally at up to 10 mg/kg/day. Prostate sparing was defined as at least a 2:1 ratio of levator ani ED50 to prostate ED50. The ED50 is defined as 50% of the maximum response above the vehicle treated castrate level. For shorter term studies (4-7 days), the maximum response is defined as the maximum response from positive control (DHT or standard SARM, S-22) treatment. For the longer term studies (7-49 days), the ED50 is defined as 50% of the eugonadal state. All research complied with the principles of laboratory animal care (NIH publication No. 85-23, revised 1985) and GlaxoSmithKline policy on animal use. Those of skill in the art will recognize that in vivo animal model studies such as the castrated male rat model studies described above are subject to variability. Accordingly, it is to be understood that the values for favorable levator ani hypertrophy and prostate sparing recited above are exemplary only. Although specific embodiments of the present invention are herein illustrated and described in detail, the invention is not limited thereto. The above detailed descriptions are provided as exemplary of the present invention and should not be construed as constituting any limitation of the invention. Modifications will be obvious to those skilled in the art, and all modifications that do not depart from the spirit of the invention are intended to be included within the scope of the appended claims. 16872949 glaxosmithkline intellectual property (no. 2) limited USA B2 Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001. Open Apr 12th, 2022 12:27PM Apr 12th, 2022 12:27PM GlaxoSmithKline Health Care Pharmaceuticals & Biotechnology
nyse:gsk GlaxoSmithKline Apr 12th, 2022 12:00AM Jul 12th, 2021 12:00AM https://www.uspto.gov?id=US11299541-20220412 Biopharmaceutical compositions The present disclosure relates to compositions, for treating interleukin 5 (IL-5) mediated diseases, and related methods. 11299541 1. A composition comprising: a) an anti-IL-5 antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, and b) acidic antibody variants of the anti-IL-5 antibody wherein ≤80% of antibodies in the composition are the acidic antibody variants, wherein the acidic antibody variants comprises: SEQ ID NO: 1, except that residue N386 of Seq ID NO: 1 is deamidated, or SEQ ID NO: 2, except that residue N31 of Seq ID NO: 2 is deamidated, or a combination thereof. 2. The composition of claim 1, wherein ≤75% of the antibodies in the composition are the acidic antibody variants. 3. The composition of claim 1, wherein 20% to 80% or 20% to 45% of the antibodies in the composition are the acidic antibody variants. 4. The composition of claim 1, wherein 1% to 15% of the antibodies in the composition are basic antibody variants. 5. The composition of claim 1, wherein residue N31 of Seq ID NO: 2 is deamidated in ≤25% of the antibodies in the composition. 6. The composition of claim 5, wherein residue N31 of Seq ID NO: 2 is deamidated in: ≤22.5%, ≤20%, ≤17.5%, ≤15%, ≤12.5, ≤10%, or ≤7.5% of the antibodies in the composition. 7. The composition of claim 1, wherein residue N386 of Seq ID NO: 1 is deamidated in ≤35% of the antibodies in the composition. 8. The composition of claim 1, wherein residue M64 of Seq ID NO: 1 is oxidized in ≤50% of the antibodies in the composition and residue W52 of Seq ID NO: 1 is oxidized in ≤3% of the antibodies in the composition. 9. The composition of claim 1, wherein residue M254 of Seq ID NO: 1 is oxidized in ≤50% of the antibodies in the composition, residue M360 of Seq ID NO: 1 is oxidized in ≤50% of the antibodies in the composition, and residue M430 of Seq ID NO: 1 is oxidized in ≤50% of the antibodies in the composition. 10. The composition of claim 1, wherein ≥50% of the antibodies in the composition comprises SEQ ID NO: 1 except that C-terminal lysine K449 is deleted. 11. The composition of claim 1, wherein ≥50% of the antibodies in the composition comprises a pyro-glutamate N-terminal. 12. The composition of claim 1, wherein ≤20% of the antibodies in the composition are aggregated antibody variants. 13. The composition of claim 1, wherein an antibody variants is measured using capillary isoelectric focusing of the composition. 14. The composition of claim 1, wherein the antibodies in the composition is at a concentration of between 75 mg/ml and 100 mg/ml. 15. The composition of claim 1, wherein the composition further comprises: a) a buffering agent selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citrate, sodium phosphate, potassium phosphate, sodium citrate, and histidine, providing a pH of between 6.8 and 7.2 or a pH of between 6.2 and 6.6; b) a sugar; c) polysorbate 80; d) EDTA; or e) a combination thereof. 16. The composition of claim 1, wherein the composition comprises an aqueous liquid formulation comprising: a) 100 mg/ml antibody; b) 15.5 mM sodium phosphate dibasic heptahydrate and 4.5 mM citric acid monohydrate at pH 6.3; c) 12% weight of sucrose to volume; d) 0.02% weight of polysorbate 80 to volume; and e) 0.05 mM EDTA. 17. The composition of claim 1, wherein the composition is lyophilized and comprises: a) 100 mg/mL antibody; b) 20 mM sodium phosphate dibasic heptahydrate at pH 6.8 to 7.2; c) 12% weight of sucrose to volume; and d) 0.05% weight of polysorbate 80 to volume. 18. The composition of claim 1, wherein the composition has at least 0.70 IL-5 specific antigen binding activity or at least 70% FcRn binding activity, compared with a reference standard composition, the reference standard composition comprising: a) an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO:2; b) 98% or more heavy chain C-terminal lysine deleted variant; c) 95% or more heavy chain N-terminal pyroglutamate variant; d) 6% or less deamidated variant; e) 4% or less methionine or cysteine oxidated variant; f) 0.1% or less tryptophan oxidated variant; and g) 0.4% or less aggregated antibody variant. 19. A pharmaceutical composition comprising the composition of claim 1, and a pharmaceutically acceptable carrier. 19 FIELD OF THE DISCLOSURE The present disclosure relates to compositions, for treating interleukin 5 (IL-5) mediated diseases, and related methods. BACKGROUND OF THE DISCLOSURE IL-5 plays a role in a number of different diseases such as asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid and eosinophilic esophagitis. These serious diseases affect hundreds of millions of people world wide. Mepolizumab is a monoclonal antibody that binds to soluble IL-5 and blocks the soluble IL-5 from binding to its receptor. The structure of IL-5 is indicative of a secreted protein and there is no evidence of any membrane-bound forms of IL-5 on any cell types. Thus, Fc effector functions are not part of the mepolizumab mechanism of action. Based on the mechanism of action and pharmacokinetic properties of mepolizumab, there are two functional domains involved in the biological activity of this monoclonal antibody. These are a) binding to IL-5 in complementary determining region (CDR) which provides the mechanism of action; and b) binding to neonatal Fc receptor (FcRn) receptor in Fc region, which determines the half-life. Through extensive characterization studies performed throughout the development of the product, it has been determined that deamidation, oxidation, and aggregation are critical quality attributes of mepolizumab. Importantly, it has been found that specific levels of these variants must be maintained to ensure appropriate biological function. Thus, a need exists for compositions suitable for maintaining the biological function of mepolizumab and for treating IL-5 mediated disease. Such compositions and related methods are provided by the present disclosure. SUMMARY OF THE DISCLOSURE One aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤80% acidic antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤80% acidic antibody variants and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variant at N31 of the light chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤55% oxidised antibody variants at M64 of the heavy chain amino acid sequence; ≤3% oxidised variant at W52 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence; ≤55% oxidised antibody variants at M64 of the heavy chain amino acid sequence, M254 of the heavy chain amino acid sequence, M430 of the heavy chain amino acid sequence; ≤3% oxidised antibody variants at W52 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.8 to 7.2, wherein the buffering agent is histidine, phosphate, citric acid, citrate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. Another aspect of the disclosure is a composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.8 to 7.2, wherein the buffering agent is phosphate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; c) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and d) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) deamidated forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) deamidated forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31; and c) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) oxidized forms of the antibody comprising a heavy chain amino acid residue oxidized at methionine 64. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) oxidized forms of the antibody comprising a heavy chain amino acid residue oxidized at methionine 64; and c) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85 and a light chain amino acid residue oxidized at methionine 4. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31; and c) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85 and a light chain amino acid residue oxidized at methionine 4. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of an amino terminal pyroglutamate residue at amino acid residue 1, a carboxy terminal glycine amino acid residue at amino acid residue 448, a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, a oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4 and an oxidized cysteine at position 220. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360, and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4 and an oxidized cysteine at position 220. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317 and a deamidated asparagine residue at position 386; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising a deamidated asparagine residue at amino acid residue 31. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360, and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one selected from the group consisting of an oxidized methionine residue at position 4 and an oxidized cysteine at position 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a main form of the antibody comprising greater than, or equal to, 20% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 20% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) acidic forms of the antibody comprising up to about 80% of the protein in the composition as measured using capillary isoelectric focusing of the composition. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Representative capillary isoelectric focusing (cIEF) electropherogram of a reference standard (RS) composition comprising mepolizumab. FIG. 2. Representative cIEF electropherograms of a reference standard composition comprising mepolizumab (control) and different batches of the compositions comprising mepolizumab subjected to three days of pH 9.0 forced degradation. FIG. 3. Representative full view size exclusion chromatography (SEC) chromatogram of a RS composition comprising mepolizumab. FIG. 4. Representative expanded view SEC chromatogram of a RS composition comprising mepolizumab. FIG. 5. Representative SEC-multi-angle light scattering (MALS) chromatogram of a RS composition comprising mepolizumab. FIG. 6. Representative SEC-MALS chromatogram of a batch of the composition comprising mepolizumab. FIG. 7. Representative SEC Chromatograms of a RS composition comprising mepolizumab and for different batches of pH 3.5 stressed composition comprising mepolizumab at Day 7. DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure provides compositions, for treating interleukin 5 (IL-5) mediated diseases, and related subject matter. The term “asthma” as used herein means an inflammatory disease of the airways characterized by reversible airflow obstruction and bronchospasm. Common symptoms include wheezing, coughing, chest tightness, and shortness of breath. In the methods of the disclosure “asthma” may be “severe eosinophilic asthma.” Subjects with severe eosinophilic asthma may have asthma and blood eosinophils greater than or equal to 300 eosinophils per μL of blood in the past 12 months. Subjects with severe eosinophilic asthma may meet, one or more of, the critera described in Table 1. TABLE 1 A subject has severe eosinophilic asthma if they meet the following criteria: 1) The subject has clinical features of severe refractory asthma similar to those indicated in the American Thoracic Society Workshop on Refractory Asthma (162 Am. J. Respir. Crit. Care Med. 2341 (2000) for ≥12 months. 2) The subject has a well-documented requirement for regular treatment with high dose ICS (inhaled corticosteroids) (i.e., ≥880 μg/day fluticasone propionate or equivalent daily), with or without maintenance OCS (oral corticosteroids), in the past 12 months. 3) The subject has a well-documented requirement for controller medication, e.g., long- acting beta-2-agonist, leukotriene receptor antagonist or theophylline in the past 12 months. 4) The subject has persistent airflow obstruction as indicated by a pre-bronchodilator FEV1 <80% predicted recorded or peak flow diurnal variability of >20% on 3 or more days. 5) The subject has airway inflammation which is likely to be eosinophilic in nature as indicated by one of the following characteristics at present or documented in the previous 12 months: An elevated peripheral blood eosinophil level of ≥300/μL, that is related to asthma or Sputum eosinophils ≥3% or Exhaled nitric oxide ≥50 ppb or Prompt deterioration of asthma control (based on documented clinical history or objective measures) following a ≤25% reduction in regular maintenance dose of inhaled or oral corticosteroid dose in the previous 12 months 8) The subject has a previously confirmed history of two or more asthma exacerbations requiring treatment with oral or systemic corticosteroids in the prior 12 months prior, despite the use of high-dose ICS and additional controller medication. For subjects receiving maintenance OCS with high-dose ICS plus controller, the OCS treatment for exacerbations had to be a two-fold or greater increase in the dose of OCS. 9) The subject has asthma as documented by either: Airway reversibility (FEV1 ≥12% and 200 mL) at present or documented in the previous 12 months or Airway hyper-responsiveness (provocative concentration causing a 20% fall in FEV1 of methacholine <8 mg/mL or provocative dose causing a 20% fall in FEV1 of histamine <7.8 μmol) documented in the prior 12 months or Airflow variability in clinic FEV1 ≥20% between two examinations documented in the prior 12 months (FEV1 recorded during an exacerbation is not valid) or Airflow variability as indicated by >20% diurnal variability in peak flow observed on 3 or more days. Importantly, subjects with severe eosinophilic asthma according to these criteria may have less than 150 eosinophils per μL of blood at the initiation of treatment. Mepolizumab is a monoclonal antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2. Mepolizumab, and antigen binding proteins, in particular antibody molecules, comprising the heavy chain CDRs and light chain CDRs of mepolizumab, may be used to treat severe eosinophilic asthma according to the methods of the disclosure. For example, mepolizumab, or related antigen binding proteins, may be indicated for add-on maintenance treatment of severe eosinophilic asthma, as identified by blood eosinophils greater than or equal to 300 cells/μL in the past 12 months and/or blood eosinophils greater than or equal to 150 cells/μL at initiation of treatment and/or blood eosinophils less than 150 cells/μL at initiation of treatment, in patients. Alternatively, mepolizumab, or related antigen binding proteins, may be indicated for add-on maintenance treatment of severe eosinophilic asthma, as identified by blood eosinophils greater than or equal to 300 cells/μL in the past 12 months and/or blood eosinophils greater than or equal to 150 cells/μL at initiation of treatment, in patients. Mepolizumab, or related antigen binding proteins, may be indicated for add-on maintenance treatment of severe eosinophilic asthma, as identified by blood eosinophils greater than or equal to 300 cells/μL in the past 12 months and/or blood eosinophils less than 150 cells/μL at initiation of treatment, in patients. Such patients may be aged 12 years and older. Mepolizumab treatment may reduce exacerbations of asthma in patients (e.g., patients with an exacerbation history). The methods of the disclosure may be used when mepolizumab treatment is indicated (i.e., such treatment with mepolizumab may be combined with the methods of the disclosure). Treatment with mepolizumab can: a) Produce a reduction in exacerbation frequency. Compared with placebo, treatment with mepolizumab, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, can reduce the rate of 1) clinically significant exacerbations, 2) exacerbations requiring hospitalization or ED visits, and 3) exacerbations requiring hospitalization. This benefit may potentially lead to reductions in morbidity and fatal events due to asthma. b) Produce a reduction in daily OCS dose: Treatment with mepolizumab, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, may allow subjects to reduce their daily dose of concomitant corticosteroid without experiencing loss of asthma control. Subjects treated with mepolizumab may achieve a median percentage reduction of 50% from baseline in daily oral corticosteroid (OCS) dose versus 0% for those treated with placebo. In addition, 54% of subjects treated with mepolizumab may achieve a reduction of OCS dose to 5.0 mg compared with 32% of subjects treated with placebo (p=0.025). c) Produce an improvement in lung function: Clinically relevant changes in pre- and post-bronchodilator FEV1 may be demonstrated with mepolizumab treatment, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, compared with placebo. Any improvements in lung function are of particular clinical importance in this population of subjects as most are on maximal asthma therapy including high dose ICS (inhaled corticosteroids) and/or OCS plus a controller medication. d) Produce an improvement in asthma control: Statistically significant and clinically relevant improvements may be observed in ACQ-5 with mepolizumab, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, compared with placebo, indicating subjects may achieve asthma control with the addition of mepolizumab to their existing asthma treatment. e) Produce an improvement in quality of life: Statistically significant and clinically relevant changes in SGRQ scores may be demonstrated with mepolizumab, such as 100 mg per subject administered subcutaneously or 75 mg per subject administered intravenously, compared with placebo. Subjects may experience marked improvement in asthma symptoms and ability of perform daily activities. f) Produce a persistence of efficacy and pharmacodynamic effect: Over a period of 32- and/or 52-week treatment durations, a sustained reduction in asthma exacerbations and blood eosinophils, and improvements in lung function, asthma control, and quality of life with no development of tolerance may be observed. and g) Produce a reduction in blood eosinophils. Treatment with compositions comprising mepolizumab, such as 100 mg of mepolizumab per subject administered subcutaneously or 75 mg per subject administered intravenously, may result in rapid reduction of blood eosinophils (approximately 80% by the first assessment at Week 4 after initial treatment; e.g., from 250-290 cells/μL to 40-60 cells/μL etc.). In the methods of the disclosure “asthma” may be “severe asthma.” Subjects with severe asthma meet the definition of severe asthma described in the European Respiratory Society/ American Thoracic Society (ERS/ATS) Guidelines for severe asthma. Thus, severe asthma is asthma which requires treatment with guideline suggested medications for Global Initiative for Asthma (GINA) steps 4-5 asthma (high dose inhaled corticosteroids [ICS] plus long acting beta2-agonist [LABA] or leukotriene modifier/theophylline) for the previous year, or systemic corticosteroids (CS) for >=50% of the previous year to maintain control of the subject's asthma. Treatment with compositions comprising mepolizumab may be used to treat severe asthma according to the methods of the disclosure. In the methods of the disclosure “asthma” may be “uncontrolled eosinophilic asthma.” Subjects with uncontrolled eosinophilic asthma meet the critera described in Table 2. TABLE 2 A subject has uncontrolled eosinophilic asthma if they meet the following criteria: 1) The subject has a history of diagnosed asthma for at least the prior 12 months. 2) The subject has been prescribed daily use of medium-dose or high-dose ICS (inhaled corticosteroid) plus LABA (long-acting beta agonists) for at least the prior 12 months. 3) The subject's dose of other asthma controller medications must be stable for at least the prior 30 days. 4) The subject has at least 2 documented asthma exacerbations in the prior 12 months that required use of a systemic corticosteroid burst. Treatment with compositions comprising mepolizumab may be used to treat uncontrolled eosinophilic asthma according to the methods of the disclosure. In the methods of the disclosure “asthma” may be “eosinophilic asthma.” Subjects with uncontrolled eosinophilic asthma meet the critera described in Table 3. TABLE 3 A subject has eosinophilic asthma if they meet the following criteria: 1) The patient has a previous diagnosis of asthma. 2) The patient has had at least 1 asthma exacerbation requiring oral, intramuscular (im), or intravenous (iv) corticosteroid use for at least 3 days in the prior 12 months. 3) The patient has a current blood eosinophil level of at least 400/μl. 4) The patient has airway reversibility of at least 12% to beta-agonist administration. 5) The patient has an ACQ score of at least 1.5. 6) The patient is taking inhaled fluticasone at a dosage of at least 440 μg, or equivalent, daily. Chronic oral corticosteroid use (no more than 10 mg/day prednisone or equivalent) is allowed. The patient's baseline asthma therapy regimen (including, but not limited to, inhaled corticosteroids, oral corticosteroids up to a maximum dose of 10 mg prednisone daily or equivalent, leukotriene antagonists, 5-lipoxygenase inhibitors, or cromolyn) must be stable for the prior 30 days. In the methods of the disclosure “asthma” may be “sub-eosinophilic asthma.” Subjects with uncontrolled eosinophilic asthma meet the critera described in Table 4. TABLE 4 A subject has sub-eosinophilic asthma if they meet the following criteria: 1) The patient has a previous diagnosis of asthma. 2) The patient has had at least 1 asthma exacerbation requiring oral, intramuscular (im), or intravenous (iv) corticosteroid use for at least 3 days in the prior 12 months. 3) The patient has a current blood eosinophil level of less than 400/μl. 4) The patient has airway reversibility of at least 12% to beta-agonist administration. 5) The patient has an ACQ score of at least 1.5. 6) The patient is taking inhaled fluticasone at a dosage of at least 440 μg, or equivalent, daily. Chronic oral corticosteroid use (no more than 10 mg/day prednisone or equivalent) is allowed. The patient's baseline asthma therapy regimen (including, but not limited to, inhaled corticosteroids, oral corticosteroids up to a maximum dose of 10 mg prednisone daily or equivalent, leukotriene antagonists, 5-lipoxygenase inhibitors, or cromolyn) must be stable for the prior 30 days. Treatment with compositions comprising mepolizumab may be used to treat sub-eosinophilic asthma and may also be used to treat sub-eosinophilic asthma according to the methods of the disclosure. The term “bullous pemphigoid” (BP) as used herein means an acute or chronic autoimmune skin disease, involving the formation of blisters, more appropriately known as bullae, at the space between the skin layers epidermis and dermis. BP is the most common autoimmune blistering skin disease. It characteristically affects the elderly (>70 years) with an annual incidence of 5 to 35 per million. The incidence of BP is dramatically increasing with an average of 17% per year. BP often starts with extremely pruritic skin lesions resembling eczema or urticaria before vesicles and blisters arise. In 10-30% of patients, BP also involves the oral mucosa. Disease severity can be determined by means of the autoimmune bullous skin disorder intensity score (ABSIS) that evaluates the involved area as well as the disease activity. The disease is due to an autoimmune response to structural components of junctional adhesion complexes leading to the damage of the dermal-epidermal junction with subepidermal blister formation. Specifically, autoreactive B and T cell responses against the hemidesmosomal antigens BP180 and BP230 have been identified. Serum levels of autoantibodies to BP180 reflect the disease severity and activity. The T cells are memory CD4+ cells producing both Th1 and Th2 cytokines, mostly IL-4, IL-5 and IL-13. IL-5 as well as eotaxin are abundantly found in blister fluids. The production of IL-5 is indeed associated with blood eosinophilia and significant eosinophil infiltration in the skin of BP patients. Eosinophils are thought to be critically implicated in blister formation by releasing toxic granule proteins (ESP, MBP) and proteolytic enzymes. The term “eosinophilic esophagitis” (EoE) as used herein means an allergic inflammatory condition of the esophagus that involves eosinophils. Symptoms are swallowing difficulty, food impaction, and heartburn. EoE is characterised by a dense infiltrate with white blood cells of the eosinophil type into the epithelial lining of the esophagus. EoE is believed to be an allergic reaction against ingested food, based on the important role eosinophils play in allergic reactions. The EoE diagnostic panel can be used to diagnose EoE. EoE can also be diagnosed if gastroesophageal reflux does not respond to a 6 week trial of twice-a-day high-dose proton-pump inhibitors (PPIs) or if a negative ambulatory pH study ruled out gastroesophageal reflux disease (GERD). Endoscopically, ridges, furrows, or rings may be seen in the oesophageal wall. Sometimes, multiple rings may occur in the esophagus, leading to the term “corrugated esophagus” or “feline esophagus” due to similarity of the rings to the cat esophagus. The presence of white exudates in esophagus is also suggestive of the diagnosis. On biopsy taken at the time of endoscopy, numerous eosinophils can typically be seen in the superficial epithelium. A minimum of 15 eosinophils per high-power field are required to make the diagnosis. Eosinophilic inflammation is not limited to the oesophagus alone, and does extend though the whole gastrointestinal tract. Profoundly degranulated eosinophils may also be present, as may microabcesses and an expansion of the basal layer. Radiologically, the term “ringed esophagus” has been used for the appearance of eosinophilic esophagitis on barium swallow studies to contrast with the appearance of transient transverse folds sometimes seen with esophageal reflux (termed “feline esophagus”). Treatment with compositions comprising mepolizumab may be used to treat COPD according to the methods of the disclosure. Subjects with “chronic obstructive pulmonary disease” (COPD) may meet one or more following criteria: a) a prior COPD diagnosis: subjects with a clinically documented history of COPD for at least 1 year in accordance with the definition by the American Thoracic Society/European Respiratory Society; b) severity of COPD: Subjects may present with the following: a measured pre and post-salbutamol Forced Expiratory Volume in one second/ Forced vital capacity (FEV1/FVC) ratio of <0.70 to confirm a diagnosis of COPD; a measured post-salbutamol FEV1>20 percent and <=80 percent of predicted normal values calculated using National Health and Nutrition Examination Survey (NHANES) III reference equations; c) a history of exacerbations: a well documented history (like medical record verification) in the 12 months of: at least two moderate COPD exacerbations. Moderate is defined as the use of systemic corticosteroids (IM, intravenous, or oral) and/or treatment with antibiotics, or at least one severe COPD exacerbation. Severe is defined as having required hospitalization. Note: At least one exacerbation must have occurred while the subject was taking Inhaled corticosteroid (ICS) plus long acting beta2-agonist (LABA) plus long acting muscarinic antagonist (LAMA). Note: Prior use of antibiotics alone does not qualify as a moderate exacerbation unless the use was specifically for the treatment of worsening symptoms of COPD; and d) concomitant COPD therapy: a well documented requirement for optimized standard of care (SoC) background therapy that includes ICS plus 2 additional COPD medications (i.e., triple therapy) for the 12 months prior and meets the following criteria. Immediately prior to visit to the healthcare provider, a minimum of 3 months of use of an inhaled corticosteroid (at a dose >=500 micrograms (mcg)/day fluticasone propionate dose equivalent plus); or LABA and LAMA. Treatment with compositions comprising mepolizumab may be used to treat COPD according to the methods of the disclosure. The term “eosinophilic granulomatosis with polyangiitis” (EGPA) as used herein means an autoimmune condition that causes inflammation of small and medium-sized blood vessels (vasculitis) in persons with a history of airway allergic hypersensitivity (atopy). EGPA may also be referred to as Churg-Strauss Syndrome (CSS) or allergic granulomatosis. EGPA usually manifests in three stages. The early (prodromal) stage is marked by airway inflammation; almost all patients experience asthma and/or allergic rhinitis. The second stage is characterized by abnormally high numbers of eosinophils (hypereosinophilia), which causes tissue damage, most commonly to the lungs and the digestive tract. The third stage consists of vasculitis, which can eventually lead to cell death and can be life-threatening. Subjects with EGPA may meet one or more following criteria: a) asthma; b) blood eosinophil levels greater than 10% of a differential white blood cell count; c) presence of mononeuropathy or polyneuropathy; d) unfixed pulmonary infiltrates; e) presence of paranasal sinus abnormalities; and e) histological evidence of extravascular eosinophils. For classification purposes, a patient shall be said to have EGPA if at least four of the preceding six criteria are positive. Treatment with compositions comprising mepolizumab may be used to treat EGPA according to the methods of the disclosure. The compositions of the disclosure may be administered to an EGPA patient in an amount of 300 mg once every 4 weeks. The term “hypereosinophilic syndrome” (HES) as used herein means a disease characterized by a persistently elevated eosinophil count (≥1500 eosinophils/mm3) in the blood for at least six months without any recognizable cause, with involvement of either the heart, nervous system, or bone marrow. Subjects with hypereosinophilic syndrome may meet one or more following criteria: a) a documented history of hypereosinophilic syndrome; b) a blood eosinophil count greater than 1500 cells for 6 months; c) signs and symptoms of organ system involvement; and d) no evidence of parasitic, allergic or other causes of eosinophilia after comprehensive evaluation. Treatment with compositions comprising mepolizumab may be used to treat hypereosinophilic syndrome according to the methods of the disclosure. The compositions of the disclosure may be administered to a hypereosinophilic syndrome patient in an amount of 300 mg once every 4 weeks. The term “nasal polyposis” as used herein means a disease characterized by the presence of polyps nasal cavity. Such polyps may be in the upper nasal cavity and/or may originate from within the ostiomeatal complex. Subjects with nasal polyposis may meet one or more following criteria: a) a documented history of nasal polyposis; or b) nasal polyps apparent on examination (e.g., endoscopic examination). Treatment with compositions comprising mepolizumab may be used to treat nasal polyposis according to the methods of the disclosure. The compositions of the disclosure may be administered to a nasal polyposis patient in an amount of 750 mg once every 4 weeks. The term “antibody” as used herein refers to molecules with an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, monoclonal, recombinant, polyclonal, chimeric, human, and humanized molecules of this type. Monoclonal antibodies may be produced by a eukaryotic cell clone expressing an antibody. Monoclonal antibodies may also be produced by a eukaryotic cell line which can recombinantly express the heavy chain and light chain of the antibody by virtue of having nucleic acid sequences encoding these introduced into the cell. Methods to produce antibodies from different eukaryotic cell lines such as Chinese Hamster Ovary cells, hybridomas or immortalized antibody cells derived from an animal (e.g., human) are well known. The antibody may be derived from rat, mouse, primate (e.g., cynomolgus, Old World monkey or Great Ape), human or other sources such as nucleic acids generated using molecular biology techniques which encode an antibody molecule. The antibody may comprise a constant region, which may be of any isotype or subclass. The constant region may be of the IgG isotype, for example, IgG1, IgG2, IgG3, IgG4 or variants thereof. The antigen binding protein constant region may be IgG1. The antigen binding protein may comprise one or more modifications selected from a mutated constant domain such that the antibody has enhanced effector functions/ADCC and/or complement activation. An antibody may be capable of binding to a target antigen. Examples, of such target antigens include human IL-5 comprising the amino acid sequence shown in SEQ ID NO: 11. Mepolizumab comprising the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2 is an example of an antibody. Mepolizumab binds human IL-5 and antagonizes its activity. Mepolizumab is a recombinant humanized monoclonal antibody (IgG1, Kappa) Mepolizumab has two light and two heavy chains. The mepolizumab heavy chain is encoded by the nucleic acid sequence shown in SEQ ID NO: 13. The mepolizumab heavy chain contains 449 amino acids with an estimated molecular mass of approximately 49 kDa. The predicted mature heavy chain amino acid sequence for mepolizumab is: (SEQ ID NO: 1) YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN* STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In the heavy chain amino acid sequence above, heavy chain frameworks and CDRs according to the Kabat definition are identified as zig-zag underlined framework1, solid underlined CDR1, zig-zag underlined framework2, solid underlined CDR2, zig-zag underlined framework3, solid underlined CDR3 and zig-zag framework4 in order from the amino proximal portion to the carboxy terminal portion of the sequence presented. In the heavy chain amino acid sequence above, an asterisk to the right of a character for a single letter amino acid code indicates the amino acid residue to the left can be a N-glycosylation site. The mepolizumab light chain is encoded by the nucleic acid sequence shown in SEQ ID NO: 14. The mepolizumab light chain contains 220 amino acids residues with an estimated molecular mass of approximately 24 kDa. The mature light chain amino acid sequence is: (SEQ ID NO: 2) KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC In the light chain amino acid sequence above, light chain frameworks and CDRs according to the Kabat definition are identified as zig-zag underlined framework1, solid underlined CDR1, zig-zag underlined framework2, solid underlined CDR2, zig-zag underlined framework3, solid underlined CDR3 and zig-zag framework4 in order from the amino proximal portion to the carboxy terminal portion of the sequence presented. The mepolizumab heavy and light chains are covalently linked by a single disulfide bond and the heavy chains are linked to each other by two disulfide bonds resulting in a typical IgG molecule. Both heavy chains can be glycosylated at asparagine 299 with complex biantennary oligosaccharides. The predicted polypeptide molecular mass is about 146 kDa and the predicted carbohydrate molecular mass is approximately 3 kDa giving a total estimated molecular mass of 149.2 kDa for mepolizumab. Mepolizumab as encoded has 1338 amino acid residues (220 amino acid residues per light chain, 449 amino acid residues per heavy chain). The main pI of mepolizumab is about 8.7-9.1. The equilibrium dissociation constant (KO for the molecular interaction of mepolizumab and human IL-5 as measured using standard surface plasmon reasonance assays is less than 2.29×10−11 M. Mepolizumab can be provided as a lyophilized powder containing the antibody and excipients which can be reconstituted with a pharmaceutically acceptable carrier (e.g., sterile water). This reconstituted pharmaceutical composition can then be administered either subcutaneously or intravenously (e.g., with further dilution). Mepolizumab can also be provided as a liquid formulation containing the antibody, excipients and a pharmaceutically acceptable carrier. This liquid pharmaceutical composition can then be administered either subcutaneously or intravenously (e.g., with further dilution). The term “antibody variant” as used herein means an antibody that differs from a parent antibody by virtue of at least one amino acid modification (e.g., by having a different amino acid side chain), post-translational modification or other modification in at least one heavy chain, light chain, or combinations of these that results in a structural change (e.g., different amino acid side chain, different post-translational modification or other modification) relative to the parent antibody. Mepolizumab is an example of a such a parent antibody. Structural changes can be determined directly by a variety of methods well know in the art such as LC-MS, direct sequencing or indirectly via methods such as isoelectric focusing and the like. Such methods are well known to those of ordinary skill in the art. The term “IL-5” as used herein means human IL-5 comprising the amino acid sequence shown in SEQ ID NO: 11. The term “specifically binds”, as used herein in relation to antigen binding proteins means that the antigen binding protein binds to a target antigen as well as a discrete domain, or discrete amino acid sequence, within a target antigen with no or insignificant binding to other (for example, unrelated) proteins. This term, however, does not exclude the fact that the antigen binding proteins may also be cross-reactive with closely related molecules (for example, those with a high degree of sequence identity or from another genera or species). The antigen binding proteins described herein may bind to human IL-5 or the human IL-5 receptor with at least 2, 5, 10, 50, 100, or 1000-fold greater affinity than they bind to closely related molecules. The binding affinity (KD) of the antigen binding protein-target antigen interaction may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less. Alternatively, the KD may be between 5 and 10 nM; or between 1 and 2 nM. The KD may be between 1 pM and 500 pM; or between 500 pM and 1 nM. The binding affinity of the antigen binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD=Kd/Ka). The binding affinity may be measured by BIACORE™, for example, by capture of the test antibody onto a protein-A coated sensor surface and flowing target antigen over this surface. Alternatively, the binding affinity can be measured by FORTEBIO, for example, with the test antibody receptor captured onto a protein-A coated needle and flowing target antigen over this surface. The Kd may be 1×10−3 Ms−1 or less, 1×10−4 Ms−1 or less, or 1×10−5 Ms−1 or less. The Kd may be between 1×10−5 Ms−1 and 1×10−4 Ms−1; or between 1×10−4 Ms−1 and 1×10−3 Ms−1. A slow Kd may result in a slow dissociation of the antigen binding protein-target antigen complex and improved neutralization of the target antigen. The term “specific antigen binding activity” as used herein means antigen binding activity as measured by Surface Plasmon Resonance (SPR). IL-5 specific binding activity may be determined by SPR using a BIACORE™ instrument, for example performed in the binding mode. It is binding activity divided by total protein (e.g., mepolizumab) content in a sample. The term “FcRn binding activity” as used herein means Neonatal Fc (FcRn) Receptor binding activity as measured by Surface Plasmon Resonance (SPR). FcRn binding may be determined using a BIACORE™ instrument. It is binding activity to the FcRn receptor, divided by the total protein concentration of the sample. The SPR method for specific antigen binding and FcRn binding uses a reference standard of mepolizumab. The mepolizumab reference standard can be used in assays to obtain system suitability and sample comparability data, to ensure methods are performing appropriately. The reference standard can allow the establishment of a calibration curve and concentrations of the samples are interpolated from the curve. For example, the reference standard is a composition comprising SEQ ID NO:1 and SEQ ID NO:2. In another embodiment, the reference standard is a composition comprising SEQ ID NO:1 and SEQ ID NO:2, and 98% or more HC C-terminal lysine deleted variant, and 95% or more HC N-terminal pyro-glutamate variant. In a further embodiment, the reference standard is a composition comprising SEQ ID NO:1 and SEQ ID NO:2, and 98% or more HC C-terminal lysine deleted variant, 95% or more HC N-terminal pyro-glutamate variant, and 6% or less deamidated variant. In another embodiment, the reference standard is a composition comprising SEQ ID NO:1 and SEQ ID NO:2, and 98% or more HC C-terminal lysine deleted variant, 95% or more HC N-terminal pyro-glutamate variant, 6% or less deamidated variant, 4% or less methionine or cysteine oxidated variant, and 0.1% tryptophan oxidated variant. In a further embodiment, the reference standard is a composition comprising SEQ ID NO:1 and SEQ ID NO:2, and 98% or more HC C-terminal lysine deleted variant, 95% or more HC N-terminal pyro-glutamate variant, 6% or less deamidated variant, 4% or less methionine or cysteine oxidated variant, 0.1% or less tryptophan oxidated variant, and 0.4% or less aggregated variant. In another embodiment, the reference standard is a composition comprising the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1. In one embodiment the reference standard is a composition comprising SEQ ID NO: 1 and SEQ ID NO:2, about 62.9% main peak, about 35.9% acidic peak, about 1.2% basic peak, about 99.6% monomer, about 0.4% aggregate, about 0% fragment, about 0.8% HC deamidated N317, about 5.5% HC deamidated N386, about 5.2% HC deamidated N31, about 0.2% HC deamidated N299, about 0.9% HC oxidised M64, about 3.5% HC oxidised M254, about 0.5% HC oxidised M360, about 0.5% HC oxidised M430, about 0.3% HC oxidised M82 and M85, about 0.2% LC oxidised M4, about 0.0% LC oxidised C220, about 0.1% HC oxidised W52, 98% or more HC C-terminal lysine deleted variant, and 95% or more HC N-terminal pyro-glutamate variant. In one embodiment the composition has a specific IL-5 binding activity of ≥0.70; and a FcRn binding activity of ≥70%. For example, the specific antigen binding is in the range of from 0.70 to 1.30; and/or the FcRn binding is in the range of from 70% to 130%, as compared to the reference standard which is set as 1.0 specific IL-5 binding activity, and 100% FcRn binding activity. IL-5 neutralization ED50 ratio is the ED50 of a reference antibody standard (e.g., a mepolizumab antibody standard comprising the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2) divided by the ED50 of an antibody sample (e.g., a mepolizumab variant sample or a sample of a manufactured batch of composition comprising a mepolizumab antibody comprising the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2). By “isolated”, it is intended that the molecule, such as an antigen binding protein or nucleic acid, is removed from the environment in which it may be found in nature. For example, the molecule may be purified away from substances with which it would normally exist in nature. For example, the mass of the molecule in a sample may be 95% of the total mass. The terms “VH” and “VL” are used herein to refer to the heavy chain variable region and light chain variable region respectively of an antigen binding protein. “CDRs” are defined as the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least one CDR and wherein the at least one CDR is CDRH3. Framework regions follow each of these CDR regions. Acceptable heavy chain variable region and light chain variable region framework 1, framework 2 and framework 3 regions are readily recognized by those of ordinary skill in the art. Acceptable heavy chain constant regions (including hinge regions) and light chain constant regions are readily recognized by those of ordinary skill in the art as well. Acceptable antibody isotypes are similarly readily recognized by those of ordinay skill in the art. Throughout this specification, amino acid residues in variable domain sequences and full length antibody sequences are numbered according to the Kabat numbering convention. Similarly, the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” used in the specification follow the Kabat numbering convention. It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full length antibody sequences. There are also alternative numbering conventions for CDR sequences, for example those set out according to the Chothia numbering convention. The structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person. Other numbering conventions for CDR sequences available to a skilled person include “AbM” (University of Bath) and “contact” (University College London) methods. The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the “minimum binding unit”. The minimum binding unit may be a sub-portion of a CDR. Table 5 below represents one definition using each numbering convention for each CDR or binding unit. The Kabat numbering scheme is used in Table 5 to number the variable domain amino acid sequence. It should be noted that some of the CDR definitions may vary depending on the individual publication used. TABLE 5 Minimum Kabat CDR Chothia CDR AbM CDR Contact CDR binding unit H1 31-35/35A/35B 26-32/33/34 26-35/35A/35B 30-35/35A/35B 31-32 H2 50-65 52-56 50-58 47-58 52-56 H3 95-102 95-102 95-102 93-101 95-101 L1 24-34 24-34 24-34 30-36 30-34 L2 50-56 50-56 50-56 46-55 50-55 L3 89-97 89-97 89-97 89-96 89-96 “Percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is the “Identities” value, expressed as a percentage, that is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair-wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query sequence may be described by a nucleic acid sequence identified in one or more claims herein. Nucleic acid sequences which may be useful, and included, in the compositions and related methods of the disclosure may have between about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% and about 100% identity to the nucleic acid sequences identified in the disclosure (e.g., nucleic acids encoding an antibody heavy chain or antibody light chain). In the disclosure, percent identity between the nucleic acid sequences described may include any discrete subrange of the percent identiy ranges recited above (e.g., any range of integer values within a particular range or discrete subvalues within a particular range). “Percent identity” between a query amino acid sequence and a subject amino acid sequence is the “Identities” value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query sequence may be described by an amino acid sequence identified in one or more claims herein. The amino acid sequences which may be useful, and included, in compositions and related methods of the disclosure may have between about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% and about 100% identity to the amino acid sequences identified in the disclosure (e.g., to an antibody heavy chain or antibody light chain). In the disclosure, percent identity between the amino acid sequences described may includes any discrete subrange of the percent identiy ranges recited above (e.g., any range of integer values within a particular range or discrete subvalues within a particular range). The terms “peptide”, “polypeptide”, “protein” and “peptide chain” each refer to a molecule comprising two or more amino acid residues. A peptide may be monomeric or polymeric. It is well recognized in the art that certain amino acid substitutions are regarded as being “conservative” Amino acids are divided into groups based on common side-chain properties and substitutions within groups that maintain all or substantially all of the binding affinity of the antigen binding protein are regarded as conservative substitutions. See Table 6. The antigen binding proteins disclosed herein can comprise such “conservative” amino acid substitutions. TABLE 6 Side chain Members Hydrophobic met, ala, val, leu, ile Neutral hydrophilic cys, ser, thr Acidic asp, glu Basic asn, gln, his, lys, arg Residues that influence gly, pro chain orientation Aromatic trp, tyr, phe The term “pharmaceutical compostion” as used herein means a composition suitable for administration to a patient. The pharmaceutical compositions described herein may comprise purified preparations of an antibody as described herein. For example, the pharmaceutical preparation may comprise a purified preparation of an antibody as described herein in combination with a pharmaceutically acceptable carrier. Typically, such pharmaceutical compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice. Examples of such carriers include sterilized carriers, such as saline, Ringers solution, or dextrose solution, optionally buffered with suitable buffers to a pH within a range of 5 to 8. Pharmaceutical compositions may be administered by injection or infusion (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, or intraportal). Such compositions are suitably free of visible particulate matter. Pharmaceutical compositions may comprise between 1 mg to 10 g of antigen binding protein, for example, between 5 mg and 1 g of antigen binding protein. Alternatively, the composition may comprise between 5 mg and 500 mg of antigen binding protein, for example, between 5 mg and 50 mg. Methods for the preparation of such pharmaceutical compositions are well known to those skilled in the art. Pharmaceutical compositions may comprise between 1 mg to 10 g of antigen binding protein in unit dosage form, optionally together with instructions for use. Pharmaceutical compositions may be lyophilized (freeze dried) for reconstitution prior to administration according to methods well known or apparent to those skilled in the art. Where antibodies have an IgG1 isotype, a chelator of copper, such as citrate (e.g., sodium citrate) or EDTA or histidine, may be added to the pharmaceutical composition to reduce the degree of copper-mediated degradation of antibodies of this isotype. Pharmaceutical compositions may also comprise a solubilizer, such as arginine, a surfactant/anti-aggregation agent such as polysorbate 80, and an inert gas such as nitrogen to replace vial headspace oxygen. The term “therapeutically effective amount” as used herein means an amount of an agent (such as an antibody or a pharmaceutical composition), which provides a therapeutic benefit in the treatment or management of one or more symptoms of a condition to be treated (such as asthma, severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis (EGPA), hypereosinophilic syndrome and nasal polyposis). Examples of such treatment or management of one or more symptoms of asthma—including severe eosinophilic asthma, uncontrolled eosinophilic asthma, eosinophilic asthma or sub-eosinophilic asthma—include 1) a reduction of the frequency of asthma exacerabations; 2) a reduction in the time to first clinically significant exacerbation requiring oral or systemic corticosteroids, hospitalisation, and/or emergency department (ED) visits; 3) a reduction in the frequency of exacerbations requiring hospitalization (including intubation and admittance to an intensive care unit) or ED visits; 4) a reduction in the time to first exacerbation requiring hospitalization or ED visit; 5) a change from baseline in clinic pre-bronchodilator FEV1; 6) a change from baseline in clinic post-bronchodilator FEV1; 7) a change from baseline in an Asthma Control Questionnaire (ACQ) score; 8) improved lung function as assessed by spirometry (e.g., vital capacity (VC), forced vital capacity (FVC), forced expiratory volume (FEV) at timed intervals of 0.5, 1.0 (FEV1), 2.0, and 3.0 seconds, forced expiratory flow 25-75% (FEF 25-75) and maximal voluntary ventilation (MVV) total lung capacity, idal volume, residual volume, expiratory reserve volume, inspiratory reserve volume, inspiratory capacity, inspiratory vital capacity, vital capacity, functional residual capacity, residual volume expressed as percent of total lung capacity, alveolar gas volume, actual volume of the lung including the volume of the conducting airway, forced vital capacity, etc.); and 9) a reduction in asthma exacerbations requiring steroids for control (such as oral steroids or steroids—like prednisone, prednisolone etc.—administered by any route). Such a reduction in asthma exacerbations requiring steroids for control may be an approximately 50% reduction in exacerbations requiring steroids (e.g., oral steroids). Therapeutically effective amounts and treatment regimes are generally determined empirically and may be dependent on factors, such as the age, weight, and health status of the patient and disease or disorder to be treated. Such factors are within the purview of the attending physician. The dosage of antigen binding protein administered to a subject is generally between 1 μg/kg to 150 mg/kg, between 0.1 mg/kg and 100 mg/kg, between 0.5 mg/kg and 50 mg/kg, between 1 and 25 mg/kg, between about 0.3 mg/kg and about 3 mg/kg or between 1 and 10 mg/kg of the subject's body weight. For example, the dose may be 10 mg/kg, 30 mg/kg, or 60 mg/kg. The dose may also be from 10 mg/kg to 110 mg/mg 15 mg/kg to 25 mg/kg or 15 mg/kg to 100 mg/kg. The antigen binding protein may be administered, for example, parenterally, subcutaneously, intravenously, or intramuscularly. Doses may also be administered on a per subject basis such as about 20 mg per subject to about 750 mg per subject, about 75 mg per subject to about 750 mg per subject, about 20 mg per subject to about 200 mg per subject. The dose may be any discrete subrange with these dosage ranges. For example, the dose (such as a dose of mepolizumab or a pharmaceutical composition comprising mepolizumab) may also be administered subcutaneously on a per subject basis such as about 100 mg per subject (e.g., once every four weeks), or 300 mg per subject (or other doses administered may be subcutaneously with provided approximately the same, or comparable, bioavailability is achieved as with intravenous administration—e.g., three doses of 100 mg per subject to achieve a total dose administered subcutaneously of 300 mg per subject). Ranges provided herein, of any type, include all values within a particular range described and values about an endpoint for a particular range. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. The administration of a dose may be by slow continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours, or from 2 to 6 hours. Such an administration may result in reduced side effects. The administration of a dose may be repeated one or more times as necessary, for example, three times daily, once every day, once every 2 days, once a week, once a every 14 days, once a month, once every 3 months, once every 4 months, once every 6 months, or once every 12 months. The antigen binding proteins may be administered by maintenance therapy, for example once a week for a period of 6 months or more. The antigen binding proteins may be administered by intermittent therapy, for example, for a period of 3 to 6 months and then no dose for 3 to 6 months, followed by administration of antigen binding proteins again for 3 to 6 months, and so on, in a cycle. For example, the dose may be administered subcutaneously, once every 14 or 28 days, in the form of multiple doses on each day of administration. In one embodiment, the dosage of the composition is 100 mg once every 4 weeks (28 days). The antigen binding protein may be administered to the subject in such a way as to target therapy to a particular site. The antigen binding protein in the methods of the disclosure may be used in combination with one or more other therapeutically active agents, such as antibodies or small molecule inhibitors By the term “treating” and grammatical variations thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate the condition of one or more of the biological manifestations of the condition, (2) to interfere with a) one or more points in the biological cascade that leads to or is responsible for the condition or b) one or more of the biological manifestations of the condition, (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, (4) to slow the progression of the condition or one or more of the biological manifestations of the condition or (5) to prevent the onset of one or more of the biological manifistations of the condition. Prophylactic therapy is also contemplated thereby. The skilled artisan will appreciate that “prevention” is not an absolute term. In medicine, “prevention” is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. The terms “individual”, “subject” and “patient” are used herein interchangeably. The subject is typically a human. The subject may also be a mammal, such as a mouse, rat, or primate (e.g., a marmoset or monkey). The subject can be a non-human animal. The antigen binding proteins, compositions and methods of the disclosure also have veterinary use. The subject to be treated may be a farm animal, for example, a cow or bull, sheep, pig, ox, goat or horse, or may be a domestic animal such as a dog or cat. The animal may be any age, or a mature adult animal. Treatment can be therapeutic, prophylactic or preventative. The subject will be one who is in need thereof. Those in need of treatment may include individuals already suffering from a particular medical disease, in addition to those who may develop the disease in the future. Thus, the methods, antigen binding proteins and compositions of the disclosure described herein can be used for prophylactic treatment or preventative treatment if specified. In this case, methods, antigen binding proteins and compositions of the disclosure can be used to prevent or delay the onset of one or more aspects or symptoms of a disease. The subject can be asymptomatic. The subject may have a genetic predisposition to the disease. A prophylactically effective amount of the antigen binding protein is administered to such an individual. A prophylactically effective amount is an amount which prevents or delays the onset of one or more aspects or symptoms of a disease described herein. The methods, antigen binding proteins and compositions of the disclosure need not affect a complete cure, or eradicate every symptom or manifestation of the disease to constitute a viable therapeutic treatment. As is recognised in the art, drugs employed as therapeutic agents in methods of treatment may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a disease in order to constitute a viable prophylactic agent. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur (for example by delaying the onset of the disease) or worsen in a subject, is sufficient. One aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤80% acidic antibody variants. In one embodiment the composition has: a) ≥0.70 IL-5 specific antigen binding; and/or b) ≥70% FcRn binding. IL-5 specific antigen binding, such as binding to human IL-5 comprising the amino acid sequence of SEQ ID NO: 11, can be measured using standard assays, such as surface plasmon resonance (e.g., BIACORE™), that are well known in the art. FcRn binding can similarly be measured using standard assays, such as surface plasmon resonance (e.g., BIACORE™), that are well known in the art. In another embodiment a) the specific antigen binding is in the range of from 0.70 to 1.30; and/or b) the FcRn binding is in the range of from 70% to 130%. In some embodiments the specific antigen binding may be in the range of about 0.9 to 1.1, 0.75 to about 1, about 0.7 to about 0.8, about 0.7, about 0.91 to about 0.95, about .994 to to about .997 or about 0.7 to about 0.9. In some embodiments the FcRn binding is in the range of from about 70% to about 100%, about 100% to about 130%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 80% to about 90%, about 80% to about 100%, about 100% to about 110%, about 110% to about 120%, about 120% to about 130%, about 80% to about 120% and about 90% to about 110%. In one embodiment, the composition comprises: ≤80% acidic antibody variants. For example, the composition may comprise ≤75%, ≤70%, ≤65%, ≤60%, ≤55%, ≤50%, or ≤45% acidic antibody variants. In another embodiment the composition comprises: ≤35% deamidated antibody variants. In another embodiment the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence. For example, the composition may comprise ≤22.5%, ≤20%, ≤17.5%, ≤15%, ≤12.5, ≤10%, or ≤7.5% deamidated antibody variants at N31 of the light chain amino acid sequence. In another embodiment the composition comprises: ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence. For example, the composition may comprise ≤32.5%, ≤30%, ≤25.5%, or ≤20%, ≤17.5%, ≤15%, ≤12.5%, ≤10%, or ≤7.5% deamidated antibody variants at N386 of the heavy chain amino acid sequence. In another embodiment the composition comprises: ≤55% oxidised antibody variants. In another embodiment the composition comprises: ≤55% oxidised antibody variant at any one or a combination of: a) M64 of the heavy chain amino acid sequence; b) M254 of the heavy chain amino acid sequence; and/or c) M430 of the heavy chain amino acid sequence. For example, the composition may comprise ≤50%, ≤45%, ≤40%, ≤35%, ≤30%, or ≤25%, ≤20%, ≤15%, ≤10%, or ≤5% oxidised antibody variants at M64, M254, and/or M430 of the heavy chain amino acid sequence. In another embodiment the composition comprises: ≤3% oxidised antibody variants at W52 of the heavy chain amino acid sequence. For example, the composition may comprise ≤2.5%, ≤2%, ≤1.5%, ≤1%, ≤0.5%, ≤0.4%, ≤0.3%, ≤0.25%, ≤0.2%, ≤0.15%, or ≤0.1% oxidised antibody variants at W52 of the heavy chain amino acid sequence. In another embodiment a deamidated antibody variant amount and/or an oxidised variant amount, is determined by peptide mapping LC-MS/MS. In another embodiment the composition comprises: ≤20% aggregated antibody variants. For example, the composition may comprise ≤17.5%, ≤15%, ≤12.5, ≤10%, ≤7.5%, ≤5%, or ≤4%, aggregated variant. The compositions may comprise less than or equal to 3%, 2%, 1% or 0.5% aggregated antibody. The composition may comprise greater than or equal to 98% monomeric antibody. In another embodiment the aggregated antibody variant comprises a dimer. Such an aggregated antibody can comprise two antibody molecules (e.g., two IgG1 antibody molecules). In another embodiment the aggregated antibody variant amount is determined by size exclusion chromatography (SEC). Methods for performing size exclusion chromatography and measuring protein molecule size are well known in the art. In another embodiment the composition comprises: ≥50% heavy chain amino acid sequence C-terminal lysine K449 deleted antibody variants. For example, the composition may comprise ≥60%, ≥70%, ≥75, ≥80%, ≥85%, ≥90%, or ≥95% heavy chain amino acid sequence C-terminal lysine K449 deleted antibody variants. In another embodiment the composition comprises: ≥50% heavy chain amino acid sequence pyro-glutamate N-terminal antibody variants. For example, the composition may comprise ≥60%, ≥70%, ≥75, ≥80%, ≥85%, ≥90%, or ≥95% heavy chain amino acid sequence pyro-glutamate N-terminal antibody variants. In another embodiment the composition comprises Host Cell Protein (HCP). The HCP may be CHO cell derived. HCP is a process-related impurity in contrast to mepolizumab product-related substances (i.e. mepolizumab plus mepolizumab variants). Industry standard acceptable limits for HCP can be up to 100 ppm (equal to 100 ng/mg). HCP content in a composition described herein may be ≤50 ng/mg, ≤40 ng/mg, ≤30 ng/mg, or ≤20 ng/mg. For example, HCP content of the composition may be ≤10 ng/mg. In a particular embodiment, HCP content of the composition may be ≤5 ng/mg or ≤2 ng/mg. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤80% acidic antibody variants and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤55% oxidised antibody variants at M64 of the heavy chain amino acid sequence; ≤3% oxidised variants at W52 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. Another aspect of the disclosure is a composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence; ≤55% oxidised antibody variants at M64 of the heavy chain amino acid sequence, M254 of the heavy chain amino acid sequence, M430 of the heavy chain amino acid sequence; ≤3% oxidised antibody variants at W52 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. The compositions of the disclosure may further comprise a buffering agent selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid, citrate, sodium phosphate, potassium phosphate, sodium citrate, and histidine, providing a pH of between 6.8 and 7.2 or a pH of from pH 6.2 to pH 6.6 with a pH value of 6.3 being preferred. The buffer in the compositions of the disclosure may be present in the range from about 10-30 mM, about 10-20 mM, about 20 mM or about 15.5 mM. For example, the buffer in the compositions of the disclosure is present at about 20 mM, or at about 15.5 mM sodium phosphate dibasic heptahydrate. The compositions of the disclosure may comprise sodium phosphate dibasic heptahydrate and citric acid buffering agents providing a pH of from 6.2 to 6.6 inclusive with a pH value of 6.3 being preferred. The sodium phosphate dibasic heptahydrate buffering agent may be present in the range from about 15-16.4 mM and the citric acid buffering agent may be present in the range from about 3.8-4.9 mM. For example, the compositions of the disclosure may comprise about 15.5 mM sodium phosphate dibasic heptahydrate and about 4.5 mM citric acid monohydrate. The compositions of the disclosure may further comprise a sugar. The compositions of the disclosure may further comprise sucrose. Sucrose may be present in the compositions of the disclosure in the range from about 5-20%; about 10-15%, about 11-13% or at about 12% weight by volume. The compositions of the disclosure may further comprise polysorbate 80. Polysorbate 80 may be present in the range from about 0.01-0.1% weight by volume. For example, polysorbate 80 may be present in the compositions of the disclosure at about 0.02% weight by volume, or at about 0.05% weight by volume. The compositions of the disclosure may further comprise EDTA. EDTA may be present in the range from about 0.01-0.1 mM. For example, EDTA may be present at about 0.05 mM. In one embodiment, the compositions of the disclosure further comprise 20 mM sodium phosphate dibasic heptahydrate, 12% weight of sucrose to volume and 0.05% weight of polysorbate 80 to volume. In another embodiment, the compositions of the disclosure further comprise 15.5 mM sodium phosphate dibasic, 3.9 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. The compositions of the disclosure may comprise an aqueous liquid formulation at pH 6.2 containing 16.1 mM sodium phosphate dibasic heptahydrate, 3.9 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. The compositions of the disclosure may comprise an aqueous liquid formulation at pH 6.2 containing 15.2 mM sodium phosphate dibasic heptahydrate, 4.8 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. The compositions of the disclosure may comprise an aqueous liquid formulation at pH 6.4 containing 15.8 mM sodium phosphate dibasic heptahydrate, 4.2 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. The compositions of the disclosure may comprise an aqueous liquid formulation at pH 6.6 containing 16.3 mM sodium phosphate dibasic heptahydrate, 3.7 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. The compositions of the disclosure may comprise an aqueous liquid formulation at pH 6.3 containing 15.5 mM sodium phosphate dibasic heptahydrate, 4.5 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. Importantly, the tangential filtration and ultrafiltration exchange step of Example 1 below may be adjusted to produce the compositions of the disclosure, such as a composition of the disclosure comprising 15.5 mM sodium phosphate dibasic heptahydrate, 4.5 mM citric citric acid monohydrate, 12% weight to volume sucrose, 0.02% weight to volume polysorbate 80, 0.05 mM EDTA at a pH of 6.3—or other such liquid formulations. Another aspect of the disclosure is a composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.8 to 7.2, wherein the buffering agent is histidine, phosphate, citric acid, citrate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. In the composition the heavy chain may comprise an amino acid sequence having at least 95%, 96%, 96.88%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1. In the composition the light chain may comprise an amino acid sequence having at least 98%, 98.63 or 99% identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid and citrate. In another embodiment the buffering agent is sodium phosphate, potassium phosphate, or sodium citrate. In another embodiment the composition further comprises a sugar, a carbohydrate and/or a salt. In another embodiment the composition comprises sucrose. Another aspect of the disclosure is a composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.8 to 7.2, wherein the buffering agent is phosphate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. In another embodiment the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid and citrate. In another embodiment the composition further comprises a sugar. In another embodiment the sugar is sucrose. In another embodiment the composition comprises polysorbate 80. In another embodiment the composition comprises one selected from a first formulation of 20 mM sodium phosphate dibasic heptahydrate, 12% weight of sucrose to volume and 0.05% weight of polysorbate 80 to volume; and a second formulation of 15.5 mM sodium phosphate dibasic heptahydrate, 3.9 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; and a third formulation of 26 mM sodium phosphate dibasic heptahydrate, 15% weight of sucrose to volume and 0.065% weight of polysorbate 80 to volume. The composition may be at a pH between about 6.8 to about 7.2, about 6.1 to about 6.5 or about 6 to about 6.6. In another embodiment the antibody has a dissociation constant equal to, or less than, about 3.5×10−11 M for human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition. The main form of the antibody may also comprise greater than, or equal to, 57.9%, 59.4%, and 60% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition. The acid forms of the antibody may also comprise greater than, or equal to, 37.6%, 37.8%, 38.4% and 39.8% of the protein in the composition as measured using capillary isoelectric focusing of the composition. The total acidic peak area determined by cIEF can be as high as 72% and still retain 0.74 IL-5 specific binding and 80% FcRn binding. In another embodiment the acidic forms of the antibody comprise at least one selected from the group consisting of a peak 65 acidic form, a peak 78 acidic form, a peak 88 acidic form and a peak 92 acidic form. In another embodiment the acidic forms of the antibody comprise at least one deamidated amino acid residue selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31. An acceptable level of deamidation on LC N31 is greater than or equal to 17%, or greater than or equal to 17.4% as measured by peptide mapping LC MS/MS). An acceptable level of deamidation on HC 386 is greater than or equal to 30% as measured by peptide mapping LC MS/MS). The acceptable upper level may be the level of a particular variant that allows the antibody molecules in the composition to retain antigen binding activity of about 0.70 to about 1.30 as measured by SPR and FcRn binding activity of about 70% to about 130% as measured by SPR or other antigen binding activity or FcRn binding activity values, or ranges, disclosed herein. In another embodiment the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. Acceptable levels of oxidation on the heavy chain residues of the antibody as measured by peptide mapping LC-MS/MS may be about 50% for HC M64, M254, and M430 and about 3% for HC W52. The acceptable upper level may be the level of a particular variant that allows the antibody molecules in the composition to retain antigen binding activity of about 0.70 to about 1.30 as measured by SPR and FcRn binding activity of about 70% to about 130% as measured by SPR or other antigen binding activity or FcRn binding activity values, or ranges, disclosed herein. In another embodiment the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. The basic form of the antibody may also comprise greater than, or equal to, 2.2% and 2.3% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the basic form of the antibody comprises a peak 112 basic form. In another embodiment the basic form of the antibody comprises a heavy chain having a carboxy terminal residue that is glycine 448. In another embodiment the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. In another embodiment the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a heavy chain amino acid residue oxidized at cysteine 222. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; c) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and d) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a heavy chain amino acid residue oxidized at cysteine 222. In another embodiment the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220, and wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) deamidated forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) deamidated forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31; and c) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. In the composition CDRH2 may comprise an amino acid sequence having at least 85% or 87.5% identity to the amino acid sequence of SEQ ID NO: 6. In the composition CDRL1 may comprise an amino acid sequence having at 93%, 94% or 94.11% identity to the amino acid sequence of SEQ ID NO: 8. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) oxidized forms of the antibody comprising a heavy chain amino acid residue oxidized at methionine 64. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) oxidized forms of the antibody comprising a heavy chain amino acid residue oxidized at methionine 64; and c) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. In the composition the heavy chain variable region may comprise an amino acid sequence having at least 90%, 95% or 95.57% identity to the amino acid sequence of SEQ ID NO: 3. In the composition the light chain variable region may comprise an amino acid sequence having at least 90%, 98% or 98.31% identity to the amino acid sequence of SEQ ID NO: 3. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85 and a light chain amino acid residue oxidized at methionine 4. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31; and c) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85 and a light chain amino acid residue oxidized at methionine 4. In another embodiment the total protein concentration is about 75 mg/mL. The total protein concentration may also be about any pair of values, or single value in the range of about 75 mg/mL to about 150 mg/mL such as about 75 mg/mL to about 100 mg/mL, about 67.3 to about 87.5 mg/mL, about 76 g protein/L to about 82 g protein/L, about 46 g protein/L to about 66 g protein/L or about 100 mg/mL. In the compositions the purity of the anti-human-IL-5 antibodies in the sample is greater than, or equal to, 97.0%, 96%, 95%, or 80%, 85%. In another embodiment the composition further comprises a) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the composition further comprises a) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and b) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the composition further comprises a) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and b) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the composition of further comprises a) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; b) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of an amino terminal pyroglutamate residue at amino acid residue 1, a carboxy terminal glycine amino acid residue at amino acid residue 448, a deamidated aparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine residue at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4 and an oxidized cysteine residue at position 220. In another embodiment the composition comprises a) about greater than or equal to 92% of the population comprises an amino terminal pyroglutamate residue at amino acid residue 1 of the antibody heavy chain, b) about greater than or equal to 90% of the population comprises a carboxy terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain, c) less than or equal to 6.0% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; d) about less than or equal to 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, e) about less than or equal to 4.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about less than or equal to 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, and g) about less than or equal to 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain. In another embodiment the composition comprises a) about 92% to about 99% of the population comprises an amino terminal pyroglutamate residue at amino acid residue 1 of the antibody heavy chain, b) about 95% to about 99.5% of the population comprises a carboxy terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain, c) about 0.3% to about 1.5% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, d) about 1.5% to about 4.5% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; e) about 0.5% to about 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, f) about 0.2% to about 1.5% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, g) about 2.5% to about 3.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, h) about 0.4% to about 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, i) about 3.3% to about 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and j) about 0.1% to about 1% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. In another embodiment the composition comprises: a) about 93.7% to about 98.6% of the population comprises an amino terminal pyroglutamate residue at amino acid residue 1 of the antibody heavy chain, b) about 97.6% to about 99.2% of the population comprises a carboxy terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain, c) about 0.4% to about 1.2% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, d) about 1.6% to about 4.2% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; e) about 0.7% to about 0.9% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, f) about 0.3% to about 1.1% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, g) about 2.6% to about 3.3% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, h) about 0.5% to about 0.7% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, i) about 3.4% to about 6.5% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and j) about 0.2% to about 0.8% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine residue at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4 and an oxidized cysteine residue at position 222. In another embodiment the composition comprises: a) about 0.3% to about 1.5% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.5% to about 4.5% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; c) about 0.5% to about 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, d) about 0.2% to about 1.5% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, e) about 2.5% to about 3.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about 0.4% to about 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, g) about 3.3% to about 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and h) about 0.1% to about 1% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. In another embodiment the composition comprises: a) about 0.4% to about 1.2% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.6% to about 4.2% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; c) about 0.7% to about 0.9% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, d) about 0.3% to about 1.1% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, e) about 2.6% to about 3.3% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about 0.5% to about 0.7% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, g) about 3.4% to about 6.5% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and h) about 0.2% to about 0.8% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of a deamidated at asparagine residue at position 299, a deamidated asparagine residue at position 317 and a deamidated asparagine residue at position 386; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising a deamidated asparagine residue at amino acid residue 31. In another embodiment the composition comprises: a) about 0.3% to about 1.5% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.5% to about 4.5% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; and c) about 3.3% to about 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain. In another embodiment the composition comprises: a) about 0.4% to about 1.2% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.6% to about 4.2% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; and c) about 3.4% to about 6.5% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain. Another aspect of the disclosure is a composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine residue at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consistine of an oxidized methionine residue at position 4 and an oxidized cysteine residue at position 220. In another embodiment the composition comprises: a) about 0.5% to about 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, b) about 0.2% to about 1.5% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, c) about 2.5% to about 3.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, d) about 0.4% to about 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, and e) about 0.1% to about 1% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. In another embodiment the composition comprises: a) about 0.7% to about 0.9% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, b) about 0.3% to about 1.1% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, c) about 2.6% to about 3.3% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, d) about 0.5% to about 0.7% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, and e) about 0.2% to about 0.8% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a main form of the antibody comprising greater than, or equal to, 20% of the protein in the composition as measured using capillary isoelectric focusing of the composition. Another aspect of the disclosure is a composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 20% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) acidic forms of the antibody comprising up to about 80% of the protein in the composition as measured using capillary isoelectric focusing of the composition. In another embodiment the composition is for the treatment of a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid and eosinophilic esophagitis. Another embodiment is a method of treating a disease in a subject comprising the steps of a) identifying a subject with a disease selected from the group consisting of of asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid and eosinophilic esophagitis; and b) administering a therapeutically effective amount of a composition according to the disclosure to the subject; whereby the disease in the subject is treated. Another embodiment is a method of producing a composition of the disclosure, comprising the steps of: a) expressing in a host cell an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence; b) growing the cells at a pH of about 6.75 to about 7.00; c) harvesting a cell culture supernatant; d) placing the cell culture supernatant in contact with a protein A resin or protein G resin to bind antibody molecules; e) eluting the antibody molecules from the resin to produce an first eluate; f) treating the first eluate at a pH of about 3.3 to about 3.7 for about 15 to about 240 minutes to produce a treated first eluate; g) placing the treated first eluate in contact with a anion exchange resin at a load pH of about 8.3 to about 8.7; h) collecting a second eluate (a flow through eluate) from the anion exchange resin and holding this for about 96 hours or less; i) treating the second eluate with guanidine and ammonium sulphate to produce a solution; j) placing the solution in contact with a hydrophobic interaction chromatographic resin bed at a load ratio of about 12 g protein/L resin to about 27 g protein/L of resin load ratio; k) eluting a third eluate comprising the antibody molecules from the hydrophobic interaction chromatographic resin with an elution gradient volume of about 9 resin bed volumes to about 11 resin bed volumes and an elution peak cut stop of about 17% of the maximum peak height to about 23% of the maximum peak height; and 1) formulating the third eluate; whereby a composition of the disclosure is produced. In the methods of the disclosure any nucleic acid sequence suitable for expression of an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence. For example, the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 may be used to express an antibody in a eukaryotic cell. Alternatively, other nucleic acid sequences with different sequences which encode (e.g., due to the use of alternative codons) the antibody heavy chain amino acid sequence as shown in SEQ ID NO: 1 or the antibody light chain amino acid sequence as shown in SEQ ID NO: 2 may be used. In the methods of the disclosure deamidation can be controlled by growing cells at a pH of about 6.75 to about 7.00. In the methods of the disclosure deamidation can be controlled by growing cells for about 12 to about 18 days for an in vitro cell age of less than or equal to 166 days. In the methods of the disclosure deamidation can be controlled by placing the treated first eluate in contact with an anion exchange resin at a load pH of about 8.3 to about 8.7 and collecting the second eluate from the anion exchange resin and holding this for about 96 hours or less. In the methods of the disclosure aggregationjcan be controlled during phenyl SEPHAROSE™ fast flow chromatography by placing the solution in contact with the hydrophobic interaction chromatographic resin bed at a load ratio of about 12 g protein/L resin to about 27 g protein/L of resin load ratio; eluting a third eluate comprising the antibody molecules from the hydrophobic interaction chromatographic resin with an elution gradient volume of about 9 resin bed volumes to about 11 resin bed volumes and an elution peak cut stop of about 17% of the maximum peak height to about 23% of the maximum peak height. Aggregation can also be limited after final filtration, filling and freezing of the pharmaceutical compositions of the disclosure to less than or equal to about 6 hours. Importantly, any of the steps of the disclosed methods may be omitted, or combined to produce the compositions of the disclosure. Upon production of the antibody, post-translational modifications may occur. This may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, deamidation (for example at an asparagine or glutamine residue), oxidation (for example at a methionine, tryptophan or free cysteine residue), disulfide bond scrambling, isomerisation (for example at an aspartic acid residue), C-terminal lysine clipping (for example from one or both heavy chains), and N-terminal glutamine cyclisation (for example in the heavy and/or light chain). The antibody composition may comprise (i) the antibody (i.e., an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2); and (ii) antibody variants that include one or more or a combination of: charge variants (e.g., acidic and basic variants), amino acid sequence variants, and antibody structural variants (e.g., aggregated and fragmented variants). Acidic or basic antibody variants can be characterised and distinguished from the antibody based on their overall acidic or basic charge. For example, the charge distribution of the antibody composition can be detected using capillary isoelectric focussing (cIEF) or ion exchange chromatography. Acidic variants may comprise deamidated antibody variants, glycated antibody variants, sialylated antibody variants, and oxidised antibody variants. Cysteine and tryptophan oxidation in the antibody variant result in a pI shift (i.e., a charge difference) and are detected with other acidic antibody variants. Methionine oxidation in the antibody variant can be monitored by a change in antigen binding, or by peptide mapping, for example by LC-MS/MS. Deamidation is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid (iso-aspartate) (iso-D) and aspartic acid (aspartate) (D) at approximately 3:1 ratio. This deamidation reaction is therefore related to isomerization of aspartate (D) to iso-aspartate. The deamidation of asparagine and the isomerisation of aspartate, both involve the intermediate succinimide. To a much lesser degree, deamidation can occur with glutamine residues in a similar manner. Deamidation can occur in a CDR, in a Fab (non-CDR region), or in the Fc region. Deamidation causes a change in the charge of the antibody, such that deamidated antibody variants are acidic compared to the antibody. The antibody composition may comprise ≤35% deamidated antibody variant. For example, N31 of the light chain may be deamidated to Iso-D, D or succinimide. The antibody composition may comprise ≤25% deamidated antibody variant at position 31 of the light chain. This can result in one amino acid change in the sequence of the light chain of the antibody, for example in ≤25% of the antibody composition. For example, N386 of the heavy chain may be deamidated to Iso-D, D or succinimide. The antibody composition may comprise ≤35% deamidated antibody variant at position 386 of the heavy chain. This can result in one amino acid change in the sequence of the heavy chain of the antibody, for example in ≤35% of the antibody composition. The composition may comprise a mixture of antibody variants. Deamidation events can be cumulative, so that two or more asparagines residues are deamidated. Therefore, the antibody composition may comprise at least one amino acid change in the sequence of the heavy chain of the antibody and/or at least one amino acid change in the sequence of the heavy chain of the antibody. For example, the antibody composition may comprise deamidated antibody variant at position 31 of the light chain and deamidated antibody variant at position 386 of the heavy chain. Oxidation can occur during production and storage (i.e., in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but may occur at tryptophan and free cysteine residues. Oxidation can occur in a CDR, in a Fab (non-CDR) region, or in the Fc region. Oxidation can cause a change in the charge of the antibody, such that oxidised antibody variants are acidic compared to the antibody. Some oxidised antibody variants have the same charge as the antibody. The antibody composition may comprise ≤55% oxidised antibody variant. For example, any one or a combination of M64, M254, and/or M430 of the heavy chain may be oxidised. The antibody composition may comprise ≤55% oxidised antibody variant at any one or a combination of M64, M254, and/or M430 of the heavy chain. For example, W52 of the heavy chain may be oxidised. The antibody composition may comprise ≤3% oxidised antibody variant at W52 of the heavy chain. The composition may comprise a mixture of antibody variants. Therefore, the antibody composition may comprise at least one amino acid change in the sequence of the heavy chain of the antibody and/or at least one amino acid change in the sequence of the heavy chain of the antibody. For example, the antibody composition may comprise deamidated antibody variant at position 31 of the light chain; and/or deamidated antibody variant at position 386 of the heavy chain; and/or oxidation at any one or a combination of M64, M254, and/or M430 and/or W52 of the heavy chain. Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (—SH). These free (unpaired) sulfhydryls (—SH) can promote shuffling. N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu) via cyclization. It is thought that most pGlu formation happens in the production bioreactor, but it can also be formed non-enzymatically, depending on pH and temperature of processing and storage conditions. Cyclization of N-terminal Q or E is commonly observed in natural human antibodies. The antibody composition described herein may comprise ≥50% pGlu at the N-terminus of the antibody. pGlu may be present in the heavy chain. This can result in one amino acid change in the sequence of the heavy or light chain of the antibody, for example in ≥50% of the antibody composition. The composition may comprise a mixture of antibody variants. Sequence changes can be cumulative, so that the composition comprises two or more sequence changes in the heavy and/or light chain. Therefore, the antibody composition may comprise at least one amino acid change in the sequence of the heavy chain of the antibody and/or at least one amino acid change in the sequence of the heavy chain of the antibody. For example, the antibody composition may comprise deamidated antibody variant at position 31 of the light chain; and/or deamidated antibody variant at position 386 of the heavy chain; and/or oxidation at any one or a combination of M64, M254, and/or M430 and/or W52 of the heavy chain; and/or pGlu at the N-terminus of the heavy and/or light chain. C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant and natural human antibodies. Variants of this process include removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Upon administration to the human subject/patient is likely to result in the removal of any remaining C-terminal lysines. The antibody composition described herein may comprise ≥50% C-terminal lysine deleted at the C-terminus of the antibody. K449 may be deleted in one or both of the heavy chains of the antibody. Thus there are two antibody variants: lysine single deletion in the heavy chain, and lysine double deletion in the heavy chain. The antibody (i.e., an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2) has both lysines intact/present. This can result in one amino acid change in the sequence of the heavy chain of the antibody, for example in ≥50% of the antibody composition. The composition may comprise a mixture of antibody variants. For example, the antibody composition may comprise deamidated antibody variant at position 31 of the light chain; and/or deamidated antibody variant at position 386 of the heavy chain; and/or oxidation at any one or a combination of M64, M254, and/or M430 and/or W52 of the heavy chain; and/or pGlu at the N-terminus of the heavy and/or light chain; and/or C-terminal lysine deleted at the C-terminus. Aggregated or fragmented antibody variants can be characterised and distinguished from the antibody based on their size. For example, the size distribution of the antibody composition can be detected using size exclusion chromatography (SEC). The antibody composition may comprise ≤20% aggregated antibody variant. The aggregated antibody variant may comprise dimer. The composition may comprise a mixture of antibody variants. For example, the antibody composition may comprise deamidated antibody variant at position 31 of the light chain; and/or deamidated antibody variant at position 386 of the heavy chain; and/or oxidation at any one or a combination of M64, M254, and/or M430 and/or W52 of the heavy chain; and/or pGlu at the N-terminus of the heavy and/or light chain; and/or C-terminal lysine deleted at the C-terminus; and/or aggregated antibody variant. The compositions described may have been subjected to, or have undergone, one or more post-translational modifications. The modification may occur in a CDR, the variable framework region, or the constant region. The modification may result in a change in charge of the molecule. The post-translational modifications and changes in primary amino acid sequence described above, do not result in significant changes in antigen binding affinity, biological activity, PK/PD, aggregation, immunogenicity, or binding to the Fc receptor, of the compositions. The compositions are substantially free of contaminating materials. The antibody composition comprising the antibody and antibody variants described above retain specific antigen binding and/or FcRn binding. For example, the antibody composition comprising the antibody and antibody variants described above has ≥0.70 IL-5 specific antigen binding; and/or ≥70% FcRn binding. Thus these levels (%) of variants can be tolerated in the antibody composition without impacting function. The compositions described herein may be produced by any number of conventional techniques. For example, the compositions may be expressed in and purified from recombinant expression systems. In one embodiment, the composition is produced by a method of culturing a host cell under conditions suitable for expression of a polypeptide comprising SEQ ID NO: 1 and SEQ ID NO:2, wherein the composition is expressed, and optionally purified, and optionally formulated within a pharmaceutical composition. A number of different expression systems and purification regimes can be used to produce the compositions. Generally, host cells are transformed with a recombinant expression vector encoding the antibody. A wide range of host cells can be employed, including Eukaryotic cell lines of mammalian origin (e.g., CHO, Perc6, HEK293, HeLa, NSO). Suitable host cells include mammalian cells such as CHO (e.g., CHOK1 and CHO-DG44). The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism (e.g., plant or animal). The host cell may be a non-human host cell. Appropriate cloning and expression vectors for use with eukaryotic or mammalian cellular hosts and methods of cloning are known in the art. The cells may be cultured under conditions that promote expression of the antibody. For example, a production bioreactor is used to culture the cells. The production bioreactor volume may be: (i) about 20,000 litres, about 10,000 litres; about 5,000 litres; about 2,000 litres; about 1,000 litres; or about 500 litres; or (ii) between 500 and 20,000 litres; between 500 and 10,000 litres; between 500 and 5,000 litres; between 1,000 and 10,000 litres, or between 2,000 and 10,000 litres. For example, the cells may be cultured in a production bioreactor at a pH of about 6.75 to pH 7.00. Alternatively, the cells may be cultured in a production bioreactor for about 12 to about 18 days. Alternatively, the cells may be cultured in a production bioreactor at a pH of about 6.75 to pH 7.00, for about 12 to about 18 days. This culture step may help to control the level of deamidated antibody variants, for example, to reduce the level of deamidated antibody variants. The composition may be recovered and purified by conventional protein purification procedures. For example, the composition may be harvested directly from the culture medium. Harvest of the cell culture medium may be via clarification, for example by centrifugation and/or depth filtration. Recovery of the composition is followed by purification to ensure adequate purity. One or more chromatography steps may be used in purification, for example one or more chromatography resins; and/or one or more filtration steps. For example affinity chromatography using resins, such as protein A, G, or L may be used to purify the composition. Alternatively, or in addition to, an ion-exchange resin such as a cation-exchange may be used to purify the composition. Alternatively, or in addition to, a hydrophobic interaction chromatographic resin may be used to purify the composition. Alternatively the purification steps comprise: an affinity chromatography resin step, followed by a cation-exchange resin step, followed by a hydrophobic interaction chromatographic resin step. For example, the harvest is placed in contact with a protein A resin. The solution comprising the composition may be eluted from the protein A resin and treated at pH 3.3 to 3.7 for 15 to 240 minutes. This protein A resin step may help to control the level of aggregated antibody variants, for example, to reduce the level of aggregated antibody variants. The solution comprising the composition may then be further clarified by depth filtration and/or dual layer filtration. Alternatively, or in addition to, an anion exchange resin may be used. The solution comprising the composition may be placed in contact with an anion exchange resin (for example Q-SEPHAROSE™ Fast Flow anion exchange chromatography) at a load pH of 8.3 to 8.7. The solution comprising the composition may be eluted from the anion exchange resin and held for 96 hours or less. This anion exchange resin step may help to control the level of deamidated antibody variants, for example, to reduce the level of deamidated antibody variants. Optionally, guanidine and/or ammonium sulphate may be added to the solution comprising the composition, and held for 15 to 240 minutes. Alternatively, or in addition to, a hydrophobic interaction chromatographic resin may be used. The solution comprising the composition may be placed in contact with a hydrophobic interaction chromatographic resin (e.g., phenyl SEPHAROSE™ fast flow chromatography) at a load ratio of 12 to 27 g protein/L resin. For example, the solution comprising the composition may be eluted using an elution gradient volume (bed volumes; BV) of about 9 to about 11. An elution peak cut stop (% of maximum peak height) of about 17 to about 23 may be used during elution from the hydrophobic interaction chromatographic resin. This hydrophobic interaction chromatographic resin step may help to control the level of aggregated antibody variants, for example, to reduce the level of aggregated antibody variants. The solution comprising the composition may then be filtered to remove virus. The solution comprising the composition may then be formulated at an antibody concentration of about 76 g protein/L to about 82 g protein/L, or to about 100 g protein/L. The solution comprising the composition may be filled into containers and frozen. Aliquots of the solution comprising the composition may be lyophilized. Lyophilizate may be reconstituted by the addition of water to produce a composition comprising 75 mg/L of protein, the monoclonal anti-IL-5 mepolizumab antibody and 20 mM sodium phosphate dibasic heptahydrate, 12% weight of sucrose to volume and 0.05% weight of polysorbate 80 to volume at a pH of from about 6.8 to about 7.2. In another embodiment the composition of the disclosure is produced using this method of producing a composition of the disclosure. Another aspect of the disclosure is a composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.2 to 6.6, wherein the buffering agent is histidine, phosphate, citric acid, citric acid monohydrate, citrate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. Another aspect of the disclosure is a composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.2 to 6.6, wherein the buffering agent is phosphate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. In another embodiment of the compositions of the disclosure the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate and citric acid. In another embodiment of the compositions of the disclosure comprise one selected from a first formulation of 16.1 mM sodium phosphate dibasic heptahydrate, 3.9 mM citric acid, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; a second formulation of 15.2 mM sodium phosphate dibasic heptahydrate, 4.8 mM citric acid, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; a third formulation of 15.8 mM sodium phosphate dibasic heptahydrate, 4.2 mM citric acid, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; a fourth formulation of 16.3 mM sodium phosphate dibasic heptahydrate, 3.7 mM citric acid, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; and a fifth formulation of 15.5 mM sodium phosphate dibasic heptahydrate, 4.5 mM citric acid, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. In summary, the disclosure includes: 1. A composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤80% acidic antibody variants. 2. The composition according to 1, wherein the composition has: a) ≥0.70 IL-5 specific antigen binding; and/or b) ≥70% FcRn binding. 3. The composition according to 2, wherein a) the specific antigen binding is in the range of from 0.70 to 1.30; and/or b) the FcRn binding is in the range of from 70% to 130%. 4. The composition according to any one of the preceding, wherein the composition comprises: ≤35% deamidated antibody variants. 5. The composition according to according to any one of the preceding, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence. 6. The composition according to according to any one of the preceding, wherein the composition comprises: ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence. 7. The composition according to any one of the preceding, wherein the composition comprises: ≤55% oxidised antibody variant at any one or a combination of: a) M64 of the heavy chain amino acid sequence; b) M254 of the heavy chain amino acid sequence; and/or c) M430 of the heavy chain amino acid sequence. 8. The composition according to any one of the preceding, wherein the composition comprises: ≤3% oxidised antibody variants at W52 of the heavy chain amino acid sequence. 9. The composition according to any one of 4 to 8, wherein a deamidated antibody variant amount and/or an oxidised variant amount, is determined by peptide mapping LC-MS/MS. 10. The composition according to any one of the preceding, wherein the composition comprises: ≤20% aggregated antibody variants. 11. The composition according to 10, wherein the aggregated antibody variant comprises a dimer. 12. The composition according to 10 or 11 wherein the aggregated antibody variant amount is determined by SEC. 13. The composition according to any one of the preceding, wherein the composition comprises: ≥50% heavy chain amino acid sequence C-terminal lysine K449 deleted antibody variants. 14. The composition according to any one of the preceding, wherein the composition comprises: ≥50% heavy chain amino acid sequence pyro-glutamate N-terminal antibody variants. 15. A composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤80% acidic antibody variants and ≤20% aggregated antibody variants. 16. A composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; and ≤20% aggregated antibody variants. 17. A composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤55% oxidised antibody variants at M64 of the heavy chain amino acid sequence; ≤3% oxidised antibody variants at W52 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. 18. A composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. 19. A composition comprising an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence, wherein the composition comprises: ≤25% deamidated antibody variants at N31 of the light chain amino acid sequence; ≤35% deamidated antibody variants at N386 of the heavy chain amino acid sequence; ≤55% oxidised antibody variants at M64 of the heavy chain amino acid sequence, M254 of the heavy chain amino acid sequence, M430 of the heavy chain amino acid sequence; ≤3% oxidised antibody variants at W52 of the heavy chain amino acid sequence; and ≤20% aggregated antibody variants. 20. A composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.8 to 7.2, wherein the buffering agent is histidine, phosphate, citric acid, citrate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. 21. The composition of 20, wherein the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid and citrate. 22. The composition of 20, wherein the buffering agent is sodium phosphate, potassium phosphate, or sodium citrate. 23. The composition of 20, wherein the composition further comprises a sugar, a carbohydrate and/or a salt. 24. The composition of 23, wherein the composition comprises sucrose. 25. A composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.8 to 7.2, wherein the buffering agent is phosphate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. 26. The composition of 25, wherein the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid and citrate. 27. The composition of 26, wherein the composition further comprises a sugar. 28. The composition of 27, wherein the sugar is sucrose. 29. The composition of 28, comprising polysorbate 80. 30. The composition of 29, comprising one selected from a first formulation of 20 mM sodium phosphate dibasic heptahydrate, 12% weight of sucrose to volume and 0.05% weight of polysorbate 80 to volume; a second formulation of 15.5 mM sodium phosphate dibasic heptahydrate, 3.9 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; and a third formulation of 26 mM sodium phosphate dibasic heptahydrate, 15% weight of sucrose to volume and 0.065% weight of polysorbate 80 to volume. 31. The composition of 29, wherein the antibody has a dissociation constant equal to, or less than, about 3.5×10−11 M for human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11. 32. The composition of 31, wherein the monoclonal antibody concentration is about 75 mg/mL or about 100 mg/mL. 33. The composition of 30, wherein the antibody has a dissociation constant equal to, or less than, about 3.5×10−11 M for human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11. 34. The composition of 33, wherein the monoclonal antibody concentration is about about 75 mg/mL or about 100 mg/mL. 35. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition. 36. The composition of 35 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 37. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition. 38. The composition of 37 wherein the acidic forms of the antibody comprise at least one selected from the group consisting of a peak 65 acidic form, a peak 78 acidic form, a peak 88 acidic form and a peak 92 acidic form. 39. The composition of 38 wherein the acidic forms of the antibody comprise at least one deamidated amino acid residue selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31. 40. The composition of 39 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 41. The composition of 39 wherein the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 42. The composition of 39 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and wherein the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 43. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. 44. The composition of 43 wherein the basic form of the antibody comprises a peak 112 basic form. 45. The composition of 44 wherein the basic form of the antibody comprises a heavy chain having a carboxy terminal residue that is glycine 448. 46. The composition of 45 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 47. The composition of 45 wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a heavy chain amino acid residue oxidized at cysteine 220. 48. The composition of 45 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a heavy chain amino acid residue oxidized at cysteine 220; and wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 49. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 50% of the protein in the composition as measured using capillary isoelectric focusing of the composition; c) acidic forms of the antibody comprising about 20% to about 45% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and d) a basic form of the antibody comprising about 1% to about 15% of the protein in the composition as measured using capillary isoelectric focusing of the composition. 50. The composition of 49 wherein the acidic forms of the antibody comprise at least one selected from the group consisting of a peak 65 acidic form, a peak 78 acidic form, a peak 88 acidic form and a peak 92 acidic form. 51. The composition of 50 wherein the acidic forms of the antibody comprise at least one deamidated amino acid residue selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31. 52. The composition of 49 wherein the basic form of the antibody comprises a peak 112 basic form. 53. The composition of 52 wherein the basic form of the antibody comprises a heavy chain having a carboxy terminal residue that is glycine 448. 54. The composition of 49 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 55. The composition of 49 wherein the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a heavy chain amino acid residue oxidized at cysteine 220. 56. The composition of 49 wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 57. The composition of 49 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a heavy chain amino acid residue oxidized at cysteine 222; and wherein the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 58. The composition of 49 wherein the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 59. The composition of 49 wherein the main form of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 60. The composition of 49 wherein the main form of the antibody comprises at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; wherein the acidic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 220, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220; and wherein the basic forms of the antibody comprise at least one oxidized amino acid residue selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 61. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) deamidated forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31. 62. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 63. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) deamidated forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue deamidated at asparagine 299, a heavy chain amino acid residue deamidated at asparagine 317, a heavy chain amino acid residue deamidated at asparagine 386 and a light chain amino acid residue deamidated at asparagine 31; and c) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85, a heavy chain amino acid residue oxidized at cysteine 222, a heavy chain amino acid residue oxidized at methionine 254, a heavy chain amino acid residue oxidized at methionine 360, a heavy chain amino acid residue oxidized at methionine 430, a light chain amino acid residue oxidized at methionine 4 and a light chain amino acid residue oxidized at cysteine 220. 64. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. 65. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52 and a heavy chain amino acid residue oxidized at methionine 64. 66. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region having the CDRH1 amino acid sequence shown in SEQ ID NO: 5, the CDRH2 amino acid sequence shown in SEQ ID NO: 6, and the CDRH3 amino acid sequence shown in SEQ ID NO: 7; and a light chain variable region having the CDRL1 amino acid sequence shown in SEQ ID NO: 8, the CDRL2 amino acid sequence shown in SEQ ID NO: 9, and the CDRL3 amino acid sequence shown in SEQ ID NO: 10; b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52 and a heavy chain amino acid residue oxidized at methionine 64; and c) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. 67. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31. 68. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; and b) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85 and a light chain amino acid residue oxidized at methionine 4. 69. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4; b) deamidated forms of the antibody comprising a light chain amino acid residue deamidated at asparagine 31; and c) oxidized forms of the antibody comprising at least one selected from the group consisting of a heavy chain amino acid residue oxidized at tryptophan 52, a heavy chain amino acid residue oxidized at methionine 64, a heavy chain amino acid residue oxidized at methionine 82, a heavy chain amino acid residue oxidized at methionine 85 and a light chain amino acid residue oxidized at methionine 4. 70. A composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of an amino terminal pyroglutamate residue at amino acid residue 1, a carboxy terminal glycine amino acid residue at amino acid residue 448, a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4 and an oxidized cysteine at position 220. 71. The composition of 70 wherein: a) about greater than or equal to 92% of the population comprises an amino terminal pyroglutamate residue at amino acid residue 1 of the antibody heavy chain, b) about greater than or equal to 90% of the population comprises a carboxy terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain, c) less than or equal to 6.0% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; d) about less than or equal to 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, e) about less than or equal to 4.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about less than or equal to 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, and g) about less than or equal to 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain. 72. The composition of 71 wherein: a) about 93.7% to about 98.6% of the population comprises an amino terminal pyroglutamate residue at amino acid residue 1 of the antibody heavy chain, b) about 97.6% to about 99.2% of the population comprises a carboxy terminal glycine amino acid residue at amino acid residue 448 of the antibody heavy chain, c) about 0.4% to about 1.2% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, d) about 1.6% to about 4.2% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; e) about 0.7% to about 0.9% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, f) about 0.3% to about 1.1% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, g) about 2.6% to about 3.3% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, h) about 0.5% to about 0.7% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, i) about 3.4% to about 6.5% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and j) about 0.2% to about 0.8% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. 73. A composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317, a deamidated asparagine residue at position 386, an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine at position 222, an oxidized methionine at position 254, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at amino acid residue 31, an oxidized methionine residue at position 4 and an oxidized cysteine at position 220. 74. The composition of 73 wherein: a) about 0.3% to about 1.5% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.5% to about 4.5% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; c) about 0.5% to about 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, d) about 0.2% to about 1.5% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, e) about 2.5% to about 3.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about 0.4% to about 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, g) about 3.3% to about 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and h) about 0.1% to about 1% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. 75. The composition of 74 wherein: a) about 0.4% to about 1.2% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.6% to about 4.2% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; c) about 0.7% to about 0.9% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, d) about 0.3% to about 1.1% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, e) about 2.6% to about 3.3% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about 0.5% to about 0.7% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, g) about 3.4% to about 6.5% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain, and h) about 0.2% to about 0.8% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. 76. A composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of a deamidated asparagine residue at position 299, a deamidated asparagine residue at position 317 and a deamidated asparagine residue at position 386; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising a deamidated asparagine residue at amino acid residue 31. 77. The composition of 76 wherein: a) about 0.3% to about 1.5% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.5% to about 4.5% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; and c) about 3.3% to about 6.6% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain. 78. The composition of 77 wherein: a) about 0.4% to about 1.2% of the population comprises a deamidated asparagine residue at position 317 of the antibody heavy chain, b) about 1.6% to about 4.2% of the population comprises a deamidated asparagine residue at position 386 of the antibody heavy chain; and c) about 3.4% to about 6.5% of the population comprises a deamidated asparagine residue at amino acid residue 31 of the antibody light chain. 79. A composition comprising a population of anti-IL-5 antibodies having a) a modified form of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 comprising at least one amino acid residue modification selected from the group consisting of an oxidized tryptophan residue at position 52, an oxidized methionine residue at position 64, an oxidized methionine residue at position 82, an oxidized methionine residue at position 85, an oxidized cysteine at position 222, an oxidized methionine at position 254, an oxidized methionine at position 360 and an oxidized methionine residue at position 430; and b) a modified form of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 comprising at least one amino acid residue modification selected from the group consisting of an oxidized methionine residue at position 4 and an oxidized cysteine at position 220. 80. The composition of 79 wherein: c) about 0.5% to about 1.5% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, d) about 0.2% to about 1.5% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, e) about 2.5% to about 3.5% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, f) about 0.4% to about 0.8% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, and g) about 0.1% to about 1% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. 81. The composition of 80 wherein: a) about 0.7% to about 0.9% of the population comprises an oxidized methionine residue at position 64 of the antibody heavy chain, b) about 0.3% to about 1.1% of the population comprises an oxidized methionine residue at position 82 of the antibody heavy chain or an oxidized methionine residue at position 85 of the antibody heavy chain, c) about 2.6% to about 3.3% of the population comprises an oxidized methionine at position 254 of the antibody heavy chain, d) about 0.5% to about 0.7% of the population comprises an oxidized methionine residue at position 430 of the antibody heavy chain, and e) about 0.2% to about 0.8% of the population comprises an oxidized methionine residue at position 4 of the antibody light chain. 82. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; and b) a main form of the antibody comprising greater than, or equal to, 20% of the protein in the composition as measured using capillary isoelectric focusing of the composition. 83. A composition comprising a) an anti-IL-5 antibody comprising a heavy chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2; b) a main form of the antibody comprising greater than, or equal to, 20% of the protein in the composition as measured using capillary isoelectric focusing of the composition; and c) acidic forms of the antibody comprising up to about 80% of the protein in the composition as measured using capillary isoelectric focusing of the composition. 84. A composition according to any of the preceding for the treatment of a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid and eosinophilic esophagitis. 85. A method of treating a disease in a subject comprising the steps of a) identifying a subject with a disease selected from the group consisting of of asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, hypereosinophilic syndrome, nasal polyposis, bullous pemphigoid and eosinophilic esophagitis; and b) administering a therapeutically effective amount of a composition according to any of the preceding to the subject; whereby the disease in the subject is treated. 86. The composition of 20 to 83, wherein the composition has: a) ≥0.70 IL-5 specific antigen binding; and/or b) ≥70% FcRn binding. 87. The composition of 20 to 83, wherein a) the specific antigen binding is in the range of from 0.70 to 1.30; and/or b) the FcRn binding is in the range of from 70% to 130%. 88. The composition of 20 to 83, wherein the composition comprises: ≤20% aggregated antibody variants. 89. The composition of 20 to 83, wherein the aggregated antibody variant comprises a dimer. 90. A method of producing a composition of 1-83, comprising the steps of: a) expressing in a host cell an antibody having a heavy chain amino acid sequence as shown in SEQ ID NO: 1 and a light chain amino acid sequence as shown in SEQ ID NO: 2, or an antibody variant having a heavy chain amino acid sequence at least 90% identical to the heavy chain amino acid sequence and/or a light chain amino acid sequence at least 90% identical to the light chain amino acid sequence; b) growing the cells at a pH of about 6.75 to about 7.00 for about 12 to about 18 days for an in vitro cell age of less than or equal to 166 days; c) harvesting a cell culture supernatant; d) placing the cell culture supernatant in contact with a protein A resin or protein G resin to bind antibody molecules; e) eluting the antibody molecules from the resin to produce an first eluate; f) treating the first eluate at a pH of about 3.3 to about 3.7 for about 15 to about 240 minutes to produce a treated first eluate; g) placing the treated first eluate in contact with a anion exchange resin at a load pH of about 8.3 to about 8.7; h) collecting a second eluate from the anion exchange resin and holding this for about 96 hours or less; i) treating the second eluate with guanidine and ammonium sulphate to produce a solution; j) placing the solution in contact with a hydrophobic interaction chromatographic resin bed at a load ratio of about 12 g protein/L resin to about 27 g protein/L of resin load ratio; k) eluting a third eluate comprising the antibody molecules from the hydrophobic interaction chromatographic resin with an elution gradient volume of about 9 resin bed volumes to about 11 resin bed volumes and an elution peak cut stop of about 17% of the maximum peak height to about 23% of the maximum peak height; and l) formulating the third eluate; whereby the composition of 1-83 is produced. 91. The composition of 1-83 produced by the method of 90. 92. A composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.2 to 6.6, wherein the buffering agent is histidine, phosphate, citric acid, citric acid monohydrate, citrate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. 93. The composition of 92, wherein the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid and citric acid monohydrate. 94. The composition of 92, wherein the buffering agent is sodium phosphate, potassium phosphate, citric acid, citric acid monohydrate or sodium citrate. 95. The composition of 92, wherein the composition further comprises a sugar, a carbohydrate and/or a salt. 96. The composition of 95, wherein the composition comprises sucrose. 97. A composition comprising a purified preparation of a monoclonal antibody and a buffering agent, wherein the composition is at a pH from 6.2 to 6.6, wherein the buffering agent is phosphate or a salt thereof, wherein the purified preparation comprises the isoforms represented by peak 65, peak 78, peak 88, peak 92, the main peak and peak 112 shown in FIG. 1, wherein the antibody comprises a heavy chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the antibody is produced by a Chinese Hamster Ovary cell. 98. The composition of 97, wherein the buffering agent is at least one selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citric acid, citric acid monohydrate and citrate. 99. The composition of 98, wherein the composition further comprises a sugar. 100. The composition of 99, wherein the sugar is sucrose. 101. The composition of 100, comprising polysorbate 80. 102. The composition of 101, comprising one selected from a first formulation of 16.1 mM sodium phosphate dibasic heptahydrate, 3.9 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; a second formulation of 15.2 mM sodium phosphate dibasic heptahydrate, 4.8 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; a third formulation of 15.8 mM sodium phosphate dibasic heptahydrate, 4.2 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; a fourth formulation of 16.3 mM sodium phosphate dibasic heptahydrate, 3.7 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA; and a fifth formulation of 15.5 mM sodium phosphate dibasic heptahydrate, 4.5 mM citric acid monohydrate, 12% weight of sucrose to volume, 0.02% weight of polysorbate 80 to volume and 0.05 mM EDTA. 103. The composition of 101, wherein the antibody has a dissociation constant equal to, or less than, about 3.5×10−11 M for human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11. 104. The composition of 101, wherein the monoclonal antibody concentration is about 75 mg/mL or about 100 mg/mL. 105. The composition of 102, wherein the antibody has a dissociation constant equal to, or less than, about 3.5×10−11 M for human interleukin-5 comprising the amino acid sequence shown in SEQ ID NO: 11. 106. A composition according to any one of the preceding wherein the antibody is at a concentration of between about 75 mg/ml to about 100 mg/ml. 107. A composition according to any one of the preceding wherein the composition further comprises one or a combination of: a) a buffering agent selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citrate, sodium phosphate, potassium phosphate, sodium citrate, and histidine, providing a pH of between 6.8 and 7.2; and/or b) a sugar; and/or c) polysorbate 80; and/or d) EDTA. 108. A composition according to any one of the preceding claims wherein the composition further comprises one or a combination of: a) a buffering agent selected from the group consisting of sodium phosphate dibasic heptahydrate, phosphate, citrate, citric acid monohydrate, sodium phosphate, potassium phosphate, sodium citrate, and histidine, providing a pH of between 6.2 and 6.6; and/or b) a sugar; and/or c) polysorbate 80; and/or d) EDTA. EXAMPLES Example 1 Preparation of a Composition Multiple batches of a composition comprising the monoclonal anti-IL-5 antibody mepolizumab were produced. An inoculum of Chinese Hamster Ovary cells stably transfected with expression vector constructs comprising the nucleic acid sequences shown SEQ ID NO: 13 and SEQ ID NO: 14 was cultured in 5000 L bioreactors containing a liquid cell culture medium. The mature antibody encoded by these nucleic acids is mepolizumab and comprises the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 2. Bioreactors were operated at a temperature of about 34.5° C. to about 35.5° C. Air and oxygen were sparged into the culture medium and a pH of about 6.75 to 7.00 was maintained. The culture duration was about 12 to 18 days. The in vitro cell age (culture days from initial thaw of master cell bank to harvest) was 166 days or less. After this, a clarified cell culture supernatant was harvested by centrifugation and depth filtration of the cell culture medium. This clarified supernatant was then subjected to protein A chromatography and impurities were allowed to flow off this chromatography column. Bound protein including antibody molecules was then eluted from the protein A column, treated at a pH of about 3.3 to 3.7 for about 15 to 240 minutes. This treated preparation was then adjusted to about pH 4.3 to 4.7 and held for about 20 to 1110 minutes. This treated preparation was then clarified through the filtration train of a depth filter and a 0.5/0.2 μm dual layer filter. The filtered preparation was then subjected to Q- SEPHAROSE™ Fast Flow anion exchange chromatography at a load pH of about 8.3 to 8.7 and eluted from the chromatography column. This eluate was then held for about 96 hours or less. Guanidine and ammonium sulfate were then added. Guanidine was added to a concentration of about 1.8 M to 2.2 M and held for about 15 to 240 minutes. This solution was then subjected to phenyl SEPHAROSE™ fast flow chromatography at a load ratio of about 12 g protein/L resin to about 27 g/L resin, an elution gradient volume (bed volumes; BV) of about 9 to about 11, and elution peak cut stop (% of maximum peak height) of about 17 to about 23. Virus filtration was then performed using a Planova 20N virus removal filter. This filtrate was then adjusted to a target concentration of about 46 g protein/L to about 66 g protein/L and the bulk drug substances (BDS) were formulated by tangential filtration and ultrafiltration exchange with a solution comprising about 20 mM sodium phosphate dibasic heptahydrate and 12% weight of sucrose to volume. This solution was then adjusted to a target concentration of 76 g protein/L to about 82 g protein/L and about 0.05% weight of polysorbate 80 to volume was added. This solution was then filtered through 0.5/0.2 μm PES filters and containers of the solution were filled and frozen. Drug product was manufactured using a sterile manufacturing process involving thawing and combining bulk containers followed by filtration of bulk into vials, lyophilization, stoppering and crimping by manufacturing processes well known in the art. The final drug product presentation is a lyophilized drug product in a single use vial. Lyophilizate from each batch produced was reconstituted by the addition of water to produce a composition comprising 100 mg/mL of protein, the monoclonal anti-IL-5 mepolizumab antibody and 26 mM sodium phosphate dibasic heptahydrate, 15% weight of sucrose to volume and 0.065% weight of polysorbate 80 to volume at a pH of from about 6.8 to about 7.2. Example 2 Characterization of the Composition Samples from the batches of composition comprising a monoclonal anti-IL-5 antibody produced as described above were characterized. Capillary isoelectric focusing (cIEF) consistently showed the presence of six antibody isoforms in the composition (e.g., composition reference standard (RS) 101245722). See FIG. 1. These isoforms are the peak 65, peak 78, peak 88, peak 92, main peak and peak 112 isoforms shown in FIG. 1. Samples of the composition were subjected to cIEF using standard methods. pI 7.9 and pI 9.46 standards were included in samples to be analyzed by cIEF. The cIEF electropherogram shown in FIG. 1 is respresentative of those for the composition from multiple batches. The electropherogram shows the composition comprises a main form, acidic forms and basic forms of the antibody. The main form can be seen in FIG. 1 and is also identified as peak 100 in some instances. The acidic forms of the antibody correspond to the peak 65, peak 78, peak 88 and peak 92 forms of FIG. 1. The basic forms of the antibody correspond to the peak 112 forms of FIG. 1. The formula used for assignment of peaks is as follows: peakname=int{(pIPeak-8)/(pIMain-8)*100} where: int=integer and pIPeak=pI of the peak to be named. Table 7 shows the peak naming convention for cIEF electropherograms of the composition comprising a monoclonal anti-IL-5 antibody. The name of the peaks determined as described here should be verified based on the observed electrophoretic/chromatographic pattern. Peaks that do not fall within the ranges described here should be processed according to the formula above. TABLE 7 Identification of cIEF peaks Peak Peak Peak Peak Peak Peak Peak Peak Peaks with Retention times 62 75 85 89 100 103 109 120 greater than Rention times 68 81 91 95 100 109 115 126 less than Report as Peak Peak Peak Peak Peak Peak Peak Peak Peak 65 78 88 92 100 106 112 123 Integral analysis of the electropherogram peaks was performed. See Table 8. TABLE 8 Total peak area in Select cIEF Electropherograms of Different Composition Batches. MDS2 Batch MDS1 Batch T0414001 T0414002 T0414003 T04L009 T04M001 T04N002 (PPQ11) (PPQ12) (PPQ13) Charge Isoforms by cIEF Peak area Peak area Peak area Peak area Peak area Peak area 61.2% for 63.9% for 60.6% for 58.1% for 61.8% for 62.3% for main; main; main; main; main; main; 37.5% for 34.4% for 38.0% for 37.1% for 34.1% for 33.0% for total acidic; total acidic; total acidic; total acidic; total acidic; tota lacidic;  1.2% for  1.6% for  1.3% for  4.9% for  4.0% for  4.7% for total basic total basic total basic total basic total basic total basic This showed the main form represented greater than or equal to 50.0% of the total peak area in the samples (with values between from about 58.1% and 62.3% being observed as well). This also showed the acidic forms represented less than or equal to 45.0% of the total peak area in the samples (with values of between from about 20% to about 45% such as 32.2% and 40.7% being observed as well). The basic forms represented from about 1% to about 15% of the total peak area in the samples (with values of between from about 1.2% to about 4.9% being observed as well). The main form, acidic forms and basic forms peak fractions produced by cIEF were then further analyzed by weak cation exchange (WCX), trypsin peptide mapping and Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS) analyses. Standard methods were used for these analyses. These WCX, trypsin peptide mapping and LC-MS/MS results showed that the main form peak fraction contained two IgG1 mAb modifications. Thus, in the antibody heavy chain amino acid sequence of SEQ ID NO: 99.6% of the N-terminal glutamine (Gln, Q) was cyclized to pyroglutamic acid (pGlu) and 99.9% of of the heavy chain (HC) C-terminal lysine (Lys, K) 449 was cleaved. Typically, the pGlu levels in the batches tested were ≥95.0% and the level of HC without C-terminal K449 levels was ≥98.0%. These WCX, trypsin peptide mapping and LC-MS/MS results also showed for the acid forms peaks that a one-Dalton mass shift characteristic of deamidation was observed. Peptide mapping LC-MS/MS demonstrated the acidic forms peaks contain a mixture of deamidated antibody species. Deamidation was predominantly observed at HC N386 of the amino acid sequence shown in SEQ ID NO: 1 and at LC N31 of the amino acid sequence shown in SEQ ID NO: 2. Lower levels of deamidation were also observed at HC N317 of the amino acid sequence shown in SEQ ID NO: 1. In its entirety, this experimental data showed asparagine residues HC N317, HC N386, HC N299 of the amino acid sequence shown in SEQ ID NO: 1 and LC N31 of the amino acid sequence shown in SEQ ID NO: 2 were susceptible to deamidation. These WCX, trypsin peptide mapping and LC-MS/MS showed for the basic forms peaks that the antibody forms in this peak had at least one heavy chain, carboxy terminal lysine amino acid residue intact. Antibody species with intact lysines, relative to other forms in which these are absent, will migrate in the basic region due to additional positive charges from these residues. Thus, the basic forms, such as peak 112, correspond to antibody forms in which one, or both, heavy chain amino acid sequences have the carboxy terminal lysine amino acid sequence shown in SEQ ID NO: 1 intact. Primary sequencing of the composition comprising a monoclonal anti-IL-5 antibody was also performed by standard LC-MS/MS techniques. These analyses examined the primary struction and amino acid sequence of the antibody molecules in the composition. In particular, these analyses showed which amino acid residues were deamidated, oxidized, cyclized or absent in the anti-IL-5 antibody and the percentage of these in the population of anti-IL-5 antibodies (e.g., expressed from the nucleic acid sequence of SEQ ID NO: 13 and the nucleic acid sequence of SEQ ID NO: 14) present in the composition. See Table 9 TABLE 9 Primary antibody sequence by peptide mapping LC-MS/MS. MDS2 Batch MDS1 Batch T0414001 T0414002 T0414003 T04L009 T04M001 T04N002 (PPQ11) (PPQ12) (PPQ13) Primary Sequence by Peptide Mapping LC-MS/NIS Deamidation Deamidation Deamidation Deamidation Deamidation Deamidation 1.0% of heavy 1.1% of HC 1.1% of HC 1.1% of HC 1.2% of HC 1.1% of HC chain (HC or N317; N317; N317; N317; N317; H) asparagine (N) 317; 1.9% of HC 2.2% of HC 1.6% of HC 1.7% of HC 1.6% of HC 1.9% of HC N386; N386; N386; N386; N386; N386; 5.8% of light 6.5% of LC 6.2% of LC 5.6% of LC 6.5% of LC 6.2% of LC chain (LC or L) N31 N31 N31 N31 N31 N31 HC 1-5 pGlu HC 1-5 pGlu HC 1-5 pGlu HC 1-5 pGlu HC 1-5 pGlu HC 1-5 pGlu 93.7%; 94.6%; 94.0%; 93.7%; 94.6%; 95.3%; HC 449 Lys HC 449 Lys HC 449 Lys HC 449 Lys HC 449 Lys HC 449 Lys Clipped Clipped Clipped Clipped Clipped Clipped 99.2% 98.4% 97.6% 99.2% 98.9% 98.5% Oxidation Oxidation = Oxidation = Oxidation = Oxidation = Oxidation = 0.9% of HC 1.0% of HC 0.8% of HC 0.7% of HC 0.8% of HC 0.7% of HC methionine M64; M64; M64; M64; M64; (M) 64; 1.1% of HC 0.7% of HC 0.8% of HC 0.7% of HC 0.7% of HC 0.7% of HC M82/85; M82/85 ; M82/85; M82/85; M82/85; M82/85; 3.0% of HC 2.9% of HC 3.1% of HC 2.6% of HC 2.7% of HC 2.7% of HC M254; M254; M254; M254; M254; M254; 0.7% of HC 0.5% of HC 0.5% of HC 0.4% of HC 0.4% of HC 0.5% of HC M360; M360; M360; M360; M360; M360; 0.6% of HC 0.6% of HC 0.6% of HC 0.5% of HC 0.5% of HC 0.6% of HC M430; M430; M430; M430; M430; M430; 0.8% of LC 0.4% of LC 0.5% of LC 0.3% of LC 0.4% of LC 0.5% of LC M4 M4 M4 M4 M4 M4 Antibody Variants Mepolizumab binds to soluble IL-5 and blocks the soluble IL-5 from binding to its receptor. The structure of IL-5 is indicative of a secreted protein and there is no evidence of any membrane-bound forms of IL-5 on any cell types. Thus, the Fc effector functions are not part of the mepolizumab Mechanism Of Action (MOA). Based on the MOA and PK properties of mepolizumab, there are two functions involved in the biological activity of this antibody: binding to IL-5 via the CDRs, and binding to FcRn receptor via the Fc region. Through the extensive characterization studies performed above and as set out below, it was determined that at least deamidaton, oxidation, and aggregation can lead to antibody variants in the composition of mepolizumab, and that these variants can impact the function of mepolizumab. Specific levels of these variants should be maintained to ensure appropriate biological function. Function is herein described within the acceptable range of 0.70-1.30 specific antigen binding activity (IL5-binding) and 70%-130% FcRn binding. Thus the steps to identify antibody variants that impact function include: (i) is the antibody variant formed, (ii) does the variant have an impact on function, and (iii) what level of variant can result in a functional composition. Function IL-5 binding: A statistical analysis was performed to calculate the acceptable antigen binding functional activity range using all drug substance (DS) and drug product (DP) release and stability data generated to date. The calculated statistical range was compared to clinical experience and evaluated based on the known impact of product related variants on potency. Based on this analysis, the acceptable antigen binding functional activity range at time of release and at the end of the shelf-life is specific antigen binding of 0.70-1.30. The IL-5 specific binding was determined by Surface Plasmon Resonance (SPR) using a BIACORE™ instrument, performed in the binding mode. This SPR assay is able to detect decreases in antigen binding that result from changes in mepolizumab and mepolizumab variants linked to potency. SPR is used to determine the specific antigen binding activity of mepolizumab. First, mepolizumab reference standard is injected over the surface of a CM5 sensor chip containing immobilized Protein A and then diluted IL-5 protein at a fixed concentration is injected, enabling the IL-5 to bind to the captured mepolizumab sample. The concentration of mepolizumab bound to IL-5, reported as functional binding of mepolizumab to IL-5, is determined from a corresponding mepolizumab reference standard calibration curve. The SPR result was reported as the functional binding concentration of mepolizumab to IL-5, divided by the total protein concentration. FcRn binding: The Neonatal Fc (FcRn) Receptor Binding activity of mepolizumab was also measured by Surface Plasmon Resonance (SPR) using a BIACORE™ instrument. The acceptable FcRn binding functional activity range was determined to be 70-130%, based on results generated to date during mepolizumab product development, known assay variables, and results generated for similar mAb products. The Fc region of mepolizumab binds to FcRn, and this interaction reflects the long serum half-life of mepolizumab (mean terminal half-life=20 days). In the SPR assay, a nitrilotriacetic acid (NTA) sensor chip containing immobilized FcRn receptors was used to capture a fixed concentration of mepolizumab. First, Ni2+ was injected at a fixed concentration and captured on a NTA sensor chip by chelation of Ni2+ through NTA. Second, FcRn receptor was injected at a fixed concentration and the 6× histidine tag at the C-terminus of the alpha chain of the FcRn receptor binds to the Ni2+ that had been previously captured. Mepolizumab that had been diluted within the standard curve concentration range was then injected over the surface of the NTA sensor chip containing captured FcRn receptor. The concentration of mepolizumab bound to the FcRn receptor was extrapolated from a corresponding mepolizumab reference standard calibration curve. The SPR result was reported as the functional binding concentration of mepolizumab to the FcRn receptor, divided by the total protein concentration. The SPR method for specific antigen binding and FcRn binding uses a reference standard of mepolizumab. The mepolizumab reference standard is simply used in assays to obtain system suitability and sample comparability data, to ensure methods are performing appropriately. The reference standard allows the establishment of a calibration curve and concentrations of the samples are interpolated from the curve. Acidic Variants Forced degradation studies were then performed to determine the impact of increased levels of acidic variants, for example deamidation, on antibody function/efficacy, i.e., antigen binding and FcRn binding activities. In pH 9.0 forced degradation studies the composition was adjusted to pH 9.0 with 6N sodium hydroxide and was incubated for 30 days at 40° C. Samples were collected at 0, 3, 7, 14 and 30 days and were compared with the unstressed composition, which was used as a control. The pH 9.0 stressed samples were then analyzed by cIEF. The results are shown in Table 10 and FIG. 1 (day 0 and day 3). The pH 9.0 stressed composition was degraded beyond the capabilities of the cIEF assay at Day 14; therefore, only results up to the day 7 time point are shown in Table 10. At a stressed condition of pH 9.0 for 3 days, the total acidic region was observed to be 74.4% and 71.9% for two different batches of the composition. TABLE 10 cIEF data summary for pH 9.0 forced degradation studies. Primary Manufacturing Area (%) Process/ Main Total Total Condition Batch Day Peak Acidic Basic Control 62.9 35.9 1.2 Elevated MDS1 0 62.5 36.1 1.4 pH 9.0 T004L003S 3 24.8 74.4 0.8 7 8.3 91.7 0.0 MDS2 0 63.7 33.3 3.0 T0413010 3 26.9 71.9 1.2 7 11.4 88.3 0.3 The pH 9.0 forced degradation study samples of the composition from different batches were then tested for specific antigen binding activity (Table 11) and FcRn binding activity (Table 12) using standard surface plasmon reasonance (SPR) methods. TABLE 11 Data summary of specific antigen binding activity (e.g., human IL-5 binding activity) measured by SPR in pH 9.0 forced degradation study samples of the composition from different batches. Primary Specific Manufacturing Antigen Process/ Binding Condition Batch Day Activity Elevated MDS1 0 0.96 pH 9.0 T04L003S 3 0.74 7 0.60 MDS2 0 0.94 T0413010 3 0.74 7 0.62 TABLE 12 FcRn binding measured by SPR in pH 9.0 forced degradation study samples of the composition from different batches. Primary Manufacturing FcRn Process/ Binding Condition Batch Day (%) Elevated pH MDS1 0 91 9.0 T04L003S 3 84 7 82 MDS2 0 86 T0413010 3 82 7 80 The IL5 specific binding activity at Day 3 (i.e., about 72-74% acidic variant) was 0.74 for both batches of the composition subjected to pH 9.0 forced degradation. The FcRn binding activities were 82% and 80% respectively for both batches of the composition subjected to pH 9.0 forced degradation. These values were within the acceptance criteria for each assay. The acceptance criterion for specific antigen binding activity is 0.70-1.30 and the acceptance criterion for FcRn binding is 70%-130%. Thus, acidic variant can be as high as about 74% to maintain function of the mepolizumab composition. Deamidation Forced degradation studies can determine which residues that appear to be susceptible to deamidation actually deamidate, and whether the deamidated variant has an impact on function, and what levels of deamidation are acceptable within a composition to maintain function. The asparagine residues which were experimentally determined to be susceptible to deamidation are HC N317, HC N386, HC N299, and LC N31. Forced degradation studies were performed to determine the impact of increased levels of deamidation LC N31 in the antibody light chain amino acid sequence shown in SEQ ID NO: 2 on antigen binding activity. In these studies the composition from different batches was adjusted to pH 9.0 with 6N sodium hydroxide and was incubated for 30 days at 40° C. Samples were collected at 0, 3, 7, 14 and 30 days and were compared with unstressed composition (e.g., reference standard) which was used as a control. The pH 9.0 stressed samples were tested by peptide mapping LC-MS/MS. The results are shown in Table 13. When mepolizumab is held at pH 9.0 for 3 days the level of deamidated LC N31 in the antibody light chain amino acid sequence shown in SEQ ID NO: 2 is 17.4% and 16.8% for different batches of the composition. See Table 13. Antigen and FcRN binding data for mepolizumab held at pH 9.0 for 3 days are presented in Table 11 and Table 12. TABLE 13 Percentage deamidation by peptide mapping LC-MS/MS in pH 9.0 forced degradation study samples of the composition from different batches at day 3. Primary Manufacturing Process/ Deamidation (%) Condition Batch HC N317 HC N386 LC N31 HC N299 control 0.8 5.5 5.2 0.2 Elevated MDS1 0.9 28.2 17.4 1.3 pH 9.0 T04L003S MDS2 1.0 27.8 16.8 1.3 T0413010 Therefore at Day 3, the specific antigen binding activity of 0.74 (Table 11), and FcRn binding activity of 84% and 82% (Table 12), show that deamidation at N31 of up to around 17%, and deamidation at N386 of up to around 28% (Table 13), can maintain a functional composition within the acceptable range of 0.70-1.30 specific antigen binding activity and 70%-130% FcRn binding. Oxidation Forced degradation studies were performed to experimentally examine the susceptibility of methionine and other amino acid residues in the antibody heavy and light chains of the composition to oxidation. Forced degradation studies can determine which residues that appear to be susceptible to oxidation actually oxidize, and whether the oxidized variant has an impact on function, and what levels of oxidation are acceptable within a composition to maintain function/efficacy (antigen binding and/or FcRn binding). Samples of the composition were incubated with 0.1% hydrogen peroxide (H2O2) for 48 hours at room temperature (RT) to induce oxidation. Samples were collected at 0, 6, 12, 24, and 48 hours. These were compared with unstressed composition (e.g., reference standard) which was used as a control. It was determined from these studies the methionine (M) residues most prone to oxidation include HC M64, HC M82, HC M85, HC M254, HC M360, HC M430 of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1 and LC M4 of the antibody light chain amino acid sequence shown in SEQ ID NO: 2. The methionine (M) residues most prone to oxidation include M64, which is located in the HC CDR2; M254 and M430, which are located in the FcRn and Protein A binding pocket in the Fc region; and M360 of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1. Methionine residues prone to oxidation to a lesser extent included HC M4, HC M82, and HC M85 of the antibody heavy chain amino acid sequence shown in SEQ ID NO: 1. In addition, LC C220 of the antibody light chain amino acid sequence shown in SEQ ID NO: 2 was determined to be prone to oxidation under chemically induced conditions. Importantly, LC C220 and HC C222 form the inter-chain disulfide bond that joins the heavy and light chains. The levels of sulfoxide resulting from methionine oxidation and of sulfonic acid resulting from cysteine oxidation were measured using peptide mapping LC-MS/MS and is summarized in Table 14. HC M64, M254, M360, and M430 were more than 70% oxidized at 48 hours after incubation with 0.1% hydrogen peroxide (H2O2). TABLE 14 Percentage oxidation determined by peptide mapping LC-MS/MS in study samples of the composition from different batches after treatment with H2O2 for 48 hours. Primary Oxidation (%) Manufac- HC turing HC HC HC HC M82 LC LC Process BDS Batch M64 M254 M360 M430 and 85 M4 C220 Control 0.9 3.5 0.5 0.5 0.3 0.2 0.0 MDS1 T04L003S 72.9 99.9 98.5 95.6 3.2 0.8 7.6 MDS2 T0413010 85.6 99.8 98.1 98.7 3.5 1.0 7.2 Specific antigen binding by the antibodies in the composition was measured by SPR. This demonstrated a greater decrease in specific antigen binding activity at 48 hours in different H2O2 stressed batches as summarized in Table 15. This decrease in antigen binding correlates with the relatively higher levels of M64 oxidation observed by peptide mapping LC-MS/MS. The specific antigen binding activities in the tested samples from the different batches of the composition decreased by approximately 15% and 32%, respectively. When mepolizumab was 85.6% oxidized specific antigen binding activity was still retained at 0.57. The linear relationship between the cumulative levels of oxidation in mepolizumab and specific antigen binding activity was used and it was determined, at worst case, that HC M64 could be approximately 50% oxidized and the antibodies in the composition would still retain the antigen binding activity in the range of 0.70-1.30. TABLE 15 Specific antigen binding activity measured by SPR in different batches of the composition treated with H2O2 for 48 hours. Specific Primary Binding Manufacturing Time Antigen Process/Batch Condition (hours) Activity MDS1 Oxidation 0 0.92 T04L0035 Control 48 0.92 0.1% H2O2 0 0.91 48 0.76 MDS2 Oxidation 0 0.96 T0413010 Control 48 0.96 0.1% H2O2 0 0.89 48 0.57 The FcRn binding activity profiles of H2O2 stressed samples from different batches of the composition were highly similar with a substantial decrease in the FcRn binding activity at 48 hours compared with the controls (untreated reference standard) as shown in Table 16. HC M254 and HC M430 are located in the Fc region and when oxidized have been shown to result in a decrease in FcRn binding. Based on peptide mapping results generated during the forced degradation studies, when the composition is chemically oxidized with H2O2 for 48 hours, the levels of oxidized HC M254 and HC M430 observed in the different batches is ≥95%. The FcRn binding results showed approximately an 80% decrease in antigen FcRn binding in the H2O2 stressed samples from different batches of the composition. When mepolizumab was ≥90% oxidized FcRn binding activity was still retained at 22%. The linear relationship between the cumulative levels of oxidation in mepolizumab and FcRn binding activity was used and it was determined, at worst case, the HC M254 and HC M430 could be 50% oxidized and the antibodies in the composition would still retain the FcRn binding activity in the range of 70%-130%. TABLE 16 FcRn binding activity measured by SPR in different batches of the composition treated with H2O2 for 48 hours. FcRn Primary Binding Manufacturing Time Activity Process/Batch Condition (hours) (%) MDS1 Oxidation 0 97 T04L003S Control 48 94 0.1% H2O2 0 79 48 22 MDS2 Oxidation 0 97 T0413010 Control 48 98 0.1% H2O2 0 77 48 17 A photo stress study was conducted to determine the impact of light induced tryptophan oxidation on the antigen binding activity of the antibodies in different batches of the composition. This showed tryptophan W52 in the antibody heavy chain is prone to oxidation. For these studies the composition from different batches was exposed to 1.8 million lux-hr of visible light over approximately 60 hours at 25° C. to induce photo stress. Samples collected at 0, 3, 7, 14, and 30 hours were compared with an unstressed reference standard of the composition which was used as a control. The levels of di-oxidation/kynureninie resulting from tryptophan oxidation were highly similar in the different batches of light exposure stressed composition as summarized in Table 17. Increases in HC W52 oxidation were detected after 60 hours of light exposure. TABLE 17 Percentage oxidation measured by peptide mapping LC-MS/MS in different batches of the composition after light exposure stress for 60 hours. Primary Manufacturing Oxidation Level (%) Process/Batch W52 (+32 Da) W52 (+4 Da) control 0.1 0.0 MDS1 T04L003S 3.3 3.4 MDS2 T0413010 3.5 4.6 Specific antigen binding activity profiles of the antibodies in the different batches of light exposure stressed composition showed a decrease in specific antigen binding activity over time. This is summarized in Table 18. When mepolizumab was approximately 7% oxidized specific antigen binding activity was still retained at 0.53. The linear relationship between the cumulative levels of tryptophan oxidation in mepolizumab and specific antigen binding activity was used and it was determined, at worst case, W52 could be 3% oxidized and the antibodies in the composition would still retain the antigen binding activity in the range of 0.70-1.30. TABLE 18 Specific Antigen Binding Activity measured by SPR in different batches of the composition after light exposure stress for 60 hours. Primary Manufacturing Specific Antigen Binding Process/Batch Condition Day Activity MDS1 T04L003S Light Exposure 0 0.89 Control 60 0.89 Light Exposed 0 0.89 60 0.53 MDS2 T0413010 Light Exposure 0 0.93 Control 60 0.93 Light Exposed 0 0.93 60 0.55 Thus, to maintain function (IL-5 binding, and/or FcRn binding), HC M64 could be up to 50% oxidized, HC M254 and HC M430 could be up to 50% oxidized, and W52 could be up to 3% oxidized. Aggregation The size distribution of the antibodies in the composition was monitored by using standard non-denaturing size exclusion chromatography (SEC) methods. Three peaks were detected in the RS composition SEC profile as shown in FIG. 3 and FIG. 4. A main peak at 7.9 minutes with a relative percentage area of 99.4% was identified as monomer; a minor peak at approximately 6.7 minutes with a relative percentage area of 0.5% was identified as aggregate. A second minor peak has been observed in some batches, eluting after the main peak which indicates the presence of fragment. Typically this peak is below the SEC assay QL of 0.1. The aggregate peak was further characterized using SEC with multi-angle light scattering (MALS) detection and analytical ultracentrifugation (AUC). Results show that the SEC-MALS profiles contain an early eluting peak (dimer) and a later eluting peak (monomer) as shown in FIG. 5 for the RS composition and FIG. 6 for a different batch of the composition. The line that cross-sects each peak represents the molar mass of the detected species and the position of the dimer peak, which is not readily visible in the chromatograms due to the low abundance of dimer in the samples. The SEC-MALS data was used to calculate the molar mass of the antibody monomers and dimers. The resulting molar mass of the monomers in the RS composition and for a different batch of the composition was comparable to the mepolizumab monomer theoretical mass of 148,760 kDa as shown in Table 19. Variability was detected in the molecular weight observed for the dimer due to the low level of this species present in the sample. TABLE 19 SEC-MALS analysis of the monomers in the RS composition and for a different batch of the composition. Molar mass (kDa) Mepolizumab Sample Monomer Dimer Reference Standard 147 304 RS 101245722 Batch T0413010 147 340 Sedimentation velocity area under the curve (AUC) integral analysis was used as a complementary technique to fraction based methods including SEC to confirm there is no perturbation of self association equilibrium or exclusion of higher order aggregate from the chromatographic separation. The results of AUC analysis demonstrated the c(s) distribution contains one dominant species (main peak), identified as monomer, with a sedimentation coefficient for both the RS batch of the composition and another batch of the composition of 2.81 S; and one aggregate peak, identified as dimer, with a sedimentation coefficient of 4.87 S for the RS batch of the composition and 5.10 S for another batch of the composition as shown in Table 20. The difference in sedimentation coefficient values between the PRS and BDS is not considered substantial and is attributed to the low abundance of dimer within the samples. The only high molecular weight species detected was dimer, which is consistent with SEC-UV and SEC-MALS results. TABLE 20 AUC Analysis of different batches of the composition Mepolizumab Sedimentation Molecular Weight Sample Coefficient kDa Abundance (n = 3) Monomer Dimer Monomer Dimer Monomer Dimer PRS 2.81 4.87 137 336 99.1% 0.9% 101245722 BDS 2.81 5.10 136 353 99.3% 0.7% T0413003 The sedimentation coefficients determined for monomer and dimer were lower than the traditionally observed values for IgG1 monoclonal antibodies. The formulation of mepolizumab contains 12% (w/v) sucrose, resulting in a highly viscous sample which causes the lower sedimentation coefficients observed for these antibody molecules in the composition. The results of the SEC-UV, SEC-MALS, and AUC analysis show that the aggregate species in the composition is antibody dimer. To investigate the impact of aggregate on antigen binding activity, a low pH study was conducted on the composition from different batches. Composition samples from the different batches were adjusted to pH 3.5 with 5N hydrochloric acid. The pH 3.5 adjusted samples were then incubated for 30 days at 40° C. to induce chemical modifications. Samples collected at 0, 3, 7, 14 and 30 days were compared with an unstressed RS sample of the composition, which was used as a control. This showed when the composition is chemically stressed at low pH 3.5, aggregation is one of the primary degradation pathways. SEC degradation profiles of pH 3.5 stressed samples of the composition are shown in FIG. 7 and summarized in Table 21. Under low pH 3.5 stress conditions, different rates of aggregation where observed between the different batches of composition. However, by day 30 both batches of the composition reach equilibrium and exhibit similar levels of aggregation. This difference in aggregation rate between the different batches is attributed to the higher level of covalent dimer versus non-covalent dimer in the batches. The slower aggregation rate observed in the MDS1 batch of the composition is attributed to the higher proportion of non-covalent dimer relative to that of the MDS2 batch; non-covalent dimer associates and dissociates until equilibrium is reached, which may slow the overall rate of aggregate formation. TABLE 21 SEC data summary for an untreated RS batch of the composition and pH stressed batches of the composition. Primary Manufacturing Area (%) Condition Process/Batch Day Monomer Aggregate Fragment control 99.6 0.4 0.0 Low pH 3.5 MDS1 0 99.0 1.0 0.0 T004L003S 3 81.4 14.3 4.2 7 59.3 35.4 5.2 14 47.9 44.4 7.7 30 36.8 51.2 12.0 MDS2 0 98.4 1.6 0.1 T0413010 3 56.5 40.5 3.1 7 48.3 46.9 4.8 14 41.9 50.7 7.4 30 34.0 54.4 11.5 Specific antigen binding activity profiles of samples of the pH 3.5 stressed batches of the composition showed a decrease in specific antigen binding activity over time as summarized in Table 22. TABLE 22 Data summary of specific antigen binding activity measured by SPR in samples of pH stressed batches of the composition. Primary Specific Antigen Binding Manufacturing Activity Condition Process/Batch Day (mg/mL) Low pH 3.5 MDS1 T04L003S 0 0.92 3 0.56 7 0.43 MDS2 T0413010 0 0.95 3 0.57 7 0.43 The FcRn binding activity profiles of samples of the pH 3.5 stressed batches of the composition showed a decrease in FcRn binding activity over time as summarized in Table 23. TABLE 3 Data summary of FcRn binding measured by SPR in pH stressed batches of the composition. Primary Manufacturing FcRn Binding Condition Process/Batch Day (%) Low pH 3.5 MDS1 T04L003S 0 90 3 54 7 46 MDS2 T0413010 0 86 3 51 7 43 In summary, Tables 21-23 show that there is approximately a 50% decrease in antigen binding and FcRn binding when there is approximately 40% aggregate present in the sample. When mepolizumab was approximately 40% aggregated, specific antigen binding activity was still retained at 0.57 and FcRn binding activity was retained at 51% (Table 22 MDS2 Day 3). There was a slightly different degradation profile for aggregate content between MDS1 and MDS2 at Day 3 (Table 21) because of the different ratios of covalent versus non-covalent dimer. The linear relationship between the aggregation in mepolizumab and specific antigen and FcRn binding activities was used from MDS2 and it was determined, at worst case, mepolizumab could be 20% aggregate and the antibodies in the composition would still retain the antigen binding activity in the range of 0.70-1.30 and FcRn binding activity of 70-130%. Therefore, it is possible for the antibodies in the composition comprising mepolizumab to be 20% aggregated and still retain IL-5 binding activity in the range of 0.70-1.30 and FcRn binding activity in the range of 70%-130%. HCP Residual CHO host cell protein levels in the mepolizumab composition are measured using an enzyme-linked immunosorbent assay (ELISA). This method uses antibodies produced against native antigens of the CHO cell line grown under conditions that mimic the production process conditions of mepolizumab. It was determined that for the mepolizumab composition, an acceptable range for HCP content is ≤10 ng/mg. This range is derived from release data generated to date and represents the true analytical and process variability. 37 different batches of drug substance had the following HCP content: 1.1 ng/mg (2 batches), 1.0 ng/mg (5 batches), 0.9 ng/mg (1 batch), 0.8 ng/mg (3 batches), 0.7 ng/mg (1 batch), 0.6 (1 batch), 0.5 ng/mg (1 batch), ≤0.5 ng/mg (4 batches), ≤1 ng/mg (19 batches). In summary, there are two predominant functions involved in the biological activity of mepolizumab: binding to IL-5 via the CDRs, and binding to FcRn receptor via the Fc region. Through the extensive characterization studies performed above, it was determined that particular deamidated antibody variants, particular oxidated antibody variants, and aggregated antibody variants, can impact the function of the composition of mepolizumab. Therefore, specific levels of these variants should be maintained to ensure appropriate function/efficacy. Example 3 Informal Sequence Listing Underlining below identifies CDR sequences, according to the Kabat definition of CDRs, in the variable heavy and variable light chain portions of the antibodies or the nucleic acid sequences encoding these CDR sequences. For example, in SEQ ID NO: 1 the frameworks and CDRs are presented as plaintext framework1, underlined CDR1, plaintext framework2, underlined CDR2, plaintext framework3, underlined CDR3 and plaintext framework4 in order from the amino proximal portion to the carboxy terminal portion of the sequences presented. Asterisks to the right of a character for a single letter amino acid code indicates the amino acid residue to the left is a N-glycosylation site. This scheme is used in SEQ ID NO:s 1-4, 11, 12 and 19-22, etc. for example Amino terminal methionine residues shown in these sequences can be cleaved. Thus, the sequences here showing an amino terminal methionine residue should also be considered to disclose the cleaved versions of these proteins lacking such an amino terminal methionine residue. Nucleic acids sequences are presented as DNA nucleic acid sequences and include “t” nucleic acid residues, the corresponding RNA sequence should also be considered as disclosed such that “t” nucleic acid residues may also be regarded as disclosing a “u” nucleic acid residue. Additionally, the 5′ proximal “atg” start codon and the 3′ proximal “taa,” “tag,” and “tga” stop codons have been omitted from the cDNA nucleic acid sequences below. This is the case for SEQ ID NO:s 31-34, etc. for example. MEPOLIZUMAB FULL LENGTH HEAVY CHAIN SEQ ID NO: 1 QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVI WASGGTDYNSALMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSS LLRLDYWGRGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYN*STYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MEPOLIZUMAB FULL LENGTH LIGHT CHAIN SEQ ID NO: 2 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPK LLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPF TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC MEPOLIZUMAB VH SEQ ID NO: 3 QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVI WASGGTDYNSALMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSS LLRLDYWGRGTPVTVSS MEPOLIZUMAB VL SEQ ID NO: 4 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPK LLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPF TFGGGTKLEIK MEPOLIZUMAB CDRH1 SEQ ID NO: 5 SYSVH MEPOLIZUMAB CDRH2 SEQ ID NO: 6 VIWASGGTDYNSALMS MEPOLIZUMAB CDRH3 SEQ ID NO: 7 DPPSSLLRLDY MEPOLIZUMAB CDRL1 SEQ ID NO: 8 KSSQSLLNSGNQKNYLA MEPOLIZUMAB CDRL2 SEQ ID NO: 9 GASTRES MEPOLIZUMAB CDRL3 SEQ ID NO: 10 QNVHSFPFT HUMAN IL-5 (MATURE PROTEIN) SEQ ID NO: 11 IPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQG IGTLESQTVQGGTVERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQE FLGVMNTEWIIES HUMAN IL-5 RECEPTOR SUBUNIT ALPHA ISOFORM 1 (MATURE PROTEIN) SEQ ID NO: 12 DLLPDEKISLLPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINA PKEDDYETRITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPP GSPGTSIVNLTCTTNTTEDNYSRLRSYQVSLHCTWLVGTDAPEDTQYFLYY RYGSWTEECQEYSKDTLGRNIACWFPRTFILSKGRDWLAVLVNGSSKHSAI RPFDQLFALHAIDQINPPLNVTAEIEGTRLSIQWEKPVSAFPIHCFDYEVK IHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWS QPIYVGNDEHKPLREWFVIVIMATICFILLILSLICKICHLWIKLFPPIPA PKSNIKDLFVTTNYEKAGSSETEIEVICYIEKPGVETLEDSVF DNA ENCODING MEPOLIZUMAB FULL LENGTH HEAVY CHAIN SEQ ID NO: 13 caggttaccctgcgtgaatccggtccggcactagttaaaccgacccagacc ctgacgttaacctgcaccgtctccggtttctccctgacgagctatagtgta cactgggtccgtcagccgccgggtaaaggtctagaatggctgggtgtaata tgggctagtggaggcacagattataattcggctctcatgtcccgtctgtcg atatccaaagacacctcccgtaaccaggttgttctgaccatgactaacatg gacccggttgacaccgctacctactactgcgctcgagatcccccttcttcc ttactacggcttgactactggggtcgtggtaccccagttaccgtgagctca gctagtaccaagggcccatcggtcttccccctggcaccctcctccaagagc acctctgggggcacagcggccctgggctgcctggtcaaggactacttcccc gaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcac accttcccggctgtcctacagtcctcaggactctactccctcagcagcgtg gtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtg aatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatct tgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggg ggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatc tcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagac cctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcc aagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc aaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaa gccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgg gaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttc tatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaac aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctc tatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttc tcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagc ctctccctgtctccgggtaag DNA ENCODING MEPOLIZUMAB FULL LENGTH LIGHT CHAIN SEQ ID NO: 14 gatatcgtgatgacccagtctccagactcgctagctgtgtctctgggcgag agggccaccatcaactgcaagagctctcagagtctgttaaacagtggaaat caaaagaactacttggcctggtatcagcagaaacccgggcagcctcctaag agctcatttacggggcgtcgactagggaatctggggtacctgaccgattca gtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcagg ctgaagatgtggcagtatactactgtcagaatgttcatagattccattcac gttcggcggagggaccaagaggagatcaaacgtactgtggcggcgccatct gtcttcatcacccgccatctgatgagcagttgaaatctggaactgcctctg agtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaa ggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagca ggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaa agcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagagtgt The present invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. 17373537 glaxosmithkline intellectual property (no.2) limited USA B2 Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001. Open Apr 12th, 2022 12:27PM Apr 12th, 2022 12:27PM GlaxoSmithKline Health Care Pharmaceuticals & Biotechnology
nyse:gsk GlaxoSmithKline Apr 5th, 2022 12:00AM Jul 16th, 2019 12:00AM https://www.uspto.gov?id=US11291682-20220405 Delivery of RNA to trigger multiple immune pathways RNA encoding an immunogen is co-delivered to non-immune cells at the site of delivery and also to immune cells which infiltrate the site of delivery. The responses of these two cell types to the same delivered RNA lead to two different effects, which interact to produce a strong immune response against the immunogen. The non-immune cells translate the RNA and express the immunogen. Infiltrating immune cells respond to the RNA by expressing type I interferons and pro-inflammatory cytokines which produce a local adjuvant effect which acts on the immunogen-expressing non-immune cells to upregulate major histocompatibility complex expression, thereby increasing presentation of the translated protein to T cells. The effects on the immune and non-immune cells can be achieved by a single delivery of a single RNA e.g. by a single injection. 11291682 1. A method of raising an immune response in a vertebrate, the method comprising administering to the vertebrate an effective amount to raise the immune response of a pharmaceutical composition comprising liposomes and messenger ribonucleic acid (mRNA) molecules, wherein: (a) (i) at least 80% of the liposomes have a diameter in the range of 20-220 nm, (ii) a polydispersity index of less than 0.2, or (iii) both (i) and (ii); (b) at least half of the mRNA molecules are encapsulated within the liposomes; and (c) the mRNA molecules encode an immunogen and comprise a 7′-methylguanosine and a first 5′ ribonucleotide, the 7′-methylguanosine being linked 5′-to-5′ to the first 5′ ribonucleotide, and the first 5′ ribonucleotide comprising a 2′-methylated ribose. 2. The method of claim 1, wherein the pharmaceutical composition is administered to skeletal muscle tissue. 3. The method of claim 1, wherein the pharmaceutical composition is administered by injection. 4. The method of claim 3, wherein injection is via a needle. 5. The method of claim 1, wherein the pharmaceutical composition is administered intravenously. 6. The method of claim 1, wherein the mRNA molecules are postive-stranded. 7. The method of claim 6, wherein the mRNA molecules further comprise genetic elements that are required for RNA replication and the mRNA molecules are self-replicating mRNA molecules. 8. The method of claim 7, wherein the self-replicating mRNA molecules lack genetic elements that encode gene products necessary for viral particle assembly. 9. The method of claim 1, wherein the immune response is against at least a bacterium polypeptide, a virus polypeptide, a fungus polypeptide, a parasite polypeptide, or a tumor polypeptide. 10. The method of claim 9, wherein the immune response produces neutralizing antibodies. 11. A pharmaceutical composition comprising liposomes and messenger ribonucleic acid (mRNA) molecules, wherein: (a) (i) at least 80% of the liposomes have a diameter in the range of 20-220 nm, (ii) a polydispersity index of less than 0.2, (iii) or both (i) and (ii); (b) at least half of the mRNA molecules are encapsulated within the liposomes; and (c) the mRNA molecules encode an immunogen and comprise a 7′-methylguanosine and a first 5′ ribonucleotide, the 7′-methylguanosine being linked 5′-to-5′ to the first 5′ ribonucleotide, and the first 5′ ribonucleotide comprising a 2′-methylated ribose. 12. The pharmaceutical composition of claim 11, wherein the mRNA molecules positive-stranded. 13. The pharmaceutical composition of claim 12, wherein the RNA molecules further comprise genetic elements that are required for RNA replication and the mRNA molecules are self-replicating mRNA molecules. 14. The method of claim 13, wherein the self-replicating mRNA molecules lack genetic elements that encode gene products necessary for viral particle assembly. 15. The method of claim 1, wherein around 95% of the mRNA molecules are encapsulated within the liposomes. 16. The pharmaceutical composition of claim 11, wherein around 95% of the mRNA molecules are encapsulated within the liposomes. 17. The method of claim 1, wherein at least 80% of the liposomes have a diameter in the range of 20-220 nm and the mRNA molecules comprise a modified nucleotide. 18. The pharmaceutical composition of claim 11, wherein at least 80% of the liposomes have a diameter in the range of 20-220 nm and the mRNA molecules comprise a modified nucleotide. 19. The method of claim 1, the mRNA molecules further comprising a poly (adenosine monophosphate) (poly(A)) tail. 20. The method of claim 19, wherein at least 80% of the liposomes have a diameter in the range of 20-220 nm and the mRNA molecules comprise a modified nucleotide. 21. The method of claim 19, wherein the liposomes further comprise 1,2-diastearoyl-sn-glycero-3-phosphocholine (DSPC). 22. The method of claim 21, wherein at least 80% of the liposomes have a diameter in the range of 20-220 nm and the mRNA molecules comprise a modified nucleotide. 23. The method of claim 19, wherein the immune response is against a virus polypeptide. 24. The method of claim 19, wherein the immunogen comprises an influenza virus immunogen, a flavivirus immunogen, a coronavirus spike polypeptide immunogen, an Epstein-Barr virus (EBV) immunogen, a cytomegalovirus (CMV) immunogen, a Varicella zoster (VZV) immunogen, or a respiratory syncytial virus (RSV) immunogen. 25. The method of claim 24, wherein the mRNA molecules comprise a modified nucleotide. 26. The pharmaceutical composition of claim 11, wherein the immunogen comprises an influenza virus immunogen, a flavivirus immunogen, a coronavirus spike polypeptide immunogen, an EBV immunogen, a CMV immunogen, a VZV immunogen, or a RSV immunogen. 27. The pharmaceutical composition of claim 26, wherein at least 80% of the liposomes have a diameter in the range of 20-220 nm and the mRNA molecules comprise a modified nucleotide. 28. The pharmaceutical composition of claim 26, wherein the mRNA molecules further comprise a poly(A) tail. 29. The pharmaceutical composition of claim 28, wherein the mRNA molecules comprise a modified nucleotide. 30. The pharmaceutical composition of claim 28, wherein the liposomes further comprise DSPC. 31. The method of claim 24, wherein the liposomes further comprise cholesterol. 32. The method of claim 31, wherein the immunogen comprises an influenza A virus immunogen. 33. The method of claim 31, wherein the immunogen comprises a flavivirus immunogen. 34. The method of claim 31, wherein the immunogen comprises a coronavirus spike polypeptide immunogen. 35. The method of claim 31, wherein the immunogen comprises a CMV immunogen. 36. The method of claim 31, wherein the immunogen comprises an EBV immunogen. 37. The method of claim 31, wherein the immunogen comprises a VZV immunogen. 38. The method of claim 31, wherein the immunogen comprises a RSV immunogen. 39. The method of claim 32, wherein the mRNA molecules comprise a modified nucleotide. 40. The method of claim 33, wherein the mRNA molecules comprise a modified nucleotide. 41. The method of claim 34, wherein the mRNA molecules comprise a modified nucleotide. 42. The method of claim 35, wherein the mRNA molecules comprise a modified nucleotide. 43. The method of claim 36, wherein the mRNA molecules comprise a modified nucleotide. 44. The method of claim 37, wherein the mRNA molecules comprise a modified nucleotide. 45. The method of claim 38, wherein the mRNA molecules comprise a modified nucleotide. 46. The method of claim 39, wherein the liposomes further comprise a polyethylene glycol-conjugated (PEG-conjugated) lipid. 47. The method of claim 40, wherein the liposomes further comprise a PEG-conjugated lipid. 48. The method of claim 41, wherein the liposomes further comprise a PEG-conjugated lipid. 49. The method of claim 42, wherein the liposomes further comprise a PEG-conjugated lipid. 50. The method of claim 43, wherein the liposomes further comprise a PEG-conjugated lipid. 51. The method of claim 44, wherein the liposomes further comprise a PEG-conjugated lipid. 52. The method of claim 45, wherein the liposomes further comprise a PEG-conjugated lipid. 53. The method of claim 46, wherein the liposomes further comprise DSPC. 54. The method of claim 47, wherein the liposomes further comprise DSPC. 55. The method of claim 48, wherein the liposomes further comprise DSPC. 56. The method of claim 49, wherein the liposomes further comprise DSPC. 57. The method of claim 50, wherein the liposomes further comprise DSPC. 58. The method of claim 51, wherein the liposomes further comprise DSPC. 59. The method of claim 52, wherein the liposomes further comprise DSPC. 60. The pharmaceutical composition of claim 28, wherein the liposomes further comprise cholesterol. 61. The pharmaceutical composition of claim 60, wherein the immunogen comprises an influenza A virus immunogen. 62. The pharmaceutical composition of claim 60, wherein the immunogen comprises a flavivirus immunogen. 63. The pharmaceutical composition of claim 60, wherein the immunogen comprises a coronavirus spike polypeptide immunogen. 64. The pharmaceutical composition of claim 60, wherein the immunogen comprises a CMV immunogen. 65. The pharmaceutical composition of claim 60, wherein the immunogen comprises an EBV immunogen. 66. The pharmaceutical composition of claim 60, wherein the immunogen comprises a VZV immunogen. 67. The pharmaceutical composition of claim 60, wherein the immunogen comprises a RSV immunogen. 68. The pharmaceutical composition of claim 61, wherein the mRNA molecules comprise a modified nucleotide. 69. The pharmaceutical composition of claim 62, wherein the mRNA molecules comprise a modified nucleotide. 70. The pharmaceutical composition of claim 63, wherein the mRNA molecules comprise a modified nucleotide. 71. The pharmaceutical composition of claim 64, wherein the mRNA molecules comprise a modified nucleotide. 72. The pharmaceutical composition of claim 65, wherein the mRNA molecules comprise a modified nucleotide. 73. The pharmaceutical composition of claim 66, wherein the mRNA molecules comprise a modified nucleotide. 74. The pharmaceutical composition of claim 67, wherein the mRNA molecules comprise a modified nucleotide. 75. The pharmaceutical composition of claim 68, wherein the liposomes further comprise a PEG-conjugated lipid. 76. The pharmaceutical composition of claim 69, wherein the liposomes further comprise a PEG-conjugated lipid. 77. The pharmaceutical composition of claim 70, wherein the liposomes further comprise a PEG-conjugated lipid. 78. The pharmaceutical composition of claim 71, wherein the liposomes further comprise a PEG-conjugated lipid. 79. The pharmaceutical composition of claim 72, wherein the liposomes further comprise a PEG-conjugated lipid. 80. The pharmaceutical composition of claim 73, wherein the liposomes further comprise a PEG-conjugated lipid. 81. The pharmaceutical composition of claim 74, wherein the liposomes further comprise a PEG-conjugated lipid. 82. The pharmaceutical composition of claim 75, wherein the liposomes further comprise DSPC. 83. The pharmaceutical composition of claim 76, wherein the liposomes further comprise DSPC. 84. The pharmaceutical composition of claim 77, wherein the liposomes further comprise DSPC. 85. The pharmaceutical composition of claim 78, wherein the liposomes further comprise DSPC. 86. The pharmaceutical composition of claim 79, wherein the liposomes further comprise DSPC. 87. The pharmaceutical composition of claim 80, wherein the liposomes further comprise DSPC. 88. The pharmaceutical composition of claim 81, wherein the liposomes further comprise DSPC. 89. The method of claim 32 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 90. The method of claim 33 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 91. The method of claim 55 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 92. The method of claim 35 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 93. The method of claim 36 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 94. The method of claim 37 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 95. The method of claim 38 comprising administering to the vertebrate two or more unit doses of the pharmaceutical composition. 96. The method of claim 89, wherein the vertebrate is a human. 97. The method of claim 90, wherein the vertebrate is a human. 98. The method of claim 91, wherein the vertebrate is a human. 99. The method of claim 92, wherein the vertebrate is a human. 100. The method of claim 93, wherein the vertebrate is a human. 101. The method of claim 94, wherein the vertebrate is a human. 102. The method of claim 95, wherein the vertebrate is a human. 102 CROSS-REFERENCE TO RELATED APPLICATIONS This application is a Continuation of U.S. patent application Ser. No. 15/725,858, filed Oct. 5, 2017, now U.S. Pat. No. 10,532,067, issued Jan. 14, 2020, which is a Divisional of U.S. patent application Ser. No. 13/808,085, filed Mar. 27, 2013, now U.S. Pat. No. 9,801,897, issued Oct. 31, 2017, which is the U.S. National Phase of International Application No. PCT/US2011/043104, filed Jul. 6, 2011 and published in English, which claims the benefit of U.S. Provisional Application No. 61/361,789, filed Jul. 6, 2010, the complete contents of which are hereby incorporated herein by reference for all purposes. TECHNICAL FIELD This invention is in the field of non-viral delivery of RNA for immunisation. BACKGROUND ART The delivery of nucleic acids for immunising animals has been a goal for several years. Various approaches have been tested, including the use of DNA or RNA, of viral or non-viral delivery vehicles (or even no delivery vehicle, in a “naked” vaccine), of replicating or non-replicating vectors, or of viral or non-viral vectors. There remains a need for further and improved nucleic acid vaccines. DISCLOSURE OF THE INVENTION According to the invention, RNA encoding an immunogen is delivered to cells to trigger multiple innate immune response pathways. The delivered RNA triggers both an endosomal innate immunity receptor (e.g. TLR7) and also a cytoplasmic innate immunity receptor (e.g. a RNA helicase such as MDA5 or RIG-I), thereby enhancing the immune response which is elicited when the RNA-encoded immunogen is expressed. Thus the invention provides a method of raising an immune response in a vertebrate, comprising administering an immunogen-encoding RNA to the vertebrate such that the RNA: (i) stimulates an endosomal innate immunity receptor; (ii) stimulates a cytoplasmic innate immunity receptor; and (iii) is translated to provide expression of the immunogen. The invention also provides an immunogen-encoding RNA for use in an in vivo method of raising an immune response in a vertebrate, wherein the method comprises administering the RNA to a vertebrate such that the RNA: (i) stimulates an endosomal innate immunity receptor; (ii) stimulates a cytoplasmic innate immunity receptor; and (iii) is translated to provide expression of the immunogen. The invention also provides the use of an immunogen-encoding RNA in the manufacture medicament for raising an in vivo immune response in a vertebrate, wherein the RNA is prepared for administration to the vertebrate after which it: (i) stimulates an endosomal innate immunity receptor; (ii) stimulates a cytoplasmic innate immunity receptor; and (iii) is translated to provide expression of the immunogen. Administration The invention involves administration of a RNA molecule to a vertebrate. The administration site will usually be muscle tissue, such as skeletal muscle. Alternatives to intramuscular administration include, but are not limited to: intradermal, intranasal, intraocular, subcutaneous, intraperitoneal, intravenous, interstitial, buccal, transdermal, or sublingual administration. Intradermal and intramuscular administration are two preferred routes. Administration can be achieved in various ways. For instance, injection via a needle (e.g. a hypodermic needle) can be used, particularly for intramuscular, subcutaneous, intraocular, intraperitoneal or intravenous administration. Needle-free injection can be used as an alternative. Intramuscular injection is the preferred way of administering RNA according to the invention. Injection into the upper arm, deltoid or thigh muscle (e.g. anterolateral thigh) is typical. The administration site includes non-immune cells, such as muscle cells (which may be multi nucleated and may be arranged into facsimiles) and/or fibroblasts. RNA enters the cytoplasm of these cells after (or while) being administered. Entry can be via endocytosis e.g. across the sarcolemma of a muscle cell, or across the cell membrane of a fibroblast. RNA escapes from the endosomes into the cytoplasm, where it can be bound by RNA helicases (e.g. in the RIG-I-like receptor family i.e. RLRs) such as RIG-I (RLR-1), MDA5 (RLR-2) and/or LGP2 (RLR-3). This binding initiates RLR-mediated signalling, thereby triggering a first innate immune pathway which enhances the immunogenic effect of the delivered RNA. Even if the delivered RNA is single-stranded, it can form double-stranded RNA either during replication or due to its secondary structure, which means that the RNA can also initiate PKR-mediated signalling, again leading to the triggering of a cytoplasmic innate immune pathway. Both RLR-mediated and PKR-mediated signalling can lead to secretion of type I interferons (e.g. interferon α and/or β) by the non-immune cells. The non-immune cells may undergo apoptosis after transfection. RLR-mediated signalling in the non-immune cell in the presence of an expressed immunogen is a potent combination for initiating an effective immune response. The administration site also includes immune cells, such as macrophages (e.g. bone marrow derived macrophages), dendritic cells (e.g. bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells), monocytes (e.g. human peripheral blood monocytes), etc. These immune cells can be present at the time of administration, but will usually infiltrate the site after administration. For example, the tissue damage caused by invasive administration (e.g. caused by a needle at the administration site) can cause immune cells to infiltrate the damaged area. These infiltrating cells will encounter the RNA which is now at the delivery site and RNA can enter the immune cells via endocytosis. Inside the endosomes the RNA can bind to TLR7 (ssRNA), TLR8 (ssRNA) or TLR3. (dsRNA), thereby triggering a second innate immune pathway. These cells may then secrete type I interferons and/or pro-inflammatory cytokines. The RNA can cause this effect via pattern-recognition receptors, such as toll-like receptors (e.g. TLR7), intracellular helicases (e.g. RIG-I), and PKR (dsRNA-dependent protein kinase). The RNA may or may not be translated by the immune cells, and so the immune cells may or may not express the immunogen. If the immunogen is expressed by the immune cell then it may be presented by the immune cell's IHC-I and/or IHC-II. If the immunogen is not expressed by the immune cell then it may instead be captured by the immune cell from other cells (e.g. non-immune cells) which had taken up RNA and expressed the immunogen, and the immunogen can thus be presented by the immune cell's MHC-II and/or MHC-I. Antigen presentation will generally occur in draining lymph nodes after immune cells have migrated away from the administration site. Thus the RNA can separately trigger two innate immune pathways: one via cytoplasmic (e.g. RLR-mediated and/or PKR-mediated) signalling and one via endosomal (e.g. TLR7-mediated) signalling. These two separate triggers create an immunostimulatory environment which enhances the immune response which is elicited when the RNA-encoded immunogen is expressed as a polypeptide. The two triggers may be provided by the same cell type or by different cell types e.g. the first trigger could be in a fibroblast whereas the second trigger could be in a plasmacytoid dendritic cell. Where the two triggers are provided by the same cell type, they may even be provided by the same single cell. Usually, however, the two triggers are provided by different cell types. In some embodiments the first trigger (RLR-mediated signalling) occurs in TLR7-negative cells and the second trigger (TLR7-mediated signalling) occurs in RIG-I-negative cells (or, more generally, in RLR-negative cells). The ability of a RNA to stimulate an endosomal innate immunity receptor such as TLR7, or to a cytoplasmic innate immunity receptor such as RIG-I, can be directly detected by known in vitro assays. Indirect detection of the RNA/receptor interaction can be based on detection of downstream events which follow receptor stimulation, such as in vitro or in vivo detection of specific cytokine signatures or gene expression signatures associated with particular receptors. It is preferred that RNA “stimulates” an endosomal innate immunity receptor or a cytoplasmic innate immunity receptor by binding to that receptor i.e. the RNA “binds to” the receptor rather than merely “stimulates” it. Assays for binding of RNAs to these receptors are known in the art. The RNA can be delivered as naked RNA (e.g. merely as an aqueous solution of RNA) but, to enhance both entry to immune and non-immune cells and also subsequent intercellular effects, the RNA is preferably administered in combination with a delivery system, such as a particulate or emulsion delivery system. Three useful delivery systems of interest are (i) liposomes (ii) non-toxic and biodegradable polymer microparticles cationic submicron oil-in-water emulsions. Liposomes are a preferred delivery system. Liposomes Various amphiphilic lipids can form bilayers in an aqueous environment to encapsulate a RNA-containing aqueous core as a liposome. These lipids can have an anionic, cationic or zwitterionic hydrophilic head group. Formation of Liposomes from anionic phospholipids dates back to the 1960s, and cationic liposome-forming lipids have been studied since the 1990s. Some phospholipids are anionic whereas other are zwitterionic and others are cationic. Suitable classes of phospholipid include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidyl-glycerols, and some useful phospholipids are listed in Table I. Useful cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-diolcyloxy-N,Ndimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are DPPC, DOPC and dodecylphosphocholine. The lipids can be saturated or unsaturated. The use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail. Liposomes can be formed from a single lipid or from a mixture of lipids. A mixture may comprise (i) a mixture of anionic lipids (ii) a mixture of cationic lipids (iii) a mixture of zwitterionic lipids (iv) a mixture of anionic lipids and cationic lipids (v) a mixture of anionic lipids and zwitterionic lipids (vi) a mixture of zwitterionic lipids and cationic lipids or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids. Similarly, a mixture may comprise both saturated and unsaturated lipids. For example, a mixture may comprise DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated). Where a mixture of lipids is used, not all of the component lipids in the mixture need to be amphiphilic e.g. one or more amphiphilic lipids can be mixed with cholesterol. The hydrophilic portion of a lipid can be PEGylated (i.e. modified by covalent attachment of a polyethylene glycol). This modification can increase stability and prevent non-specific adsorption of the liposomes. For instance, lipids can be conjugated to PEG using techniques such as those disclosed in reference 1 and 2. Various lengths of PEG can be used e.g. between 0.5-8 kDa. A mixture of DSPC, PEG-DMG and cholesterol is used in the examples. Liposomes are usually divided into three groups: multilamellar vesicles (MLV); small unilamellar vesicles (SUV), and large unilamellar vesicles (LUV). MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments. SUVs and LUVs have a single bilayer encapsulating an aqueous core; SUVs typically have a diameter ≤50 nm, and LUVs have a diameter >50 nm. Liposomes useful with of the invention are ideally LUVs with a diameter in the range of 50-220 nm. For a composition comprising a population of LUVs with different diameters: (i) at least 80% by number should have diameters in the range of 20-220 nm, (ii) the average diameter (Zav, by intensity) of the population is ideally in the range of 40-200 nm, and/or (iii) the diameters should have polydispersity index <0.2. The liposome/RNA complexes of reference 37 are expected to have a diameter in the range of 600-800 nm and to have a high polydispersity. Techniques for preparing suitable liposomes are well known in the art e.g. see references 3 to 5. One useful method is described in reference 6 and involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification. Preferred liposomes of the invention are obtainable by this mixing process. RNA is preferably encapsulated within the liposomes, and so the liposome forms a outer layer around an aqueous RNA-containing core. This encapsulation has been found to protect RNA from RNase digestion. The liposomes can include some external RNA (e.g. on the surface of the liposomes), but at least half of the RNA (and ideally all of it) is encapsulated. Polymeric Microparticles Various polymers can form microparticles to encapsulate or adsorb RNA. The use of a substantially non-toxic polymer means that a recipient can safely receive the particles, and the use of a biodegradable polymer means that the particles can be metabolised after delivery to avoid long-term persistence. Useful polymers are also sterilisable, to assist in preparing pharmaceutical grade formulations. Suitable non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof. In some embodiments, the microparticles are formed from poly(α-hydroxy acids), such as a poly(lactides) (“PLA”), copolymers of lactide and glycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those having a lactide/glycolide molar ratio ranging, for example, from 20:80 to 80:20 e.g. 25:75, 40:60, 45:55, 50:50, 55:45, 60:40, 75:25. Useful PLG polymers include those having a molecular weight between, for example, 5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000, 30,000-40,000, 40,000-50,000 Da. The microparticles ideally have a diameter in the range of 0.02 μm to 8 μm. For a composition comprising a population of microparticles with different diameters at least 80% by number should have diameters in the range of 0.03-7 μm. Techniques for preparing suitable microparticles are well known in the art e.g. see references 5, 7 (in particular chapter 7) and 8. To facilitate adsorption of RNA, a microparticle may include a cationic surfactant and/or lipid e.g. as disclosed in references 9 & 10. An alternative way of making polymeric microparticles is by molding and curing e.g. as disclosed in reference 11. Microparticles of the invention can have a zeta potential of between 40-100 mV. One advantage of microparticles over liposomes is that they are readily lyophilized stable storage. RNA can be adsorbed to the microparticles, and adsorption is facilitated by including cationic materials (e.g. cationic lipids) in the microparticle. Oil-in-Water Cationic Emulsions Oil-in-water emulsions are known for adjuvanting influenza vaccines e.g. the MF59™ adjuvant in the FLUADT™ product, and the AS03 adjuvant in the PREPANDRIX™ product. RNA delivery according to the present invention can utilise an oil-in-water emulsion, provided that the emulsion includes one or mote cationic molecules. For instance, a cationic lipid can be included in the emulsion to provide a positive droplet surface to which negatively-charged RNA can attach. The emulsion comprises one or more oils. Suitable oil(s) include those from, for example, an animal (such as fish) or a vegetable source. The oil is ideally biodegradable metabolisable) and biocompatible. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolisable and so may be used. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolisable oils which may be readily recovered. For example, cod liver oil shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Preferred emulsions comprise squalene, a shark liver oil which is a branched, unsaturated terpenoid (C30H50; [(CH3)2C[═CHCH2CH2C(CH3)]2═CHCH2—]2; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN 7683-64-9). Squalane, the saturated analog to squalene, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Other useful oils are the tocopherols, particularly in combination with squalene. Where the oil phase of an emulsion includes a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols are preferred. D-α-tocopherol and DL-α-tocopherol can both be used. A preferred α-tocopherol is DL-α-tocopherol. An oil combination comprising squalene and a tocopherol (e.g. DL-α-tocopherol) can be used. The oil in the emulsion may comprise a combination of oils e.g., squalene and at least one further oil. The aqueous component of the emulsion can be plain water (e.g. w.f.i.) or can include further components e.g. solutes. For instance, it may include salts to form a buffer e.g. citrate or phosphate salts, such as sodium salts. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. A buffered aqueous phase is preferred, and buffers will typically be included in the 5-20 mM range. The emulsion also includes a cationic lipid. Preferably this lipid is a surfactant so that it can facilitate formation and stabilisation of the emulsion. Useful cationic lipids generally contains a nitrogen atom that is positively charged under physiological conditions e.g. as a tertiary or quaternary amine. This nitrogen can be in the hydrophilic head group of an amphiphilic surfactant. Useful cationic lipids include, but are not limited to: 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), 3′-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol (DC Cholesterol), dimethyldioctadecyl-ammonium (DDA e.g. the bromide), 1,2-Dimyristoyl-3-Trimethyl-AmmoniumPropane (DMTAP), dipalmitoyl(Cl6:0)trimethyl ammonium propane (DPTAP), disteamyltrimethylammonium propane (DSTAP). Other useful cationic lipids are: benzalkonium chloride (BAK), benzethonium chloride, cetramide (which contains tetradecyltrimethylammonium bromide and possibly small amounts of dedecyltrimethylammonium bromide and hexadecyltrimethyl ammonium bromide), cetylpyridinium chloride (CPC), cetyl trimethylammonium chloride (CTAC), N,N′,N′-polyoxyethylene (10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammonium methoxide, ceryldimethylethylammonium bromide, dimethyldioctadecyl ammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl trioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3tetramethylbutyl)-phenoxyl-ethoxy)ethyl]-benzenemetha-naminium chloride (DEBDA), dialkyldimetylammonium salts, [1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonium) propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3-(dimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2 dioleoyl 3-succinyl-sn-glycerol choline ester, cholesteryl (4′-trimethylammonium) butanoate), N-alkyl pyridinium salts (e.g. cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic bolaform electrolytes (Cl2Me6; Cl2BU6), dialkylglycetylphosphorylcholine, lysolecithin, L-α dioleoylphosphatidylethanolamine, cholesterol hemisuccinate choline ester, lipopolyamines, including but not limited to dioctadecylamidoglycylspermine (DOGS), dipalmitoyl phosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine (LPLL, LPDL), poly (L (or D)-lysine conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate ester with pendant amino group (C12GluPhCnN+), ditetradecyl glutamate ester with pendant amino group (C12GluPhCnN+), cationic derivatives of cholesterol, including but not limited to cholesteryl-3 β-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3 β-oxysuccinamidoethylene-dimethylamine, cholesteryl-3 β-carboxyamidoethylenetrimethylammonium salt, and cholesteryl-3 β-carboxyamidoethylenedimethylamine. Other useful cationic lipids are described in refs. 12 & 13. The cationic lipid is preferably biodegradable (metabolisable) and biocompatible. In addition to the oil and cationic lipid, an emulsion can include a non-ionic surfactant and/or a zwitterionic surfactant. Such surfactants include, but are not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (BO), propylene oxide (PO), and/or butylene oxide (BC)), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40), phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for including in the emulsion are polysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. Mixtures of these surfactants can be included in the emulsion e.g. Tween 80/Span 85 mixtures, or Tween 80/Triton-X100 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol. Useful mixtures can comprise a surfactant with a HLB value in the range of 10-20 (e,g. polysorbate 80, with a HLB of 15.0) and a surfactant with a HLB value in the range of 1-10 (e.g. sorbitan trioleate, with a HLB of 1.8). Preferred amounts of oil (% by volume) in the final emulsion are between 2-20% e.g. 5-15%, 6-14%, 7-13%, 8-12%, A squalene content of about 4-6% or about 9-11% is particularly useful. Preferred amounts of surfactants (% by weight) in the final emulsion are between 0.001% and 8%. For example: polyoxyethylene, sorbitan esters (such as polysorbate 80) 0.2 to 4%, in particular between 0.4-0.6%, between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%, between 1.9-2.1%, about 2%, or 0.85-0.95%, or about 1%; sorbitan esters (such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5% or about 1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%. The absolute amounts of oil and surfactant, and their ratio, can be varied within wide limits while still forming an emulsion. A skilled person can easily vary the relative proportions of the components to obtain a desired emulsion, but a weight ratio of between 4:1 and 5:1 for oil and surfactant is typical (excess oil). An important parameter for ensuring immunostimulatory activity of an emulsion, particularly in large animals, is the oil droplet size (diameter). The most effective emulsions have a droplet size in the submicron range. Suitably the droplet sizes will be in the range 50-750 nm. Most usefully the average droplet size is less than 250 nm e.g. less than 200 nm, less than 150 nm. The average droplet size is usefully in the range of 80-180 nm. Ideally, at least 80% (by number) of the emulsion's oil droplets are less than 250 nm in diameter, and preferably at least 90%. Apparatuses for determining the average droplet size in an emulsion, and the size distribution, are commercially available. These these typically use the techniques of dynamic light scattering and/or single-particle optical sensing e.g. the Accusizer™ and Nicomp™ series of instruments available from Particle Sizing Systems (Santa Barbara, USA), or the Zetasizer™ instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan). Ideally, the distribution of droplet sizes (by number) has only one maximum i.e. there is a single population of droplets distributed around an average (mode), rather than having two maxima. Preferred emulsions have a polydispersity of <0.4 e.g. 0.3, 0.2, or less. Suitable emulsions with submicron droplets and a narrow size distribution can be obtained by the use of microfluidisation. This technique reduces average oil droplet size by propelling streams of input components through geometrically fixed channels at high pressure and high velocity. These streams contact channel walls, chamber walls and each other. The results shear, impact and cavitation forces cause a reduction in droplet size. Repeated steps of microfluidisation can be performed until an emulsion with a desired droplet size average and distribution are achieved. As an alternative to microfluidisation, thermal methods can be used to cause phase inversion, as disclosed in reference 14. These methods can also provide a submicron emulsion with a tight particle size distribution. Preferred emulsions can be filter sterilised i.e. their droplets can pass through a 220 nm filter. As well as providing a sterilisation, this procedure also removes any large droplets in the emulsion. In certain embodiments, the cationic lipid in the emulsion is DOTAP. The cationic oil-in-water emulsion may comprise from about 0.5 mg/ml to about 25 mg/ml DOTAP. For example, the cationic oil-in-water emulsion may comprise DOTAP at from about 0.5 mg/ml to about 25 mg/ml, from about 0.6 mg/ml to about 25 mg/ml, from about 0.7 mg/ml to about 25 mg/ml, from about 0.8 mg/ml to about 25 mg/ml, from about 0.9 mg/ml to about 25 mg/ml, from about 1.0 mg/ml to about 25 mg/ml, from about 1.1 mg/ml to about 25 mg/ml, from about 1.2 mg/ml to about 25 mg/ml, from about 1.3 mg/ml to about 25 mg/ml, from about 1.4 mg/ml to about 25 mg/ml, from about 1.5 mg/ml to about 25 mg/ml, from about 1.6 mg/ml to about 25 mg/ml, from about 1.7 mg/ml to about 25 mg/ml, from about 0.5 mg/ml to about 24 mg/ml, from about 0.5 mg/ml to about 22 mg/ml, from about 0.5 mg/ml to about 20 mg/ml, from about 0.5 mg/ml to about 18 mg/ml, from about 0.5 mg/ml to about 15 mg/ml, from about 0.5 mg/ml to about 12 mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 0.5 mg/ml to about 5 mg/ml, from about 0.5 mg/ml to about 2 mg/ml, from about 0.5 mg/ml to about 1.9 mg/ml, from about 0.5 mg/ml to about 1.8 mg/ml, from about 0.5 mg/ml to about 1.7 mg/ml, from about 0.5 mg/ml to about 1.6 mg/ml, from about 0.6 mg/ml to about 1.6 mg/ml, from about 0.7 mg/ml to about 1.6 mg/ml., from about 0.8 mg/ml to about 1.6 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 12 mg/ml, about 18 mg/ml, about 20 mg/ml, about 21.8 mg/ml, about 24 mg/ml, etc. In an exemplary embodiment, the cationic oil-in-water emulsion comprises from about 0.8 mg/ml to about 1.6 mg/ml DOTAP, such as 0.8 mg/ml, 1.2 mg/ml, 1.4 mg/ml or 1.6 mg/ml. In certain embodiments, the cationic lipid is DC Cholesterol. The cationic oil-in-water emulsion may comprise DC Cholesterol at from about 0.1 mg/ml to about 5 mg/ml DC Cholesterol. For example, the cationic oil-in-water emulsion may comprise DC Cholesterol from about 0.1 mg/ml to about 5 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5 mg/ml to about 5 mg/ml, from about 0.62 mg/ml to about 5 mg/ml, from about 1 mg/ml to about 5 mg/ml, from about 1.5 mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.46 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, from about 4.5 mg/ml to about 5 mg/ml, from about 0.1 mg/ml to about 4.92 mg/ml, from about 0.1 mg/ml to about 4.5 mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.46 mg/ml, from about 0.1 mg/ml to about 2 tug/ml, from about 0.1 mg/ml to about 1.5 from about 0.1 mg/ml to about 1 mg/ml, from about 0.1 mg/ml to about 0.62 mg/ml, about 0.15 mg/ml, about 0.3 mg/ml, about 0.6 mg/ml, about 0.62 mg/ml, about 0.9 mg/ml, about 1.2 mg/ml, about 2.46 mg/ml, about 4.92 mg/ml, etc. In an exemplary embodiment, the cationic oil-in-water emulsion comprises from about 0.62 mg/ml to about 4.92 mg/ml DC Cholesterol, such as 2.46 mg/ml. In certain embodiments, the cationic lipid is DDA. The cationic oil-in-water emulsion may comprise from about 0.1 mg/ml to about 5 mg/ml DDA. For example, the cationic oil-in-water emulsion may comprise. DDA at from about 0.1 mg/ml to about 5 mg/ml, from about 0.1 to about 4.5 mg/ml, from about 0.1 mg/ml to about 4 mg/ml, from about 0.1 mg/ml to about 3.5 mg/ml, from about 0.1 mg/ml to about 3 mg/ml, from about 0.1 mg/ml to about 2.5 mg/ml, front about 0.1 mg/ml to about 2 mg/ml, from about 0.1 mg/ml to about 1.5 mg/ml, from about 0.1 mg/ml to about 1.45 mg/ml, from about 0.2 mg/ml to about 5 mg/ml, from about 0.3 mg/ml to about 5 mg/ml, from about 0.4 mg/ml to about 5 mg/ml, from about 0.5 mg/ml to about 5 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, from about 0.73 mg/ml to about 5 mg/ml, from about 0.8 mg/ml to about 5 mg/ml, from about 0.9 mg/ml to about 5 mg/ml, from about 1.0 mg/ml to about 5 mg/ml, from about 1.2 mg/ml to about 5 mg/ml, from about 1.45 mg/ml to about 5 mg/ml, from about 2 mg/ml to about 5 mg/ml, from about 2.5 mg/ml to about 5 mg/ml, from about 3 mg/ml to about 5 mg/ml, from about 3.5 mg/ml to about 5 mg/ml, from about 4 mg/ml to about 5 mg/ml, from about 4.5 mg/ml to about 5 mg/ml, about 1.2 mg/ml, about 1.45 mg/ml, etc. Alternatively, the cationic oil-in-water emulsion may comprise DDA at about 20 mg/ml, about 21 mg/ml, about 21.5 mg/ml, about 21.6 mg/ml, about 25 mg/ml. In an exemplary embodiment, the cationic oil-in-water emulsion comprises from about 0.73 mg/ml to about 1.45 mg/ml DDA, such as 1.45 mg/ml. Certain preferred compositions of the invention for administration to a patient comprise squalene, span 85, polysorbate 80, and DOTAP. For instance: squalene may be present at 5-15 mg/ml; span 85 may be present at 0.5-2 mg/ml; polysorbate 80 may be present at 0.5-2 mg/ml; and DOTAP may be present at 0.1-10 mg/ml. The emulsion can include the same amount (by volume) of span 85 and polysorbate 80. The emulsion can include more squalene than surfactant. The emulsion can include more squalene than DOTAP. The RNA The invention involves in vivo delivery of RNA which encodes an immunogen. The RNA triggers two separate innate immunity pathways and is also translated, leading to expression of the immunogen. The RNA is +-stranded, and so it can be translated without needing any intervening replication steps such as reverse transcription. Preferred +-stranded RNAs are self-replicating. A self-replicating RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself). A self-replicating RNA molecule is thus typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded immunogen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the immunogen. The overall results of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded immunogen becomes a major polypeptide product of the cells. One suitable system for achieving self-replication is to use an alphavirus-based RNA replicon. These +-stranded replicons are translated after delivery to a cell to give of a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic −-strand copies of the +-strand delivered RNA. These −-strand transcripts can themselves be transcribed to give further copies of the +-stranded parent RNA and also to give a subgenomic transcript which encodes the immunogen. Translation of the subgenomic transcript thus leads to in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons can use a replicase from a sindbis virus, a semliki forest virus, an eastern equine encephalitis virus, a venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons [15]. A preferred self-replicating RNA molecule thus encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen. The polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4. Whereas natural alphavirus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, it is preferred that a self-replicating RNA molecule of the invention does not encode alphavirus structural proteins. Thus a preferred self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses are absent from self-replicating RNAs of the invention and their place is taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins. Thus a self-replicating RNA molecule useful with the invention may have two open reading frames. The first (5′) open reading frame encodes a replicase; the second (3′) open reading frame encodes an immunogen. In some embodiments the RNA may have additional (e.g. downstream) open reading frames e.g. to encode further immunogens (see below) or to encode accessory polypeptides. A self-replicating RNA molecule can have a 5′ sequence which is compatible with the encoded replicase. Self-replicating RNA molecules can have various lengths but they are typically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or 9000-12000 nucleotides. Thus the RNA is longer than seen in siRNA delivery. A RNA molecule useful with the invention may have a 5′ cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5′ nucleotide of a RNA molecule useful with the invention may have a 5′ triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5′-to-5′ bridge. A 5′ triphosphate can enhance RIG-I binding. A RNA molecule may have a 3′ poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3′ end. A RNA molecule useful with the invention will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during replication of a single-stranded RNA or within the secondary structure of a single-stranded RNA. A RNA molecule useful with the invention can conveniently be prepared by in vitro transcription (IVT), IVT can use a (cDNA) template created and propagated in plasmid form in bacteria, or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the replicon's poly-A is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5′ nucleotide(s) and in some embodiments these requirements must be matched with the requirements of the encoded replicase, to ensure that the IVT-transcribed RNA can function efficiently as a substrate for its self-encoded replicase. As discussed in reference 16, the self-replicating RNA can include (in addition to any 5′ cap structure) one or more nucleotides having a modified nucleobase. Thus the RNA can comprise m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2′-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine), t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamolyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2′-O-ribosyladenosine (phosphate)); I (inosine); m11 (1-methylinosine); m'Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm (5,2-O-dimethylcytidine); ac4Cm. (N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-O-trimethylguanosine); Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G*(archacosine); D (dihydrouridine); m5Um (5,2′-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); memo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylinethyl-2-O-methyluridine); mem5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); nem5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5 U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcylidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2′-O-methylcytidine); mlGm (1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S-faurinomethyl-2-thiouridine)); imG-14 (4-dimethyl guanosine); imG2 (isoguanosine); or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5 methyluracil 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or an abasic nucleotide. For instance, a self-replicating RNA can include one or more modified pyrimidine nucleobases, such as pseudouridine and/or 5-methylcytosine residues. In some embodiments, however, the RNA includes no modified nucleobases, and may include no modified nucleotides i.e. all of the nucleotides in the RNA are standard A, C, G and U ribonucleotides (except for any 5′ cap structure, which may include a 7′-methylguanosine). In other embodiments, the RNA may include a 5′ cap comprising a 7′-methylguanosine, and the first 1, 2 or 3 5′ ribonucleotides may be methylated at the 2′ position of the ribose. A RNA used with the invention ideally includes only phosphodiester linkages between nucleosides, but in some embodiments it can contain phosphoramidate, phosphorothioate, and/or methylphosphonate linkages. Ideally, administered RNA includes fewer than 10 different species of RNA e.g. 5, 4, 3, or 2 different species; most preferably, a composition includes a single RNA species i.e. all RNA molecules in the composition (e.g. within a liposome) have the same sequence and same length. The Immunogen RNA molecules used with the invention encode a polypeptide immunogen. After administration of the RNA the immunogen is translated in vivo and can elicit an immune response in the recipient. The immunogen may elicit an immune response against a bacterium, a virus, a fungus or a parasite (or, in some embodiments, against an allergen; and in other embodiments, against a tumor antigen). The immune response may comprise an antibody response (usually including IgG) and/or a cell-mediated immune response. The polypeptide immunogen will typically elicit an immune response which recognises the corresponding bacterial, viral, fungal or parasite (or allergen or tumour) polypeptide, but in some embodiments the polypeptide may act as a mimotope to elicit an immune response which recognises a bacterial, viral, fungal or parasite saccharide. The immunogen will typically be a surface polypeptide e.g. an adhesin, a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, etc. RNA molecules can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If immunogens are expressed as separate polypeptides then one or more of these may be provided with an upstream IRES or an additional viral promoter element. Alternatively, multiple immunogens may be expressed from a polyprotein that encodes individual immunogens fused to a short autocatalytic protease (e.g. foot-and-mouth disease virus 2A protein), or as inteins. Unlike references 37 and 17, the RNA encodes an immunogen. For the avoidance of doubt, the invention does not encompass RNA which encodes a firefly luciferase or which encodes a fusion protein of E.coli β-galactosidase or which encodes a green fluorescent protein (GFP). Also, the RNA is not total mouse thymus RNA. In some embodiments the immunogen elicits an immune response against one of these bacteria: Neisseria meningitidis: useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding protein. A combination of three useful polypeptides is disclosed in reference 18. Streptococcus pneumoniae: useful polypeptide immunogens are disclosed in reference 19. These include, but are not limited to, the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase precursor (spr0057), spr0096, General stress protein GSP-781 (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA. Streptococcus pyogenes: useful immunogens include, but are not limited to, the polypeptides disclosed in references 20 and 21. Moraxella catarrhalis. Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3. Staphylococcus aureus: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 22, such as a hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and/or the sta011 lipoprotein. Clostridium tetani: the typical immunogen is tetanus toxoid. Corynebacterium diphtheriae: the typical immunogen is diphtheria toxoid. Haemophilus influenzae: Usefill immunogens include, but are not limited to, the polypeptides disclosed in references 23 and 24. Pseudomonas aeruginosa Streptococcus agalactiae: useful immunogens include, but are not limited to, the polypeptides disclosed in reference 20. Chlamydia trachomatis: Useful immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as disclosed in reference 25. LcrE [26] and HtrA [27] are two preferred immunogens. Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 28. Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and/or urease [29]. Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E.coli (UPEC) and meningitis/sepsis-associated E.coli (MNEC). Useful UPEC polypeptide immunogens are disclosed in references 30 and 31. Useful MNEC immunogens are disclosed in reference 32. A useful immunogen for several E.coli types is AcfD [33]. Bacillus anthracis Yersinia pestis: Useful immunogens include, but are not limited to, those disclosed in references 34 and 35. Staphylococcus epidermis Clostridium perfringens or Clostridium botulinums Legionella pneumophila Coxiella burnetii Brucella, such as B.abortus, B.canis, B.melitensis, B.neotomae, B.ovis, B.suis, B.pinnipediae. Francisella, such as F.novicida, F.philomiragia, F.tularensis. Neisseria gonorrhoeae Treponema pallidum Haemophilus ducreyi Enterococcus faecalis or Enterococcus faeciumi Staphylococcus saprophyticus Yersinia enterocolitica Mycobacterium tuberculosis Rickettsia Listeria monocytogenes Vibrio cholerae Salmonella typhi Borrelia burgdorferi Porphyromonas gingivalis Kiebsiella In some embodiments the immunogen elicits an immune response against one of these viruses: Orthomyxovirus: Useful immunogens can be from an influenza A, B or C virus, such as the hemagglutinin, neuraminidase or matrix M2 proteins. Where the immunogen is an influenza A virus hemagglutinin it may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. Paramyxoviridae viruses: Viral immunogens include, but are not limited to, those derived from Pneumoviruses (e.g. respiratory syncytial virus, RSV), Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g. measles). Poxviridae: Viral immunogens include, but are not limited to, those derived from Orthopoxvirus such as Variola vera, including but not limited to, Variola major and Variola minor. Picornavirus: Viral immunogens include, but are not limited to, those derived from Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In one embodiment, the enterovirus is a poliovirus e.g. a type 1, type 2 and/or type 3 poliovirus. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a coxsackie A or B virus. Bunyavirus: Viral immunogens include, but are not limited to, those derived from an Orthobunyavirus, such as California encephalitis virus, a Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever virus. Heparnavirus: Viral immunogens include, hut are not limited to, those derived from a Heparnavirus, such as hepatitis A virus (HAV). Filovirus: Viral immunogens include, but are not limited to, those derived from a Filovirus, such as an Ebola virus (including a Zaire, ivory Coast, Reston or Sudan ebolavirus) or a Marburg virus. Togavirus: Viral immunogens include, but are not limited to, those derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus. This includes rubella virus. Flavivirus: Viral immunogens include, but are not limited to, those derived from a Flavivirus, such as Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus. Pestivirus: Viral immunogens include, but are not limited to, those derived from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV). Hepadnavirus: Viral immunogens include, but are not limited to, those derived from a Hepadnavirus, such as Hepatitis B virus. A composition can include hepatitis B virus surface antigen (HBsAg). Other hepatitis viruses: A composition can include an immunogen from a hepatitis C virus, delta hepatitis virus hepatitis E virus, or hepatitis G virus. Rhabdovirus: Viral immunogens include, but are not limited to, those derived from a Rhabdovirus, such as a Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV). Caliciviridae: Viral immunogens include, but are not limited to, those derived from Calciviridae, such as Norwalk virus (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus. Coronavirus: Viral immunogens include, but are not limited to, those derived from a SARS coronavirus, avian infectious bronchitis (IBV), Mouse hepatitis virus (MEV), and Porcine transmissible gastroenteritis virus (TGEV). The coronavirus immunogen may be a spike polypeptide. Retrovirus: Viral immunogens include, but are not limited to, those derived from an Oncovirus, Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus. Reovirus: Viral immunogens include, but are not limited to, those derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Parvovirus: Viral immunogens include, but are not limited to, those derived from Parvovirus B19. Herpesvirus: Viral immunogens include, but are not limited to, those derived from a human herpesvirus, such as, by way of example only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and 2), Varicella-zoster Virus (VZV), Epstein-Barr virus (EBV, Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). Papovaviruses: Viral immunogens include, but are not limited to, those derived from Papillomaviruses and Polyomaviruses. The (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g, from one or more of serotypes 6, 11, 16 and/or 18. Adenovirus: Viral immunogens include those derived from adenovirus serotype 36 (Ad-36). In some embodiments, the immunogen elicits an immune response against a virus which infects fish, such as: infectious salmon anemia virus (TSAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), koi herpesvirus, salmon picoma-like virus (also known as picorna-like virus of atlantic salmon), landlocked salmon virus (LSV), atlantic salmon rotavirus (ASR), trout strawberry disease virus (TSD), coho salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV). Fungal immunogens may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton favifornie; or from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastontyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi; the less common are Brachiola spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp., Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp. and Cladosporium ssp. In some embodiments the immunogen elicits an immune response against a parasite from the Plasmodium genus, such as P.falciparum, P.vivax, P.malariae or P.ovale. Thus the invention may be used for immunising against malaria. In some embodiments the immunogen elicits an immune response against a parasite from the Caligidae family, particularly those from the Lepeophtheirus and Caligus genera e.g. sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi. In some embodiments the immunogen elicits an immune response against: pollen allergens (tree-, herb, weed-, and grass pollen allergens); insect or arachnid allergens (inhalant, saliva and venom allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera venom allergens); animal hair and dandruff allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales, Pinales and piatanaceac including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), plane tree (Platanus), the order of Poales including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including herbs of the genera Ambrosia, Artemisia, and Parietaria. Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those from mammals such as cat, dog and horse, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (Apidae), wasps (Vespidea), and ants (Formicoidae). In some embodiments the immunogen is a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-1/Melan A, gp100, MClR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein-1/TRPI and tyrosinase related protein-2/TRP2 (associated with, e.g., melanoma); (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer; (1) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6, TAG-72, TLP, TPS, and the like. Pharmaceutical Compositions RNA will be administered as a component in a pharmaceutical composition for immunising subjects against various diseases. These compositions will typically include a pharmaceutically acceptable carrier in addition to the RNA, often as part of a delivery system as described above. A thorough discussion of pharmaceutically acceptable carriers is available in reference 36. A pharmaceutical composition of the invention may include one or more small molecule immunopotentiators. For example, the composition may include a TLR2 agonist (e.g. Pam3CSK4), TLR4 agonist (e.g. an aminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist (e.g. imiquimod), a TLR8 agonist (e.g. resiquimod) and/or a TLR9 agonist (e.g. IC31). Any such agonist ideally has a molecular weight of <2000 Da. Where a RNA is encapsulated, in some embodiments such agonist(s) are also encapsulated with the RNA, but in other embodiments they are unencapsulated. Where a RNA is adsorbed to a particle, in some embodiments such agonist(s) are also adsorbed with the RNA, but in other embodiments they are unadsorbed. Pharmaceutical compositions of the invention may include the particles in plain water (e.g. w.f.i.) or in a buffer e.g. a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will typically be included in the 5-20 mM range. Pharmaceutical compositions of the invention may have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0. Compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9 mg/ml. Compositions of the invention may include metal ion chelators. These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis. Thus a composition may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typically present at between 10-500 μM e.g. 0.1 mM. A citrate salt, such as sodium citrate, can also act as a chelator, while advantageously also providing buffering activity. Pharmaceutical compositions of the invention may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between 290-310 mOsm/kg. Pharmaceutical compositions of the invention may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free compositions are preferred, and preservative-free vaccines can be prepared. Pharmaceutical compositions of the invention are preferably sterile. Pharmaceutical compositions of the invention are preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. Pharmaceutical compositions of the invention are preferably gluten free. Pharmaceutical compositions of the invention may be prepared in unit dose form. In some embodiments a unit dose may have a volume of between 0.1-1.0 ml e.g. about 0.5 ml. The compositions may be prepared as injectables, either as solutions or suspensions. The composition may be prepared for pulmonary administration e.g. by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops. Injectables for intramuscular administration are typical. Compositions comprise an immunologically effective amount of RNA, as well as any other components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The RNA content of compositions of the invention will generally be expressed in terms of the amount of RNA per dose. A preferred dose has ≤10 μg RNA, and expression can be seen at much lower levels e.g. ≤1 μg/dose, ≤100 ng/dose, ≤10 ng/dose, ≤1 ng/dose, etc. The invention also provides a delivery device (e.g. syringe, nebuliser, sprayer, inhaler, dermal patch, etc.) containing a pharmaceutical composition of the invention. This device can be used to administer the composition to a vertebrate subject. RNAs are not delivered in combination with ribosomes and so pharmaceutical compositions of the invention are ribosome-free. Methods of Treatment and Medical Uses RNA delivery according to the invention is for eliciting an immune response in vivo against an immunogen of interest. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. The method may raise a booster response. By raising an immune response the vertebrate can be protected against various diseases and/or infections e.g. against bacterial and/or viral diseases as discussed above. RNA-containing compositions are immunogenic, and are more preferably vaccine compositions. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (e.g. horses, cattle, deer, goats, pigs). Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. Vaccines prepared according to the invention may be used to treat both children and adults. Thus a human patient may be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients for receiving the vaccines are the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65 years), the young (e.g. ≤5 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population. Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue; unlike reference 37, intraglossal injection is not typically used with the present invention), or mucosally, such as by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 ml. The invention may be used to elicit systemic and/or mucosal immunity, preferably to elicit an enhanced systemic and/or mucosal immunity. Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.), In one embodiment, multiple doses may be administered approximately 6 weeks, 10 weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and 14 weeks, as often used in the World Health Organisation's Expanded Program on Immunisation (“EPI”). In an alternative embodiment, two primary doses are administered about two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, e.g. about 6, 8, 10 or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the third primary dose, e.g. about 6, 8, 10, or 12 months after the third primary dose. General Embodiments In some embodiments of the invention, the RNA includes no modified nucleotides (see above). In other embodiments the RNA can optionally include at least one modified nucleotide, provided that one or more of the following features (already disclosed above) is also required: A. Where the RNA is delivered with a liposome, the liposome comprises DSDMA, DODMA, DLinDMA and/or DLenDMA. B. Where the RNA is encapsulated in a liposome, the hydrophilic portion of a lipid in the liposome is PEGylated. C. Where the RNA is encapsulated in a liposome, at least 80% by number of the liposomes have diameters in the range of 20-220 nm. D. Where the RNA is delivered with a microparticle, the microparticle is a non-toxic and biodegradable polymer microparticle. E. Where the RNA is delivered with a microparticle, the microparticles have a diameter in the range of 0.02 μm to 8 μm. F. Where the RNA is delivered with a microparticle, at least 80% by number of the microparticles have a diameter in the range of 0.03-7 μm. G. Where the RNA is delivered with a microparticle, the composition is lyophilized. H. Where the RNA is delivered with an emulsion, the emulsion comprises a biodegradable oil (e.g. squalene). I. Where the RNA is delivered with an emulsion, the emulsion includes one or more cationic molecules e.g. one or more cationic lipids. J. The RNA has a 3′ poly-A tail, and the immunogen can elicits an immune response in vivo against a bacterium, a virus, a fungus or a parasite. K. The RNA is delivered in combination with a metal ion chelator with a delivery system selected from (i) liposomes (ii) non-toxic and biodegradable polymer microparticles (iii) cationic submicron oil-in-water emulsions. General The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 38-44, etc. The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y. The term “about” in relation to a numerical value x is optional and means, for example, x±10%. The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. References to charge, to cations, to anions, to zwitterions, etc., are taken at pH 7. TLR3 is the Toll-like receptor 3. It is a single membrane-spanning receptor which plays a key role in the innate immune system. Known TLR3 agonists include poly(LC) “TLR3” is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:11849. The RefSeq sequence for the human TLR3 gene is GI:2459625. TLR7 is the Toll-like receptor 7. It is a single membrane-spanning receptor which plays a key role in the innate immune system. Known TLR7 agonists include e.g. imiquimod. “TLR7” is the approved IIGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15631. The RefSeq sequence for the human TLR7 gene is GI:67944638. TLR8 is the Toll-like receptor 8. It is a single membrane-spanning receptor which plays a key role in the innate immune system. Known TLR8 agonists include e.g. resiquimod. “TLR8” is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15632. The RefSeq sequence for the human TLR8 gene is GI:20302165. The RIG-I-like receptor (“RLR”) family includes various RNA helicases which play key roles in the innate immune system[45]. RLR-1 (also known as RIG-I or retinoic acid inducible gene I) has two caspase recruitment domains near its N-terminus. The approved HGNC name for the gene encoding the RLR-1 helicase is “DDX58” (for DEAD (Asp-Glu-Ala-Asp) box polypeptide 58) and the unique HGNC ID is HGNC:19102. The RefSeq sequence for the human RLR-1 gene is GI:77732514. RLR-2 (also known as MDA5 or melanoma differentiation-associated gene 5) also has two caspase recruitment domains near its N-terminus. The approved HGNC name for the gene encoding the RLR-2 helicase is “IFIH1” (for interferon induced with helicase C domain 1) and the unique HGNC ID is HGNC:18873. The RefSeq sequence for the human RLR-2 gene is GI: 27886567, RLR-3 (also known as LGP2 or laboratory of genetics and physiology 2) has no caspase recruitment domains. The approved HGNC name for the gene encoding the RLR-3 helicase is “DHX58” (for DEXH (Asp-Glu-X-His) box polypeptide 58) and the unique HGNC ID is HGNC:29517. The RefSeq sequence for the human RLR-3 gene is GI:149408121. PKR is a double-stranded RNA-dependent protein kinase. It plays a key role in the innate immune system. “EIF2AK2” (for eukaryotic translation initiation factor 2-alpha kinase 2) is the approved HGNC name for the gene encoding this enzyme, and its unique HGNC ID is HGNC:9437. The RetSeq sequence for the human PKR gene is GI:208431825. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a gel with stained RNA. Lanes show (1) markers (2) naked replicon (3) replicon after RNase treatment (4) replicon encapsulated in liposome (5) liposome after RNase treatment (6) liposome treated with RNase then subjected to phenol/chloroform extraction. FIG. 2 is an electron micrograph of liposomes. FIG. 3 shows protein expression (as relative light units, RLU) at days 1, 3 and 6 after delivery of RNA as a virion-packaged replicon (squares), naked RNA (triangles), or as microparticles (circles). FIG. 4 shows a gel with stained RNA. Lanes show (1) markers (2) naked replicon (3) replicon encapsulated in liposome (4) liposome treated with RNase then subjected to phenol/chloroform extraction. FIG. 5 shows protein expression at days 1, 3 and 6 after delivery of RNA as a virion-packaged replicon (squares), as naked RNA (diamonds), or in liposomes (+=0.1 μg, x=1 μg). FIG. 6 shows protein expression at days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA. FIG. 7 shows anti-F IgG titers in animals receiving virion-packaged replicon (VRP or VSRP), 1 μg naked RNA, and 1 μg liposome-encapsulated RNA. FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1 μg naked RNA, and 0.1 g or 1 μg liposome-encapsulated RNA. FIG. 9 shows neutralising antibody titers in animals receiving VRP or either 0.1 g or 1 μg liposome-encapsulated RNA. FIG. 10 shows expression levels after delivery of a replicon as naked RNA (circles), liposome-encapsulated RNA (triangle & square), or as a lipoplex (inverted triangle). FIG. 11 shows F-specific IgG titers (2 weeks after second dose) after delivery of a replicon as naked RNA (0.01-1 μg), liposome-encapsulated RNA (0.01-10 μg) or packaged as a virion (VRP, 106 infectious units or IU). FIG. 12 shows F-specific IgG titers (circles) and PRNT titers (squares) after delivery of a replicon as naked RNA (1 μg), liposome-encapsulated RNA (0.1 or 1 μg), or packaged as a virion (VRP, 106 IU). Titers in naïve mice are also shown. Solid lines show geometric means. FIG. 13 shows intracellular cytokine production after restimulation with synthetic peptides representing the major epitopes in the F protein, 4 weeks after a second dose. The y-axis shows the % cytokine+ of CD8+CD4−. FIG. 14 shows F-specific IgG titers (mean log10 titers±std dev over 63 days (FIG. 14A) and 210 days (FIG. 14B) after immunisation of calves. The four lines are easily distinguished at day 63 and are, from bottom to top: PBS negative control; liposome-delivered RNA; emulsion-delivered RNA; and the “Triangle 4” product. FIG. 15 shows (A) IFN-β and (B) IL-6 released by fibroblasts. The graphs include two sets of 4 bars. The left quartet are for control mice; the right quartet are for RNA-immunised mice. The 4 bars in each quartet, from left to right, show data from rig-i+/−, rig-i −/−, mda5 +/− and mda5 −/− mice. Figures are pg/ml. FIG. 16 shows (A) IL-6 and (B) IFNα (pg/ml) released by pDC. There are 4 pairs of bars, from left to right: control; immunised with RNA-1-DOTAP; immunised with RNA-t-lipofeetamine; and immunised with RNA in liposomes. In each pair the black bar is wild-type mice, grey is rsq1 mutant. MODES FOR CARRYING OUT THE INVENTION RNA Replicons Various replicons are used below. In general these are based on a hybrid alphavirus genome with non-structural proteins from venezuelan equine encephalitis virus (VEEV), a packaging signal from sindbis virus, and a 3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10 kb long and has a poly-A tail. Plasmid DNA encoding alphavirus replicons (named: pT7-mvEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for synthesis of RNA in vitro. The replicons contain the alphavirus genetic elements required for RNA replication but lack those encoding gene products necessary for particle assembly; the structural proteins are instead replaced by a protein of interest (either a reporter, such as SEAP or GFP, or an immunogen, such as full-length RSV F protein) and so the replicons are incapable of inducing the generation of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA facilitates the synthesis of the replicon RNA in vitro and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A)-tail generates the correct 3′-end through its self-cleaving activity. Following linearization of the plasmid DNA downstream of the HDV ribozyme with a suitable restriction endonuclease, run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage derived DNA-dependent RNA polymerase. Transcriptions were performed for 2 hours at 37° C. in the presence of 7.5 nM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion). Following transcription the template DNA was digested with TURBO DNase (Ambion). The replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. Uncapped RNA was capped post-transcriptionally with Vaccinia Capping Enzyme (VCE) using the ScriptCap m7G Capping System (Epicentre Biotechnologies) as outlined in the user manual; replicons capped in this way are given the “v” prefix e.g. vA317 is the A317 replicon capped by VCE. Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the RNA samples was determined by measuring OD26nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis. PLG Adsorption Microparticles were made using 500 mg of PLG RG503 (50:50 lactide/glycolide molar ratio, MW ˜30 kDa) and 20 mg DOTAP using an Omni Macro Homogenizer. The particle suspension was shaken at 150 rpm overnight and then filtered through a 40 μm sterile filter for storage at 2-8° C. Self-replicating RNA was adsorbed to the particles. To prepare 1 mL of PLG/RNA suspension the required volume of PLG particle suspension was added to a vial and nuclease-free water was added to bring the volume to 900 μL. 100 μL RNA (10 μg/mL) was added dropwise to the PLG suspension, with constant shaking. PLG/RNA was incubated at room temperature for 30 min. For 1 mL of reconstituted suspension, 45 mg mannitol, 15 mg sucrose and 250-500 μg of PVA were added. The vials were frozen at −80° C. and lyophilized. To evaluate RNA adsorption, 100 μL particle suspension was centrifuged at 10,000 rpm for 5 min and supernatant was collected. PLG/RNA was reconstituted using 1 mL nuclease-free water. To 100 μL particle suspension (1 μg RNA), ling heparin sulfate was added. The mixture was vortexed and allowed to sit at room temperature for 30 min for RNA desorption. Particle suspension was centrifuged and supernatant was collected. For RNAse stability, 100 μL particle suspension was incubated with 6.4 mAU of RNase A at room temperature for 30 min. RNAse was inactivated with 0.126 mAU of Proteinase K at 55° C. for 10 min. 1 mg of heparin sulfate was added to desorb the RNA followed by centrifugation. The supernatant samples containing RNA were mixed with formaldehyde load dye, heated at 65° C. for 10 min and analyzed using a 1% denaturing gel (460 ng RNA loaded per lane). To assess expression, Balb/c mice were immunized with 1 μg RNA in 100 μL intramuscular injection volume (50 μL/leg) on day 0. Sera were collected on days 1, 3 and 6. Protein expression was determined using a chemiluminescence assay. As shown in FIG. 3, expression was higher when RNA was delivered by PLG (triangles) than without any delivery particle (circles). Cationic Nanoemulsion An oil-in-water emulsion was prepared by microfluidising squalene, span 85, polysorbate 80, and varying amounts of DOTAP. Briefly, oil soluble components (squalene, span 85, cationic lipids, lipid surfactants) were combined in a beaker, lipid components were dissolved in organic solvent. The resulting lipid solution was added directly to the oil phase. The solvent was allowed to evaporate at room temperature for 2 hours in a fume hood prior to combining the aqueous phase and homogenizing the sample to provide a homogeneous feedstock. The primary emulsions were passed three to five times through a Microfluidizer with an ice bath cooling coil. The batch samples were removed from the unit and stored at 4° C. This emulsion is thus similar to the commercial MF59 adjuvant, hut supplemented by a cationic DOTAP to provide a cationic nanoemulsion (“CNE”). The final composition of emulsion “CNE1.7” was squalene (4.3% by weight), span 85 (0.5% by weight), polysorbate 80 (0.5% by weight), DOTAP (1.4 mg/ml), in 1.0 mM citrate buffer, pH 6.5. RNA adsorbs to the surface of the oil droplets in these cationic emulsions. To adsorb RNA a RNA solution is diluted to the appropriate concentration in RNase free water and then added directly into an equal volume of emulsion while vortexing lightly. The solution is allowed to sit at room temperature for approximately 2 hours to allow adsorption. The resulting solution is diluted to the required RNA concentration prior to administration. Liposomal Encapsulation RNA was encapsulated in Liposomes made by the method of references 6 and 46. The liposomes were made of 10% DSPC (zwitterionic), 40% DlinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). These proportions refer to the % moles in the total liposome. DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of reference 1. DSPC (1,2-Diastearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol), ammonium salt), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol (3β-[N-(N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids. Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these were mixed with 2 ml of buffer followed by 1 hour of equilibration. The mixture was diluted with 6 ml buffer then filtered. The resulting product contained liposomes, with ˜95% encapsulation efficiency. For example, in one particular method, fresh lipid stock solutions were prepared in ethanol, 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with plenty of MilliQ water before use to decontaminate the vials of RNases. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used had a 2 mm internal diameter and a 3 mm outer diameter; obtained from Idex Health Science). The outlet from the T mixer was also PEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 h. 4 ml of the mixture was loaded into a 5 cc syringe, which was connected to a piece of FEP tubing and in another 5 cc syringe connected to an equal length of FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation). Before using this membrane for the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL, of 100 mM citrate buffer (pH 6) were successively passed through it. Liposomes were warmed for 10 min at 37° C. before passing through the membrane. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using by tangential flow filtration before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez) and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes with a 100 kD pore size cutoff and 8 cm2 surface area were used. For in vitro and in vivo experiments formulations were diluted to the required RNA concentration with 1×PBS. FIG. 2 shows an example electron micrograph of liposomes prepared by these methods. These liposomes contain encapsulated RNA encoding full-length RSV F antigen. Dynamic light scattering of one batch showed an average diameter of 141 nm (by intensity) or 78 nm (by number). The percentage of encapsulated RNA and RNA concentration were determined by Quant-iT RiboGreen. RNA reagent kit (Invitrogen), following manufacturer's instructions. The ribosomal RNA standard provided in the kit was used to generate a standard curve. Liposomes were diluted 10× or 100× in 1×TE buffer (from kit) before addition of the dye. Separately, liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% Triton X before addition of the dye (to disrupt the liposomes and thus to assay total RNA). Thereafter an equal amount of dye was added to each solution and then ˜180 μL of each solution after dye addition was loaded in duplicate into a 96 well tissue culture plate. The fluorescence (Ex 485 nm, Em 528 am) was read on a microplate reader. All liposome formulations were dosed in vivo based on the encapsulated amount of RNA. Encapsulation in liposomes was shown to protect RNA from RNase digestion. Experiments used 3.8 mAU of RNasc A per microgram of RNA, incubated flor 30 minutes at room temperature. RNase was inactivated with Proteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to 25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added to extract the RNA from the lipids into the aqueous phase. Samples were mixed by vortexing for a few seconds and then placed on a centrifuge for 15 minutes at 12 k RPM. The aqueous phase (containing the RNA) was removed and used to analyze the RNA. Prior to loading (400 ng RNA per well) all the samples were incubated with formaldehyde loading dye, denatured for 10 minutes at 65° C. and cooled to room temperature. Ambion Millennium markers were used to approximate the molecular weight of the RNA construct. The gel was run at 90 V. The gel was stained using 0.1% SYBR gold according to the manufacturer's guidelines in water by rocking at room temperature for 1 hour. FIG. 1 shows that RNase completely digests RNA in the absence of encapsulation (lane 3). RNA is undetectable after encapsulation (lane 4), and no change is seen if these liposomes are treated with RNase (lane 4). After RNase-treated liposomes are subjected to phenol extraction, undigested RNA is seen (lane 6). Even after 1 week at 4° C. the RNA could be seen without any fragmentation (FIG. 4, arrow). Protein expression in vivo was unchanged after 6 weeks at 4° C. and one freeze-thaw cycle. Thus liposome-encapsulated RNA is stable. To assess in vivo expression of the RNA a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon, rather than an immunogen. Expression levels were measured in sera diluted 1:4 in 1× Phospha-Light dilution buffer using a chemiluminescent alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group) were injected intramuscularly on day 0, 50 μl per leg with 0.1 μg or 1 μg RNA dose. The same vector was also administered without the liposomes RNase free 1× PBS) at 1 μg. Virion-packaged replicons were also tested, Virion-packaged replicons used herein (referred to as “VRPs”) were obtained by the methods of reference 47, where the alphavirus replicon is derived from the mutant VEEV or a chimera derived from the genome of VEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbis virus packaging signal (PS), packaged by co-electroporating them into BHK cells with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes. As shown in FIG. 5, encapsulation increased SEAP levels by about ½ log at the 1 μg dose, and at day 6 expression from a 0.1 μg encapsulated dose matched levels seen with 1 μg unencapsulated dose, By day 3 expression levels exceeded those achieved with VRPs (squares). Thus expressed increased when the RNA was formulated in the liposomes relative to the naked RNA control, even at a 10× lower dose. Expression was also higher relative to the VRP control, but the kinetics of expression were very different (see FIG. 5). Delivery of the RNA with electroporation resulted in increased expression relative to the naked RNA control, but these levels were lower than with liposomes. To assess whether the effect seen in the liposome groups was due merely to the liposome components, or was linked to the encapsulation, the replicon was administered in encapsulated form (with two different purification protocols, 0.1 μg RNA), or mixed with the liposomes after their formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as naked RNA (1 μg). FIG. 10 shows that the lipoplex gave the lowest levels of expression, showing that shows encapsulation is essential for potent expression. Further SEAP experiments showed a clear dose response in vivo, with expression seen after delivery of as little as 1 μgRNA (FIG. 6). Further experiments comparing expression from encapsulated and naked replicons indicated that 0.01 μg encapsulated RNA was equivalent to 1 μg of naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a 12-fold higher expression at day 6; at a 0.1 μg dose levels were 24-fold higher at day 6. Rather than looking at average levels in the group, individual animals were also studied. Whereas several animals were non-responders to naked replicons, encapsulation eliminated non-responders. Further experiments replaced DlinDMA with DOTAP. Although the DOTAP liposomes gave better expression than naked replicon, they were inferior to the DlinDMA liposomes (2- to 3-fold difference at day 1). To assess in vivo immunogenicity a replicon was constructed to express full-length F protein from respiratory syncytial virus (RSV). This was delivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), or packaged in virions (106 IU; “VRP”) at days 0 and 21. FIG. 7 shows anti-F TgG titers 2 weeks after the second dose, and the liposomes clearly enhance immunogenicity. FIG. 8 shows titers 2 weeks later, by which point there was no statistical difference between the encapsulated RNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group. Neutralisation titers (measured as 60% plaque reduction, “PRNT60”) were not significantly different in these three groups 2 weeks after the second dose (FIG. 9). FIG. 12 shows both IgG and PRNT titers 4 weeks after the second dose. FIG. 13 confirms that the RNA elicits a robust CD8 T cell response. Further experiments compared F-specific IgG titers in mice receiving VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. Titer ratios (VRP:liposome) at various times after the second dose were as follows: 2 weeks 4 weeks 8 weeks 0.1 μg 2.9 1.0 1.1   1 μg 2.3 0.9 0.9 Thus the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery. Further experiments showed superior F-specific IgG responses with a 10 μg dose, equivalent responses for 1 μg and 0.1 μg doses, and a lower response with a 0.01 μg dose. FIG. 11 shows IgG titers in mice receiving the replicon in naked form at 3 different doses, in liposomes at 4 different doses, or as VRP (106 IU). The response seen with 1 μgliposome-encapsulated RNA was statistically insignificant (ANOVA) when compared to VRP, but the higher response seen with 10 μg liposome-encapsulated RNA was statistically significant (p<0.05) when compared to both of these groups. A further study confirmed that the 0.1 μg of liposome-encapsulated RNA gave much higher anti-F IgG responses (15 days post-second dose) than 0.1 μg of delivered DNA, and even was more immunogenic than 20 μg plasmid DNA encoding the F antigen, delivered by electroporation (Elgen™ DNA Delivery System, Inovio). A further study was performed in cotton rats (Sigmodon hispidis) instead of mice. At a 1 μg dose liposome encapsulation increased F-specific IgG titers by 8.3-fold compared to naked RNA and increased PRT titers by 9.5-fold. The magnitude of the antibody response was equivalent to that induced by 5×106 IU VRP. Both naked and liposome-encapsulated RNA were able to protect the cotton rats from RSV challenge (1×105 plaque forming units), reducing lung viral load by at least 3.5 logs. Encapsulation increased the reduction by about 2-fold. A large-animal study was performed in cattle. Cows were immunised with 66 μg of replicon encoding full-length RSV F protein at days 0, 21, 86 & 146, formulated either inside liposomes or with the CNE17 emulsion. PBS alone was used as a negative control, and a licensed vaccine was used as a positive control (“Triangle 4” from Fort Dodge, containing killed virus). FIG. 14 shows F-specific IgG titers over the first 63 days. The RNA replicon was immunogenic in the cows using both delivery systems, although it gave lower titers than the licensed vaccine. All vaccinated cows showed F-specific antibodies after the second dose, and titers were very stable from the period of 2 to 6 weeks after the second dose and were particularly stable for the RNA vaccines). The titers with the liposome delivery system were more tightly clustered than with the emulsion. The data from this study provide proof of concept for RNA replicon RSV vaccines in large animals, with two of the five calves in the emulsion-adjuvanted group demonstrating good neutralizing antibody titers after the third vaccination, as measured by the complement-independent HRSV neutralization assay. In a complement-enhanced HRSV neutralization assay all vaccinated calves had good neutralizing antibody titers after the second RNA vaccination regardless of the formulation. Furthermore, both RNA vaccines elicited F-specific serum IgG titers that were detected in a few calves after the second vaccination and in all calves after the third vaccination. MF59-adjuvanted RSV-F was able to boost the IgG response in all previously vaccinated calves, and to boost complement-independent HRSV neutralization titers of calves previously vaccinated with RNA. Mechanism of Action Bone marrow derived dendritic cells (pDC) were obtained from wild-type mice or the “Resq” (rsq1) mutant strain. The mutant strain has a point mutation at the amino terminus of its TLR7 receptor which abolishes TLR7 signalling without affecting ligand binding [48]. The cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000 or inside a liposome. As shown in FIG. 16, IL-6 and INFα were induced in WT cells but this response was almost completely abrogated in mutant mice. These results shows that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferons and pro-inflammatory cytokines. The involvement of TLR7 was further investigated by comparing responses in wild type (WT) C57BL/6 mice and in the “Resq” mutant strain. Mice (5 per group) were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with 1 μg self-replicating. RNA (“vA317”, encoding the surface fusion glycoprotein of RSV) formulated in liposomes (40% DlinDMA, 10% DSPC, 48% cholesterol, 2% PEG-DMG conjugate), or with 2 μg of RSV-F protein adjuvanted with aluminum hydroxide. Serum was collected for immunological analysis on days 14 (2wp1) and 35 (2wp2).F-specific serum IgG titers (GMT) were as follows: RNA vaccine Protein vaccine Day WT Resq WT Resq Total IgG 14 1038 145 2324 2601 35 9038 1224 27211 17150 IgG 1 14 25 25 3657 2974 35 125 125 34494 26459 IgG 2c 14 1941 211 25 25 35 35804 2080 125 125 With the protein vaccine, F-specific serum IgG titers were comparable between the wild type and Resq C56BL/6 mice i.e. immunogenicity of the protein vaccine was not dependent on TLR7. In contrast, the self-replicating RNA formulated in liposomes showed a 7-fold decrease in F-specific serum IgG titers after both vaccinations, indicating at least a partial dependence on TLR7 for the immunogenicity of the RNA vaccine. The results also show that the RNA vaccine can elicit primarily a Thl-type immune response. Further experiments were performed with the same RNA and the same mutant mice. Mice were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with 1 μg of the RNA replicon, formulated either with a submicron cationic oil-in-water nanoemulsion (squalene, span 85, polysorbate 80, DOTAP) or with liposomes (40% DlinDIVIA, 10% DSPC, 48% cholesterol, 2% PEG-conjugated DMG). For comparison, 2 μg of alum-adjuvanted F protein was used, Sera were collected for immunological analysis on days 14 (2wp1) and 35 (2wp2). F-specific serum IgG, IgG1 and IgG2c titers (GMT) were as follows: RNA + liposome RNA + CNE Protein vaccine Day WT Resq WT Resq WT Resq Total 14 718 401 849 99 2795 2295 IgG 35 2786 1650 1978 374 41519 33327 IgG 1 14 25 25 136 76 3410 3238 35 125 125 195 183 38150 48040 IgG 2c 14 1605 849 136 76 25 25 35 14452 3183 7567 335 125 125 These results confirm the previous findings that, unlike the protein vaccine, the RNA vaccine shows at least a partial dependence on TLR7 for its immunogenicity, particularly with the emulsion adjuvant. Further Innate Immunity Receptors and Cytokine Responses As shown above, a delivered replicon can stimulate wild-type mouse dendritic cells to secrete IFN-α and IL-6, but the same response is not seen in dendritic cells from mice which carry the Resq mutation in TLR7. Similarly, Lipofectamine-delivered vA317 replicons can stimulate wild-type mouse fibroblasts to secrete high levels of IFN-(3 and IL-6, but the replicons stimulate much lower levels of these cytokines in fibroblasts which lack MDA5 or RIG-I i.e. cytoplasmic RNA receptors (see FIG. 15). These fibroblasts are non-immune cells which do not respond to TLR7 ligands. Mouse embryonic fibroblasts (MEFs) from RIG-I and MDA5 knockout mice (+/−) were stimulated with replicon RNA formulated with lipofectamine 2000. Heterozygous littermates (+/−) were used as controls. The RNA stimulates IL-6 and IFN-β in the heterozygous mice but in the knockout mice the activation is almost completely abrogated. Thus these helicases are important for RNA recognition in non-immune cells. In general, liposome-delivered RNA replicons were shown to induce several serum cytokines within 24 hours of intramuscular injection (IFN-α, IP-10 (CXCL-10), IL-6, KC, IL-5, IL-13, MCP-1, and MIP-a), whereas only MIP-1 was induced by naked RNA and liposome alone induced only 1L-6. IFN-α was shown to contribute to the immune response to liposome-encapsulated RSV-F-encoding replicon because an anti-IFNα receptor (IFNAR1) antibody reduced F-specific serum IgG a 10-fold reduction after 2 vaccinations. Expression Kinetics Experiments on expression kinetics used RNA encoding GFP or the SEAP reporter enzyme. The “vA306” replicon encodes SEAP; the “vA17” replicon encodes GFP; the “vA336” replicon encodes GFP but cannot self-replicate; the “vA336*” replicon is the same as vA336 but was prepared with 10% of uridines replaced with 5-methyluridine; the “vA336**” replicon is the same as va336 but 100% of its uridine residues are M5U. BALB/c mice were given bilateral intramuscular vaccinations (50 μL per leg) on day 0. Animals, 35 total, were divided into 7 groups (5 animals per group) and were immunised as follows: Group 1 Naïve control. Group 2 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP) formulated in liposomes. Group 3 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336, 1.0 μg, GFP) formulated in liposomes. Group 4 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336*, 1.0 μg GFP) formulated in liposomes. Group 5 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336**, 1.0 μg, GFP) formulated in liposomes. Group 6 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with empty liposomes at the same lipid dose as groups 2-5. Group 7 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP) formulated in liposomes. These experiments aimed to see if host responses to RNA might limit protein expression. Thus expression was followed for only 6 days, before an adaptive response (antibodies, T cells) would be apparent. Serum SEAP activity (relative light units) at days 0, 3 and 6 were as follows (GMT): Day 1 Day 3 Day 6 1 898 1170 2670 2 1428 4219 28641 3 1702 9250 150472 4 1555 8005 76043 5 1605 8822 91019 6 10005 14640 93909 7 1757 6248 53497 Replication-competent RNA encoding GFP suppressed the expression of SEAP more than replication-defective GFP RNA, suggesting a strong host defence response against replicating RNA which leads to suppression of SEAP expression. It is possible that interferons induced in response to the GFP RNA suppressed the expression of SEAP. Under the host response/suppression model, blocking host recognition of RNA would be expected to lead to increased SEAP expression, but 5′ methylation of U residues in the GFP RNA was not associated with increased SEAP, suggesting that host recognition of RNA was insensitive to 5′ methylation. It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. TABLE 1 useful phospholipids DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA 1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC 1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE 1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG 1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA 1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC 1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE 1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLPS 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA 1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DMPS 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA 1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DOPS 1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA 1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG 1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DPPS 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA 1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC 1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE 1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG 1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DSPS 1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPC Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPC Hydrogenated Soy PC LYSOPC MYRISTIC 1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC 1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC 1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin MPPC 1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine MSPC 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC 1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC 1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE 1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . .] PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC 1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC 1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC 1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine REFERENCES [1] Heyes et al. 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[42] Ausubel et al. (eds) (2002) Short protocols in molecular biology, 5th edition (Current Protocols). [43] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press). [44] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton Graham eds., 1997, Springer Verlag). [45] Yoneyama & Fujita (2007) Cytokine & Growth Factor Reviews 18:545-51. [46] Maurer et al. (2001) Biophysical Journal, 80: 2310-2326. [47] Perri et al, (2003) J Virol 77:10394-10403. [48] Iavarone et al. (2011) J Immunol 186; 4213-22. 16512541 glaxosmithkline biologicals sa USA B2 Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001. Open Apr 5th, 2022 05:12PM Apr 5th, 2022 05:12PM GlaxoSmithKline Health Care Pharmaceuticals & Biotechnology
nyse:gsk GlaxoSmithKline Apr 5th, 2022 12:00AM Jul 6th, 2011 12:00AM https://www.uspto.gov?id=US11291635-20220405 Virion-like delivery particles for self-replicating RNA molecules Nucleic acid immunisation is achieved by delivering a self-replicating RNA encapsulated within a small particle. The RNA encodes an immunogen of interest, and the particle may deliver this RNA by mimicking the delivery function of a natural RNA virus. Thus the invention provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material encapsulating a self-replicating RNA molecule which encodes an immunogen. These particles are useful as components in pharmaceutical compositions for immunising subjects against various diseases. 11291635 1. A non-virion particle for in vivo delivery of RNA to a vertebrate cell, said particle comprising a delivery material and a self-replicating RNA molecule that encodes a polypeptide immunogen, wherein (a) said non-virion particle does not comprise a protein capsid, said delivery material is a liposome, and the RNA is encapsulated in the liposome (a “LNP”); (b) the RNA molecule does not comprise modified nucleotides, other than a 5′cap, is not packaged with structural proteins as a virion, and cannot induce production of RNA-containing virions; (c) said non-virion particle can deliver RNA to a vertebrate cell in vivo; and (d) wherein the immunogen is translated in vivo and can elicit an immune response against said immunogen. 2. The particle of claim 1, wherein the self-replicating RNA molecule encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen. 3. The particle of claim 2, wherein the RNA molecule comprises two open reading frames, the first of which encodes an alphavirus replicase and the second of which encodes the immunogen. 4. The particle of claim 1, wherein the RNA molecule is 9000-12000 nucleotides long. 5. The particle of claim 1, wherein the immunogen can elicit an immune response in vivo against a bacterium, a virus, a fungus or a parasite. 6. The particle of claim 5, wherein the immunogen can elicit an immune response in vivo against respiratory syncytial virus glycoprotein F. 7. A pharmaceutical composition comprising a particle of claim 1. 7 This application is the U.S. National Phase of International Application No. PCT/US2011/043103, filed Jul. 6, 2011 and published in English, which claims the benefit of US Provisional Application No. 61/361,828, filed Jul. 6, 2010, the complete contents of which are hereby incorporated herein by reference for all purposes. TECHNICAL FIELD This invention is in the field of non-viral delivery of self-replicating RNAs for immunisation. BACKGROUND ART The delivery of nucleic acids for immunising animals has been a goal for several years. Various approaches have been tested, including the use of DNA or RNA, of viral or non-viral delivery vehicles (or even no delivery vehicle, in a “naked” vaccine), of replicating or non-replicating vectors, or of viral or non-viral vectors. There remains a need for further and improved nucleic acid vaccines and, in particular, for improved ways of delivering nucleic acid vaccines. DISCLOSURE OF THE INVENTION According to the invention, nucleic acid immunisation is achieved by delivering a self-replicating RNA encapsulated within and/or adsorbed to a small particle. The RNA encodes an immunogen of interest, and the particle may deliver this RNA by mimicking the delivery function of a natural virus. Thus the invention provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material encapsulating a self-replicating RNA molecule which encodes an immunogen. The invention also provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material on which a self-replicating RNA molecule which encodes an immunogen is adsorbed. These particles are useful as components in pharmaceutical compositions for immunising subjects against various diseases. The combination of utilising a non-virion particle to deliver a self-replicating RNA provides a way to elicit a strong and specific immune response against the immunogen while delivering only a low dose of RNA. Moreover, these particles can readily be manufactured at a commercial scale. The Particle Particles of the invention are non-virion particles i.e. they are not a virion. Thus the particle does not comprise a protein capsid. By avoiding the need to create a capsid particle, the invention does not require a packaging cell line, thus permitting easier up-scaling for commercial production and minimising the risk that dangerous infectious viruses will inadvertently be produced. Instead of encapsulating RNA in a virion, particles of the invention are formed from a delivery material. Various materials are suitable for forming particles which can deliver RNA to a vertebrate cell in vivo. Two delivery materials of particular interest are (i) amphiphilic lipids which can form liposomes and (ii) non-toxic and biodegradable polymers which can form microparticles. Where delivery is by liposome, RNA should be encapsulated; where delivery is by polymeric microparticle, RNA can be encapsulated or adsorbed. A third delivery material of interest is the particulate reaction product of a polymer, a crosslinker, a RNA, and a charged monomer. Thus one embodiment of a particle of the invention comprises a liposome encapsulating a self-replicating RNA molecule which encodes an immunogen, whereas another embodiment comprises a polymeric microparticle encapsulating a self-replicating RNA molecule which encodes an immunogen, and another embodiment comprises a polymeric microparticle on which a self-replicating RNA molecule which encodes an immunogen is adsorbed. In all three cases the particles preferably are substantially spherical. In a fourth embodiment a particle of the invention comprises the particulate reaction product of a polymer, a crosslinker, self-replicating RNA molecule which encodes an immunogen, and a charged monomer. These particles are formed within molds and so can be created with any shape including, but not limited to, spheres. RNA can be encapsulated within the particles (particularly if the particle is a liposome). This means that RNA inside the particles is (as in a natural virus) separated from any external medium by the delivery material, and encapsulation has been found to protect RNA from RNase digestion. Encapsulation can take various forms. For example, in some embodiments (as in a unilamellar liposome) the delivery material forms a outer layer around an aqueous RNA-containing core, whereas in other embodiments (e.g. in molded particles) the delivery material forms a matrix within which RNA is embedded. The particles can include some external RNA (e.g. on the surface of the particles), but at least half of the RNA (and ideally all of it) is encapsulated. Encapsulation within liposomes is distinct from, for instance, the lipid/RNA complexes disclosed in reference 1. RNA can be adsorbed to the particles (particularly if the particle is a polymeric microparticle). This means that RNA is not separated from any external medium by the delivery material, unlike the RNA genome of a natural virus. The particles can include some encapsulated RNA (e.g. in the core of a particle), but at least half of the RNA (and ideally all of it) is adsorbed. Liposomes Various amphiphilic lipids can form bilayers in an aqueous environment to encapsulate a RNA-containing aqueous core as a liposome. These lipids can have an anionic, cationic or zwitterionic hydrophilic head group. Formation of liposomes from anionic phospholipids dates back to the 1960s, and cationic liposome-forming lipids have been studied since the 1990s. Some phospholipids are anionic whereas other are zwitterionic and others are cationic. Suitable classes of phospholipid include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidyl-glycerols, and some useful phospholipids are listed in Table 1. Useful cationic lipids include, but are not limited to, dioleoyl trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are DPPC, DOPC and dodecylphosphocholine. The lipids can be saturated or unsaturated. The use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail. Liposomal particles of the invention can be formed from a single lipid or from a mixture of lipids. A mixture may comprise (i) a mixture of anionic lipids (ii) a mixture of cationic lipids (iii) a mixture of zwitterionic lipids (iv) a mixture of anionic lipids and cationic lipids (v) a mixture of anionic lipids and zwitterionic lipids (vi) a mixture of zwitterionic lipids and cationic lipids or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids. Similarly, a mixture may comprise both saturated and unsaturated lipids. For example, a mixture may comprise DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated). Where a mixture of lipids is used, not all of the component lipids in the mixture need to be amphiphilic e.g. one or more amphiphilic lipids can be mixed with cholesterol. The hydrophilic portion of a lipid can be PEGylated (i.e. modified by covalent attachment of a polyethylene glycol). This modification can increase stability and prevent non-specific adsorption of the liposomes. For instance, lipids can be conjugated to PEG using techniques such as those disclosed in reference 2 and 3. Various lengths of PEG can be used e.g. between 0.5-8 kDa. A mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is used in the examples. Liposomal particles are usually divided into three groups: multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles (LUV). MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments. SUVs and LUVs have a single bilayer encapsulating an aqueous core; SUVs typically have a diameter ≤50 nm, and LUVs have a diameter >50 nm. Liposomal particles of the invention are ideally LUVs with a diameter in the range of 50-220 nm. For a composition comprising a population of LUVs with different diameters: (i) at least 80% by number should have diameters in the range of 20-220 nm, (ii) the average diameter (Zav, by intensity) of the population is ideally in the range of 40-200 nm, and/or (iii) the diameters should have a polydispersity index <0.2. The liposome/RNA complexes of reference 1 are expected to have a diameter in the range of 600-800 nm and to have a high polydispersity. Techniques for preparing suitable liposomes are well known in the art e.g. see references 4 to 6. One useful method is described in reference 7 and involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification. Preferred liposomes of the invention are obtainable by this mixing process. Polymeric Microparticles Various polymers can form microparticles to encapsulate or adsorb RNA according to the invention. The use of a substantially non-toxic polymer means that a recipient can safely receive the particles, and the use of a biodegradable polymer means that the particles can be metabolised after delivery to avoid long-term persistence. Useful polymers are also sterilisable, to assist in preparing pharmaceutical grade formulations. Suitable non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates or polyester-amides, and combinations thereof. In some embodiments, the microparticles are formed from poly(α-hydroxy acids), such as a poly(lactides) (“PLA”), copolymers of lactide and glycolide such as a poly(D,L-lactide-co-glycolide) (“PLG”), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those having a lactide/glycolide molar ratio ranging, for example, from 20:80 to 80:20 e.g. 25:75, 40:60, 45:55, 50:50, 55:45, 60:40, 75:25. Useful PLG polymers include those having a molecular weight between, for example, 5,000-200,000 Da e.g. between 10,000-100,000, 20,000-70,000, 30,000-40,000, 40,000-50,000 Da. The microparticles ideally have a diameter in the range of 0.02 μm to 8 μm. For a composition comprising a population of microparticles with different diameters at least 80% by number should have diameters in the range of 0.03-7 μm. Techniques for preparing suitable microparticles are well known in the art e.g. see references 6, 8 (in particular chapter 7) and 9. To facilitate adsorption of RNA, a microparticle may include a cationic surfactant and/or lipid e.g. as disclosed in references 10 & 11. Microparticles of the invention can have a zeta potential of between 40-100 mV. One advantage of microparticles over liposomes is that they are readily lyophilised for stable storage. Molded Particles A third delivery material of interest is the particulate reaction product of a polymer, a crosslinker, a self-replicating RNA which encodes an immunogen, and a charged monomer. These four components can be mixed as a liquid, placed in a mold (e.g. comprising a perfluoropolyether), and then cured to form the particles according to the mold's shape and dimensions. Details of a suitable production method are disclosed in ref. 12. These methods provide a biodegradable crosslinked oligomeric polymer nanoparticle. Ideally the particles have a largest cross-sectional dimension of ≤5 μm. They may have an overall positive charge. Suitable polymers include, but are not limited to: a poly(acrylic acid); a poly(styrene sulfonate); a carboxymethylcellulose (CMC); a poly(vinyl alcohol); a poly(ethylene oxide); a poly(vinyl pyrrolidone); a dextran; a poly(vinylpyrolidone-co-vinyl acetate-co-vinyl alcohol). A preferred polymer is a poly(vinyl pyrrolidinone). The amount of polymer for forming the particles can be between 2-75 wt % e.g. 10-60 wt %, 20-60 wt %. Suitable crosslinkers can include a disulfide and/or ketal. For example, the crosslinker can comprise poly(epsilon-caprolactone)-b-tetraethylene glycol-b-poly(epsilon-capro lactone)dimethacrylate, poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-capro lactone)dimethacrylate, poly(lactic acid)-b-tetraethylene glycol-b-poly(lactic acid)dimethacrylate, poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid)dimethacrylate, poly(glycolic acid)-b-tetraethylene glycol-b-poly(glycolic acid)dimethacrylate, poly(gly colic acid)-b-poly(ethylene glycol)-b-poly(gly colic acid)dimethacrylate, poly(epsilon-caprolactone)-b-tetraethylene glycol-b-poly(epsilon-caprolactone)diacrylate, poly(epsilon-caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-caprolactone)diacrylate, poly(lactic acid)-b-tetraethylene glycol-b-poly(lactic acid)diacrylate, poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid)diacrylate, poly(glycolic acid)-b-tetraethylene glycol-b-poly(glycolic acid)diacrylate, poly(glycolic acid)-b-poly(ethylene glycol)-b-poly(glycolic acid)diacrylate, silane, silicon containing methacrylates, or dimethyldi(methacryloyloxy-1-ethoxy)silane. The amount of crosslinker for forming the particles can be between 10-25 wt % e.g. 10-60 wt %, 20-60 wt %. Charged monomers can be cationic or anionic. These include, but are not limited to: [2-(acryloyloxy)ethyl]trimethyl ammonium chloride (AETMAC) and 2-aminoethyl methacrylate hydrochloride (AEM-HCl). The amount of charged monomer for forming the particles can be between 2-75 wt %. The amount of RNA for forming the particles can be between 0.25-20 wt %. A pre-cure mixture inside a mold can include an initiator. For instance, the mold can include ≤1 wt % initiator, ≤0.5 wt % initiator, or ≤0.1 wt % initiator. Between 0.1-0.5% initiator is useful. Photoinitiators such as DEAP and DPT are useful e.g. for use with ultraviolet curing. The invention can use any of the materials disclosed in Table 1 or Examples 1-15 of reference 12, except that the siRNA components therein will be replaced by self-replicating RNAs as herein. The RNA Particles of the invention include a self-replicating RNA molecule which (unlike siRNA) encodes an immunogen. After in vivo administration of the particles, RNA is released from the particles and is translated inside a cell to provide the immunogen in situ. Unlike reference 13, the RNA in particles of the invention is self-replicating. A self-replicating RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself). A self-replicating RNA molecule is thus typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded immunogen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the immunogen. The overall results of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded immunogen becomes a major polypeptide product of the cells. One suitable system for achieving self-replication in this manner is to use an alphavirus-based replicon. These replicons are +-stranded RNAs which lead to translation of a replicase (or replicase-transcriptase) after delivery to a cell. The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic −-strand copies of the +-strand delivered RNA. These −-strand transcripts can themselves be transcribed to give further copies of the +-stranded parent RNA and also to give a subgenomic transcript which encodes the immunogen. Translation of the subgenomic transcript thus leads to in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons [14]. A preferred self-replicating RNA molecule thus encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen. The polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4. Whereas natural alphavirus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, it is preferred that the self-replicating RNA molecules of the invention do not encode alphavirus structural proteins. Thus a preferred self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses are absent from self-replicating RNAs of the invention and their place is taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins. Thus a self-replicating RNA molecule useful with the invention may have two open reading frames. The first (5′) open reading frame encodes a replicase; the second (3′) open reading frame encodes an immunogen. In some embodiments the RNA may have additional (e.g. downstream) open reading frames e.g. to encode further immunogens (see below) or to encode accessory polypeptides. A preferred self-replicating RNA molecule has a 5′ cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA. In some embodiments the 5′ sequence of the self-replicating RNA molecule must be selected to ensure compatibility with the encoded replicase. A self-replicating RNA molecule may have a 3′ poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3′ end. Self-replicating RNA molecules can have various lengths but they are typically 5000-25000 nucleotides long e.g. 8000-15000 nucleotides, or 9000-12000 nucleotides. Thus the RNA is longer than seen in siRNA delivery. Self-replicating RNA molecules will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during replication of a single-stranded RNA or within the secondary structure of a single-stranded RNA. The self-replicating RNA can conveniently be prepared by in vitro transcription (IVT). IVT can use a (cDNA) template created and propagated in plasmid form in bacteria, or created synthetically (for example by gene synthesis and/or polymerase chain-reaction (PCR) engineering methods). For instance, a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the self-replicating RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the replicon's poly-A is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5′ nucleotide(s) and in some embodiments these requirements must be matched with the requirements of the encoded replicase, to ensure that the IVT-transcribed RNA can function efficiently as a substrate for its self-encoded replicase. As discussed in reference 15, the self-replicating RNA can include (in addition to any 5′ cap structure) one or more nucleotides having a modified nucleobase. Thus the RNA can comprise m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2′-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2′-O-methyladenosine); ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6 isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6.-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2′-O-ribosyladenosine (phosphate)); I (inosine); m11 (1-methylinosine); m′Im (1,2′-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C(N4-acetylcytidine); f5C (5-fonnylcytidine); m5 Cm (5,2-O-dimethylcytidine); ac4 Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2′-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2′-O-dimethylguanosine); m22Gm (N2,N2,2′-β-trimethylguanosine); Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine); m5Um (5,2′-β-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2′-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2′-O-methylinosine); m4C(N4-methylcytidine); m4 Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6 Am (N6,T-O-dimethyladenosine); rn62 Am (N6,N6,O-2-trimethyladenosine); m2′7G (N2,7-dimethylguanosine); m2′2′7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5 Cm (5-formyl-2′-O-methylcytidine); m1Gm (1,2′-O-dimethylguanosine); m′Am (1,2-O-dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG-14 (4-demethyl guanosine); imG2 (isoguanosine); or ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or an abasic nucleotide. For instance, a self-replicating RNA can include one or more modified pyrimidine nucleobases, such as pseudouridine and/or 5-methylcytosine residues. In some embodiments, however, the RNA includes no modified nucleobases, and may include no modified nucleotides i.e. all of the nucleotides in the RNA are standard A, C, G and U ribonucleotides (except for any 5′ cap structure, which may include a 7′-methylguanosine). In other embodiments, the RNA may include a 5′ cap comprising a 7′-methylguanosine, and the first 1, 2 or 3 5′ ribonucleotides may be methylated at the 2′ position of the ribose. A RNA used with the invention ideally includes only phosphodiester linkages between nucleosides, but in some embodiments it can contain phosphoramidate, phosphorothioate, and/or methylphosphonate linkages. The amount of RNA per particle can vary, and the number of individual self-replicating RNA molecules per particle can depend on the characteristics of the particle being used. In general, a particle may include from 1-500 RNA molecules. For a liposome the number of RNA molecules is typically ≤50 per liposome e.g. <20, <10, <5, or 1-4. For a polymeric microparticle the number of RNA molecules will depend on the particle diameter but may be ≤50 per particle (e.g. <20, <10, <5, or 1-4) or from 50-200 per particle. Ideally, a particle includes fewer than 10 different species of RNA e.g. 5, 4, 3, or 2 different species; most preferably, a particle includes a single RNA species i.e. all RNA molecules in the particle have the same sequence and same length. The Immunogen Self-replicating RNA molecules used with the invention encode a polypeptide immunogen. After administration of the particles the immunogen is translated in vivo and can elicit an immune response in the recipient. The immunogen may elicit an immune response against a bacterium, a virus, a fungus or a parasite (or, in some embodiments, against an allergen; and in other embodiments, against a tumor antigen). The immune response may comprise an antibody response (usually including IgG) and/or a cell-mediated immune response. The polypeptide immunogen will typically elicit an immune response which recognises the corresponding bacterial, viral, fungal or parasite (or allergen or tumour) polypeptide, but in some embodiments the polypeptide may act as a mimotope to elicit an immune response which recognises a bacterial, viral, fungal or parasite saccharide. The immunogen will typically be a surface polypeptide e.g. an adhesin, a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, etc. Self-replicating RNA molecules can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If immunogens are expressed as separate polypeptides then one or more of these may be provided with an upstream IRES or an additional viral promoter element. Alternatively, multiple immunogens may be expressed from a polyprotein that encodes individual immunogens fused to a short autocatalytic protease (e.g. foot-and-mouth disease virus 2A protein), or as inteins. Unlike references 1 and 16, the RNA encodes an immunogen. For the avoidance of doubt, the invention does not encompass RNA which encodes a firefly luciferase or which encodes a fusion protein of E. coli β-galactosidase or which encodes a green fluorescent protein (GFP). Also, the RNA is not total mouse thymus RNA. In some embodiments the immunogen elicits an immune response against one of these bacteria: Neisseria meningitidis: useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding protein. A combination of three useful polypeptides is disclosed in reference 17. Streptococcus pneumoniae: useful polypeptide immunogens are disclosed in reference 18. These include, but are not limited to, the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase precursor (spr0057), spr0096, General stress protein GSP-781 (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA. Streptococcus pyogenes: useful immunogens include, but are not limited to, the polypeptides disclosed in references 19 and 20. Moraxella catarrhalis. Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3. Staphylococcus aureus: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 21, such as a hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and/or the sta011 lipoprotein. Clostridium tetani: the typical immunogen is tetanus toxoid. Cornynebacterium diphtheriae: the typical immunogen is diphtheria toxoid. Haemophilus influenzae: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 22 and 23. Pseudomonas aeruginosa Streptococcus agalactiae: useful immunogens include, but are not limited to, the polypeptides disclosed in reference 19. Chlamydia trachomatis: Useful immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as disclosed in reference 24. LcrE [25] and HtrA [26] are two preferred immunogens. Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 27. Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and/or urease [28]. Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and meningitis/sepsis-associated E. coli (MNEC). Useful UPEC polypeptide immunogens are disclosed in references 29 and 30. Useful MNEC immunogens are disclosed in reference 31. A useful immunogen for several E. coli types is AcfD [32]. Bacillus anthracia Yersinia pestis: Useful immunogens include, but are not limited to, those disclosed in references 33 and 34. Staphylococcus epidermis Clostridium perfringens or Clostridium botulinums Legionella pneumophila Coxiella burnetii Brucella, such as B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, B. pinnipediae. Francisella, such as F. novicida, F. philomiragia, F. tularensis. Neisseria gonorrhoeae Treponema pallidum Haemophilus ducreyi Enterococcus faecalis or Enterococcus faecium Staphylococcus saprophyticus Yersinia enterocolitica Mycobacterium tuberculosis Rickettsia Listeria monocytogenes Vibrio cholerae Salmonella typhi Borrelia burgdorferi Porphyromonas gingivalis Klebsiella In some embodiments the immunogen elicits an immune response against one of these viruses: Orthomyxovirus: Useful immunogens can be from an influenza A, B or C virus, such as the hemagglutinin, neuraminidase or matrix M2 proteins. Where the immunogen is an influenza A virus hemagglutinin it may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. Paramyxoviridae viruses: Viral immunogens include, but are not limited to, those derived from Pneumoviruses (e.g. respiratory syncytial virus, RSV), Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g. measles). Poxyiridae: Viral immunogens include, but are not limited to, those derived from Orthopoxvirus such as Variola vera, including but not limited to, Variola major and Variola minor. Picornavirus: Viral immunogens include, but are not limited to, those derived from Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In one embodiment, the enterovirus is a poliovirus e.g. a type 1, type 2 and/or type 3 poliovirus. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a coxsackie A or B virus. Bunyavirus: Viral immunogens include, but are not limited to, those derived from an Orthobunyavirus, such as California encephalitis virus, a Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever virus. Heparnavirus: Viral immunogens include, but are not limited to, those derived from a Heparnavirus, such as hepatitis A virus (HAV). Filovirus: Viral immunogens include, but are not limited to, those derived from a Filovirus, such as an Ebola virus (including a Zaire, Ivory Coast, Reston or Sudan ebolavirus) or a Marburg virus. Togavirus: Viral immunogens include, but are not limited to, those derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus. This includes rubella virus. Flavivirus: Viral immunogens include, but are not limited to, those derived from a Flavivirus, such as Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus. Pestivirus: Viral immunogens include, but are not limited to, those derived from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV). Hepadnavirus: Viral immunogens include, but are not limited to, those derived from a Hepadnavirus, such as Hepatitis B virus. A composition can include hepatitis B virus surface antigen (HBsAg). Other hepatitis viruses: A composition can include an immunogen from a hepatitis C virus, delta hepatitis virus, hepatitis E virus, or hepatitis G virus. Rhabdovirus: Viral immunogens include, but are not limited to, those derived from a Rhabdovirus, such as a Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV). Caliciviridae: Viral immunogens include, but are not limited to, those derived from Calciviridae, such as Norwalk virus (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus. Coronavirus: Viral immunogens include, but are not limited to, those derived from a SARS coronavirus, avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV). The coronavirus immunogen may be a spike polypeptide. Retrovirus: Viral immunogens include, but are not limited to, those derived from an Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus. Reovirus: Viral immunogens include, but are not limited to, those derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Parvovirus: Viral immunogens include, but are not limited to, those derived from Parvovirus B19. Herpesvirus: Viral immunogens include, but are not limited to, those derived from a human herpesvirus, such as, by way of example only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and 2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). Papovaviruses: Viral immunogens include, but are not limited to, those derived from Papillomaviruses and Polyomaviruses. The (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from one or more of serotypes 6, 11, 16 and/or 18. Adenovirus: Viral immunogens include those derived from adenovirus serotype 36 (Ad-36). In some embodiments, the immunogen elicits an immune response against a virus which infects fish, such as: infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), koi herpesvirus, salmon picorna-like virus (also known as picorna-like virus of atlantic salmon), landlocked salmon virus (LSV), atlantic salmon rotavirus (ASR), trout strawberry disease virus (TSD), coho salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV). Fungal immunogens may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme; or from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi; the less common are Brachiola spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp. In some embodiments the immunogen elicits an immune response against a parasite from the Plasmodium genus, such as P. falciparum, P. vivax, P. malariae or P. ovale. Thus the invention may be used for immunising against malaria. In some embodiments the immunogen elicits an immune response against a parasite from the Caligidae family, particularly those from the Lepeophtheirus and Caligus genera e.g. sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi. In some embodiments the immunogen elicits an immune response against: pollen allergens (tree-, herb, weed-, and grass pollen allergens); insect or arachnid allergens (inhalant, saliva and venom allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera venom allergens); animal hair and dandruff allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), plane tree (Platanus), the order of Poales including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including herbs of the genera Ambrosia, Artemisia, and Parietaria. Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those from mammals such as cat, dog and horse, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (Apidae), wasps (Vespidea), and ants (Formicoidae). In some embodiments the immunogen is a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase related protein-2/TRP2 (associated with, e.g., melanoma); (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer; (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like. Pharmaceutical Compositions Particles of the invention are useful as components in pharmaceutical compositions for immunising subjects against various diseases. These compositions will typically include a pharmaceutically acceptable carrier in addition to the particles. A thorough discussion of pharmaceutically acceptable carriers is available in reference 35. A pharmaceutical composition of the invention may include one or more small molecule immunopotentiators. For example, the composition may include a TLR2 agonist (e.g. Pam3CSK4), a TLR4 agonist (e.g. an aminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist (e.g. imiquimod), a TLR8 agonist (e.g. resiquimod) and/or a TLR9 agonist (e.g. IC31). Any such agonist ideally has a molecular weight of <2000 Da. Where a RNA is encapsulated, in some embodiments such agonist(s) are also encapsulated with the RNA, but in other embodiments they are unencapsulated. Where a RNA is adsorbed to a particle, in some embodiments such agonist(s) are also adsorbed with the RNA, but in other embodiments they are unadsorbed. Pharmaceutical compositions of the invention may include the particles in plain water (e.g. w.f.i.) or in a buffer e.g. a phosphate buffer, a Tris buffer, a borate buffer, a succinate buffer, a histidine buffer, or a citrate buffer. Buffer salts will typically be included in the 5-20 mM range. Pharmaceutical compositions of the invention may have a pH between 5.0 and 9.5 e.g. between 6.0 and 8.0. Compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 9 mg/ml. Compositions of the invention may include metal ion chelators. These can prolong RNA stability by removing ions which can accelerate phosphodiester hydrolysis. Thus a composition may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typically present at between 10-500 μM e.g. 0.1 mM. A citrate salt, such as sodium citrate, can also act as a chelator, while advantageously also providing buffering activity. Pharmaceutical compositions of the invention may have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between 290-310 mOsm/kg. Pharmaceutical compositions of the invention may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Mercury-free compositions are preferred, and preservative-free vaccines can be prepared. Pharmaceutical compositions of the invention are preferably sterile. Pharmaceutical compositions of the invention are preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. Pharmaceutical compositions of the invention are preferably gluten free. Pharmaceutical compositions of the invention may be prepared in unit dose form. In some embodiments a unit dose may have a volume of between 0.1-1.0 ml e.g. about 0.5 m1. The compositions may be prepared as injectables, either as solutions or suspensions. The composition may be prepared for pulmonary administration e.g. by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration e.g. as spray or drops. Injectables for intramuscular administration are typical. Compositions comprise an immunologically effective amount of particles, as well as any other components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The particle and RNA content of compositions of the invention will generally be expressed in terms of the amount of RNA per dose. A preferred dose has ≤100 μg RNA (e.g. from 10-100 μg, such as about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg), but expression can be seen at much lower levels e.g. ≤1 μg/dose, ≤100 μg/dose, ≤10 μg/dose, ≤1 μg/dose, etc The invention also provides a delivery device (e.g. syringe, nebuliser, sprayer, inhaler, dermal patch, etc.) containing a pharmaceutical composition of the invention. This device can be used to administer the composition to a vertebrate subject. Particles of the invention do not include ribosomes. Methods of Treatment and Medical Uses In contrast to the particles disclosed in reference 16, particles and pharmaceutical compositions of the invention are for in vivo use for eliciting an immune response against an immunogen of interest. The invention provides a method for raising an immune response in a vertebrate comprising the step of administering an effective amount of a particle or pharmaceutical composition of the invention. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. The method may raise a booster response. The invention also provides a particle or pharmaceutical composition of the invention for use in a method for raising an immune response in a vertebrate. The invention also provides the use of a particle of the invention in the manufacture of a medicament for raising an immune response in a vertebrate. By raising an immune response in the vertebrate by these uses and methods, the vertebrate can be protected against various diseases and/or infections e.g. against bacterial and/or viral diseases as discussed above. The particles and compositions are immunogenic, and are more preferably vaccine compositions. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (e.g. horses, cattle, deer, goats, pigs). Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably a teenager or an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. Vaccines prepared according to the invention may be used to treat both children and adults. Thus a human patient may be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients for receiving the vaccines are the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65 years), the young (e.g. ≤5 years old), hospitalised patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, or immunodeficient patients. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population. Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or to the interstitial space of a tissue; unlike reference 1, intraglossal injection is not typically used with the present invention). Alternative delivery routes include rectal, oral (e.g. tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 ml. The invention may be used to elicit systemic and/or mucosal immunity, preferably to elicit an enhanced systemic and/or mucosal immunity. Dosage can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one embodiment, multiple doses may be administered approximately 6 weeks, 10 weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and 14 weeks, as often used in the World Health Organisation's Expanded Program on Immunisation (“EPI”). In an alternative embodiment, two primary doses are administered about two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the second primary dose, e.g. about 6, 8, 10 or 12 months after the second primary dose. In a further embodiment, three primary doses are administered about two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one or more booster doses about 6 months to 1 year after the third primary dose, e.g. about 6, 8, 10, or 12 months after the third primary dose. GENERAL EMBODIMENTS In some embodiments of the invention, the RNA includes no modified nucleotides (see above). In other embodiments the RNA can optionally include at least one modified nucleotide, provided that one or more of the following features (already disclosed above) is also required: A. Where the RNA is delivered with a liposome, the liposome comprises DSDMA, DODMA, DLinDMA and/or DLenDMA. B. Where the RNA is encapsulated in a liposome, the hydrophilic portion of a lipid in the liposome is PEGylated. C. Where the RNA is encapsulated in a liposome, at least 80% by number of the liposomes have diameters in the range of 20-220 nm. D. Where the RNA is delivered with a microparticle, the microparticle is a non-toxic and biodegradable polymer microparticle. E. Where the RNA is delivered with a microparticle, the microparticles have a diameter in the range of 0.02 μm to 8 μm. F. Where the RNA is delivered with a microparticle, at least 80% by number of the microparticles have a diameter in the range of 0.03-7 μm. G. Where the RNA is delivered with a microparticle, the composition is lyophilised. H. The RNA has a 3′ poly-A tail, and the immunogen can elicits an immune response in vivo against a bacterium, a virus, a fungus or a parasite. I. The RNA is delivered in combination with a metal ion chelator with a delivery system selected from (i) liposomes (ii) non-toxic and biodegradable polymer microparticles. General The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 36-42, etc. The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y. The term “about” in relation to a numerical value x is optional and means, for example, x±10%. The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. References to charge, to cations, to anions, to zwitterions, etc., are taken at pH 7. TLR3 is the Toll-like receptor 3. It is a single membrane-spanning receptor which plays a key role in the innate immune system. Known TLR3 agonists include poly(I:C). “TLR3” is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:11849. The RefSeq sequence for the human TLR3 gene is GI:2459625. TLR7 is the Toll-like receptor 7. It is a single membrane-spanning receptor which plays a key role in the innate immune system. Known TLR7 agonists include e.g. imiquimod. “TLR7” is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15631. The RefSeq sequence for the human TLR7 gene is GI:67944638. TLR8 is the Toll-like receptor 8. It is a single membrane-spanning receptor which plays a key role in the innate immune system. Known TLR8 agonists include e.g. resiquimod. “TLR8” is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15632. The RefSeq sequence for the human TLR8 gene is GI:20302165. The RIG-I-like receptor (“RLR”) family includes various RNA helicases which play key roles in the innate immune system[43]. RLR-1 (also known as RIG-I or retinoic acid inducible gene I) has two caspase recruitment domains near its N-terminus. The approved HGNC name for the gene encoding the RLR-1 helicase is “DDX58” (for DEAD (Asp-Glu-Ala-Asp) box polypeptide 58) and the unique HGNC ID is HGNC:19102. The RefSeq sequence for the human RLR-1 gene is GI:77732514. RLR-2 (also known as MDA5 or melanoma differentiation-associated gene 5) also has two caspase recruitment domains near its N-terminus. The approved HGNC name for the gene encoding the RLR-2 helicase is “IFIH1” (for interferon induced with helicase C domain 1) and the unique HGNC ID is HGNC:18873. The RefSeq sequence for the human RLR-2 gene is GI: 27886567. RLR-3 (also known as LGP2 or laboratory of genetics and physiology 2) has no caspase recruitment domains. The approved HGNC name for the gene encoding the RLR-3 helicase is “DHX58” (for DEXH (Asp-Glu-X-His) box polypeptide 58) and the unique HGNC ID is HGNC:29517. The RefSeq sequence for the human RLR-3 gene is GI:149408121. PKR is a double-stranded RNA-dependent protein kinase. It plays a key role in the innate immune system. “EIF2AK2” (for eukaryotic translation initiation factor 2-alpha kinase 2) is the approved HGNC name for the gene encoding this enzyme, and its unique HGNC ID is HGNC:9437. The RefSeq sequence for the human PKR gene is GI:208431825. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a gel with stained RNA. Lanes show (1) markers (2) naked replicon (3) replicon after RNase treatment (4) replicon encapsulated in liposome (5) liposome after RNase treatment (6) liposome treated with RNase then subjected to phenol/chloroform extraction. FIG. 2 is an electron micrograph of liposomes. FIG. 3 shows protein expression (as relative light units, RLU) at days 1, 3 and 6 after delivery of RNA as a virion-packaged replicon (squares), naked RNA (triangles), or as microparticles (circles). FIG. 4 shows a gel with stained RNA. Lanes show (1) markers (2) naked replicon (3) replicon encapsulated in liposome (4) liposome treated with RNase then subjected to phenol/chloroform extraction. FIG. 5 shows protein expression at days 1, 3 and 6 after delivery of RNA as a virion-packaged replicon (squares), as naked RNA (diamonds), or in liposomes (+=0.1 μg, x=1 μg). FIG. 6 shows protein expression at days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA. FIG. 7 shows anti-F IgG titers in animals receiving virion-packaged replicon (VRP or VSRP), 1 μg naked RNA, and 1 μg liposome-encapsulated RNA. FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1 μg naked RNA, and 0.1 g or 1 μg liposome-encapsulated RNA. FIG. 9 shows neutralising antibody titers in animals receiving VRP or either 0.1 g or 1 μg liposome-encapsulated RNA. FIG. 10 shows expression levels after delivery of a replicon as naked RNA (circles), liposome-encapsulated RNA (triangle & square), or as a lipoplex (inverted triangle). FIG. 11 shows F-specific IgG titers (2 weeks after second dose) after delivery of a replicon as naked RNA (0.01-1 μg), liposome-encapsulated RNA (0.01-10 μg), or packaged as a virion (VRP, 106 infectious units or IU). FIG. 12 shows F-specific IgG titers (circles) and PRNT titers (squares) after delivery of a replicon as naked RNA (1 μg), liposome-encapsulated RNA (0.1 or 1 μg), or packaged as a virion (VRP, 106 IU). Titers in naïve mice are also shown. Solid lines show geometric means. FIG. 13 shows intracellular cytokine production after restimulation with synthetic peptides representing the major epitopes in the F protein, 4 weeks after a second dose. The y-axis shows the % cytokine+ of CD8+CD4−. FIG. 14 shows F-specific IgG titers (mean log10 titers±std dev) over 63 days (FIG. 14A) and 210 days (FIG. 14B) after immunisation of calves. The three lines are easily distinguished at day 63 and are, from bottom to top: PBS negative control; liposome-delivered RNA; and the “Triangle 4” product. FIG. 15 shows anti-HIV serum IgG titers in response to naked (“RNA”) or liposome-encapsulated (“LNP”) RNA, or to DNA delivered by electroporated into muscle. FIG. 16 shows IgG titers in 13 groups of mice. Each circle is an individual mouse, and solid lines show geometric means. The dotted horizontal line is the assay's detection limit. The 13 groups are, from left to right, A to M as described below. FIG. 17 shows (A) IL-6 and (B) IFNα (pg/ml) released by pDC. There are 4 pairs of bars, from left to right: control; immunised with RNA+DOTAP; immunised with RNA+lipofectamine; and immunised with RNA in liposomes. In each pair the black bar is wild-type mice, grey is rsq1 mutant. MODES FOR CARRYING OUT THE INVENTION RNA Replicons Various replicons are used below. In general these are based on a hybrid alphavirus genome with non-structural proteins from venezuelan equine encephalitis virus (VEEV), a packaging signal from sindbis virus, and a 3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10 kb long and has a poly-A tail. Plasmid DNA encoding alphavirus replicons (named: pT7-mVEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for synthesis of RNA in vitro. The replicons contain the alphavirus genetic elements required for RNA replication but lack those encoding gene products necessary for particle assembly; the structural proteins are instead replaced by a protein of interest (either a reporter, such as SEAP or GFP, or an immunogen, such as full-length RSV F protein) and so the replicons are incapable of inducing the generation of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA facilitates the synthesis of the replicon RNA in vitro and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A)-tail generates the correct 3′-end through its self-cleaving activity. Following linearization of the plasmid DNA downstream of the HDV ribozyme with a suitable restriction endonuclease, run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage derived DNA-dependent RNA polymerase. Transcriptions were performed for 2 hours at 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion). Following transcription the template DNA was digested with TURBO DNase (Ambion). The replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. Uncapped RNA was capped post-transcriptionally with Vaccinia Capping Enzyme (VCE) using the ScriptCap m7G Capping System (Epicentre Biotechnologies) as outlined in the user manual; replicons capped in this way are given the “v” prefix e.g. vA317 is the A317 replicon capped by VCE. Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the RNA samples was determined by measuring OD260nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis. PLG Adsorption Microparticles were made using 500 mg of PLG RG503 (50:50 lactide/glycolide molar ratio, MW ˜30 kDa) and 20 mg DOTAP using an Omni Macro Homogenizer. The particle suspension was shaken at 150 rpm overnight and then filtered through a 40 μm sterile filter for storage at 2-8° C. Self-replicating RNA was adsorbed to the particles. To prepare 1 mL of PLG/RNA suspension the required volume of PLG particle suspension was added to a vial and nuclease-free water was added to bring the volume to 900 μL. 100 μL RNA (10 μg/mL) was added dropwise to the PLG suspension, with constant shaking. PLG/RNA was incubated at room temperature for 30 min. For 1 mL of reconstituted suspension, 45 mg mannitol, 15 mg sucrose and 250-500 μg of PVA were added. The vials were frozen at −80° C. and lyophilized. To evaluate RNA adsorption, 100 μL particle suspension was centrifuged at 10,000 rpm for 5 min and supernatant was collected. PLG/RNA was reconstituted using 1 mL nuclease-free water. To 100 μL particle suspension (1 μg RNA), 1 mg heparin sulfate was added. The mixture was vortexed and allowed to sit at room temperature for 30 min for RNA desorption. Particle suspension was centrifuged and supernatant was collected. For RNAse stability, 100 μL particle suspension was incubated with 6.4 mAU of RNase A at room temperature for 30 min. RNAse was inactivated with 0.126 mAU of Proteinase K at 55° C. for 10 min. 1 mg of heparin sulfate was added to desorb the RNA followed by centrifugation. The supernatant samples containing RNA were mixed with formaldehyde load dye, heated at 65° C. for 10 min and analyzed using a 1% denaturing gel (460 ng RNA loaded per lane). To assess expression, Balb/c mice were immunized with 1 μg RNA in 100 μL intramuscular injection volume (50 μL/leg) on day 0. Sera were collected on days 1, 3 and 6. Protein expression was determined using a chemiluminescence assay. As shown in FIG. 3, expression was higher when RNA was delivered by PLG (triangles) than without any delivery particle (circles). Liposomal Encapsulation RNA was encapsulated in liposomes made by the method of references 7 and 44. The liposomes were made of 10% DSPC (zwitterionic), 40% DlinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). These proportions refer to the % moles in the total liposome. DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of reference 2. DSPC (1,2-Diastearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme. Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol), ammonium salt), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, chloride salt) and DC-chol (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids. Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these were mixed with 2 ml of buffer followed by 1 hour of equilibration. The mixture was diluted with 6 ml buffer then filtered. The resulting product contained liposomes, with ˜95% encapsulation efficiency. For example, in one particular method, fresh lipid stock solutions were prepared in ethanol. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with plenty of MilliQ water before use to decontaminate the vials of RNases. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used had a 2 mm internal diameter and a 3 mm outer diameter; obtained from Idex Health Science). The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 h. 4 ml of the mixture was loaded into a 5 cc syringe, which was connected to a piece of FEP tubing and in another 5 cc syringe connected to an equal length of FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation). Before using this membrane for the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) were successively passed through it. Liposomes were warmed for 10 min at 37° C. before passing through the membrane. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using by tangential flow filtration before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez) and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes with a 100 kD pore size cutoff and 8 cm2 surface area were used. For in vitro and in vivo experiments formulations were diluted to the required RNA concentration with 1×PBS. Further liposome manufacturing methods are disclosed below. FIG. 2 shows an example electron micrograph of liposomes prepared by these methods. These liposomes contain encapsulated RNA encoding full-length RSV F antigen. Dynamic light scattering of one batch showed an average diameter of 141 nm (by intensity) or 78 nm (by number). The percentage of encapsulated RNA and RNA concentration were determined by Quant-iT RiboGreen RNA reagent kit (Invitrogen), following manufacturer's instructions. The ribosomal RNA standard provided in the kit was used to generate a standard curve. Liposomes were diluted 10× or 100× in 1×TE buffer (from kit) before addition of the dye. Separately, liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% Triton X before addition of the dye (to disrupt the liposomes and thus to assay total RNA). Thereafter an equal amount of dye was added to each solution and then ˜180 μL of each solution after dye addition was loaded in duplicate into a 96 well tissue culture plate. The fluorescence (Ex 485 nm, Em 528 nm) was read on a microplate reader. All liposome formulations were dosed in vivo based on the encapsulated amount of RNA. Encapsulation in liposomes was shown to protect RNA from RNase digestion. Experiments used 3.8 mAU of RNase A per microgram of RNA, incubated for 30 minutes at room temperature. RNase was inactivated with Proteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to 25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added to extract the RNA from the lipids into the aqueous phase. Samples were mixed by vortexing for a few seconds and then placed on a centrifuge for 15 minutes at 12 k RPM. The aqueous phase (containing the RNA) was removed and used to analyze the RNA. Prior to loading (400 ng RNA per well) all the samples were incubated with formaldehyde loading dye, denatured for 10 minutes at 65° C. and cooled to room temperature. Ambion Millennium markers were used to approximate the molecular weight of the RNA construct. The gel was run at 90 V. The gel was stained using 0.1% SYBR gold according to the manufacturer's guidelines in water by rocking at room temperature for 1 hour. FIG. 1 shows that RNase completely digests RNA in the absence of encapsulation (lane 3). RNA is undetectable after encapsulation (lane 4), and no change is seen if these liposomes are treated with RNase (lane 4). After RNase-treated liposomes are subjected to phenol extraction, undigested RNA is seen (lane 6). Even after 1 week at 4° C. the RNA could be seen without any fragmentation (FIG. 4, arrow). Protein expression in vivo was unchanged after 6 weeks at 4° C. and one freeze-thaw cycle. Thus liposome-encapsulated RNA is stable. To assess in vivo expression of the RNA a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon, rather than an immunogen. Expression levels were measured in sera diluted 1:4 in 1× Phospha-Light dilution buffer using a chemiluminescent alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group) were injected intramuscularly on day 0, 50 μl per leg with 0.1 μg or 1 μg RNA dose. The same vector was also administered without the liposomes (in RNase free 1×PBS) at 1 μg. Virion-packaged replicons were also tested. Virion-packaged replicons used herein (referred to as “VRPs”) were obtained by the methods of reference 45, where the alphavirus replicon is derived from the mutant VEEV or a chimera derived from the genome of VEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbis virus packaging signal (PS), packaged by co-electroporating them into BHK cells with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes. As shown in FIG. 5, encapsulation increased SEAP levels by about ½ log at the 1 μg dose, and at day 6 expression from a 0.1 μg encapsulated dose matched levels seen with 1 μg unencapsulated dose. By day 3 expression levels exceeded those achieved with VRPs (squares). Thus expressed increased when the RNA was formulated in the liposomes relative to the naked RNA control, even at a 10× lower dose. Expression was also higher relative to the VRP control, but the kinetics of expression were very different (see FIG. 5). Delivery of the RNA with electroporation resulted in increased expression relative to the naked RNA control, but these levels were lower than with liposomes. Further SEAP experiments showed a clear dose response in vivo, with expression seen after delivery of as little as 1 ng RNA (FIG. 6). Further experiments comparing expression from encapsulated and naked replicons indicated that 0.01 μg encapsulated RNA was equivalent to 1 μg of naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a 12-fold higher expression at day 6; at a 0.1 μg dose levels were 24-fold higher at day 6. Rather than looking at average levels in the group, individual animals were also studied. Whereas several animals were non-responders to naked replicons, encapsulation eliminated non-responders. Further experiments replaced DlinDMA with DOTAP. Although the DOTAP liposomes gave better expression than naked replicon, they were inferior to the DlinDMA liposomes (2- to 3-fold difference at day 1). To assess in vivo immunogenicity a replicon was constructed to express full-length F protein from respiratory syncytial virus (RSV). This was delivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), or packaged in virions (106 IU; “VRP”) at days 0 and 21. FIG. 7 shows anti-F IgG titers 2 weeks after the second dose, and the liposomes clearly enhance immunogenicity. FIG. 8 shows titers 2 weeks later, by which point there was no statistical difference between the encapsulated RNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group. Neutralisation titers (measured as 60% plaque reduction, “PRNT60”) were not significantly different in these three groups 2 weeks after the second dose (FIG. 9). FIG. 12 shows both IgG and PRNT titers 4 weeks after the second dose. FIG. 13 confirms that the RNA elicits a robust CD8 T cell response. Further experiments compared F-specific IgG titers in mice receiving VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. Titer ratios (VRP: liposome) at various times after the second dose were as follows: 2 weeks 4 weeks 8 weeks 0.1 μg   2.9 1.0 1.1 1 μg 2.3 0.9 0.9 Thus the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery. Further experiments showed superior F-specific IgG responses with a 10 μg dose, equivalent responses for 1 μg and 0.1 μg doses, and a lower response with a 0.01 μg dose. FIG. 11 shows IgG titers in mice receiving the replicon in naked form at 3 different doses, in liposomes at 4 different doses, or as VRP (106 IU). The response seen with 1 μg liposome-encapsulated RNA was statistically insignificant (ANOVA) when compared to VRP, but the higher response seen with 10 μg liposome-encapsulated RNA was statistically significant (p<0.05) when compared to both of these groups. A further study confirmed that the 0.1 μg of liposome-encapsulated RNA gave much higher anti-F IgG responses (15 days post-second dose) than 0.1 μg of delivered DNA, and even was more immunogenic than 20 μg plasmid DNA encoding the F antigen, delivered by electroporation (Elgen™ DNA Delivery System, Inovio). Mice showed few visual signs of distress (weight loss, etc.) after receiving liposome-encapsulated RNA replicon, although a transient weight loss of 3-4% was seen after a second dose of 10 μg RNA. In contrast, delivery of 10 μg liposome-encapsulated DNA led to 8-10% weight loss. Mechanism of Action Bone marrow derived dendritic cells (pDC) were obtained from wild-type mice or the “Resq” (rsq1) mutant strain. The mutant strain has a point mutation at the amino terminus of its TLR7 receptor which abolishes TLR7 signalling without affecting ligand binding [46]. The cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000 or inside a liposome. As shown in FIG. 17, IL-6 and INFα were induced in WT cells but this response was almost completely abrogated in mutant mice. These results shows that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferons and pro-inflammatory cytokines. In general, liposome-delivered RNA replicons were shown to induce several serum cytokines within 24 hours of intramuscular injection (IFN-α, IP-10 (CXCL-10), IL-6, KC, IL-5, IL-13, MCP-1, and MIP-a), whereas only MIP-1 was induced by naked RNA and liposome alone induced only IL-6. IFN-α was shown to contribute to the immune response to liposome-encapsulated RSV-F-encoding replicon because an anti-IFNα receptor (IFNAR1) antibody reduced F-specific serum IgG a 10-fold reduction after 2 vaccinations. Liposome-delivered RNA replicons have generally been seen to elicit a balanced IgG1:IgG2a subtype profile in mice, sometimes with a higher IgG2a/IgG1 ratio than seen with electroporated DNA or with protein/MF59 immunizations (i.e. a Th1-type immune response). Liposome Manufacturing Methods In general, eight different methods have been used for preparing liposomes according to the invention. These are referred to in the text as methods (A) to (H) and they differ mainly in relation to filtration and TFF steps. Details are as follows: (A) Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts, San Diego, Calif.) and washed with plenty of MilliQ water before use to decontaminate the vials of RNases. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL of citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science, Oak Harbor, Wash.) using FEP tubing (fluorinated ethylene-propylene; al FEP tubing has a 2 mm internal diameter×3 mm outer diameter, supplied by Idex Health Science). The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of FEP tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 hour. 4 ml of the mixture was loaded into a 5 cc syringe, which was connected to a piece of FEP tubing and in another 5 cc syringe connected to an equal length of FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation, AnnArbor, Mich., USA). Before passing the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) were successively passed through the Mustang membrane. Liposomes were warmed for 10 min at 37° C. before passing through the membrane. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using TFF before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes (part number P/N: X1AB-100-20P) with a 100 kD pore size cutoff and 8 cm2 surface area were used. For in vitro and in vivo experiments, formulations were diluted to the required RNA concentration with 1×PBS. (B) As method (A) except that, after rocking, 226.7 μL of the stock was added to 1.773 mL ethanol to make a working lipid stock solution of 2 mL, thus modifying the lipid:RNA ratio. (C) As method (B) except that the Mustang filtration was omitted, so liposomes went from the 20 mL glass vial into the TFF dialysis. (D) As method (C) except that the TFF used polyethersulfone (PES) hollow fiber membranes (part number P-C1-100E-100-01N) with a 100 kD pore size cutoff and 20 cm2 surface area. (E) As method (D) except that a Mustang membrane was used, as in method (A). (F) As method (A) except that the Mustang filtration was omitted, so liposomes went from the 20 mL glass vial into the TFF dialysis. (G) As method (D) except that a 4 mL working solution of RNA was prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Then four 20 mL glass vials were prepared in the same way. Two of them were used for the RNA working solution (2 mL in each vial) and the others for collecting the lipid and RNA mixes, as in (C). Rather than use T mixer, syringes containing RNA and the lipids were connected to a Mitos Droplet junction Chip (a glass microfluidic device obtained from Syrris, Part no. 3000158) using PTFE tubing (0.03 inches internal diameter× 1/16 inch outer diameter) using a 4-way edge connector (Syrris). Two RNA streams and one lipid stream were driven by syringe pumps and the mixing of the ethanol and aqueous phase was done at the X junction (100 μm×105 μm) of the chip. The flow rate of all three streams was kept at 1.5 mL/min, hence the ratio of total aqueous to ethanolic flow rate was 2:1. The tube outlet was positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 h. Then the mixture was loaded in a 5 cc syringe, which was fitted to another piece of the PTFE tubing; in another 5 cc syringe with equal length of PTFE tubing, an equal volume of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 3 mL/min flow rate using a syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using TFF, as in (D). (H) As method (A) except that the 2 mL working lipid stock solution was made by mixing 120.9 μL of the lipid stock with 1.879 mL ethanol. Also, after mixing in the T mixer the liposomes from the 20 mL vial were loaded into Pierce Slide-A-Lyzer Dialysis Cassette (Thermo Scientific, extra strength, 0.5-3 mL capacity) and dialyzed against 400-500 mL of 1×PBS overnight at 4° C. in an autoclaved plastic container before recovering the final product. BHK Expression Liposomes with different lipids were incubated with BHK cells overnight and assessed for protein expression potency. From a baseline with RV05 lipid expression could be increased 18× by adding 10% 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to the liposome, 10× by adding 10% 18:2 (cis) phosphatidylcholine, and 900× by instead using RV01. In general, in vivo studies showed that unsaturated lipid tails tend to enhance IgG titers raised against encoded antigens. RSV Immunogenicity The vA317 self-replicating replicon encoding RSV F protein was administered to BALB/c mice, 4 or 8 animals per group, by bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with the replicon (1 μg) alone or formulated as liposomes with DlinDMA (“RV01”) or DOTAP (“RV13”). The RV01 liposomes had 40% DlinDMA, 10% DSPC, 48% cholesterol and 2% PEG-DMG, but with differing amounts of RNA. The RV13 liposomes had 40% DOTAP, 10% DPE, 48% cholesterol and 2% PEG-DMG. For comparison, naked plasmid DNA (20 μg) expressing the same RSV-F antigen was delivered either using electroporation or with RV01(10) liposomes (0.1 μg DNA). Four mice were used as a naïve control group. Liposomes were prepared by method (D) or method (B). For some liposomes made by method (D) a double or half amount of RNA was used. The Z average particle diameter, polydispersity index and encapsulation efficiency of the liposomes were as follows: RV Zav (nm) pdI % encapsulation Preparation RV01 (10) 158.6 0.088 90.7 (A) RV01 (08) 156.8 0.144 88.6 (A) RV01 (05) 136.5 0.136 99   (B) RV01 (09) 153.2 0.067 76.7 (A) RV01 (10) 134.7 0.147   87.8 * (A) RV13 (02) 128.3 0.179 97   (A) * For this RV01(10) formulation the nucleic acid was DNA not RNA Serum was collected for antibody analysis on days 14, 36 and 49. Spleens were harvested from mice at day 49 for T cell analysis. F-specific serum IgG titers (GMT) were as follows: RV Day 14 Day 36 Naked DNA plasmid 439 6712 Naked A317 RNA 78 2291 RV01 (10) 3020 26170 RV01 (08) 2326 9720 RV01 (05) 5352 54907 RV01 (09) 4428 51316 RV01 (10) DNA 5 13 RV13 (02) 644 3616 The proportion of T cells which are cytokine-positive and specific for RSV F51-66 peptide are as follows, showing only figures which are statistically significantly above zero: CD4+CD8− CD4−CD8+ RV IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα Naked 0.04 0.07 0.10 0.57 0.29 0.66 DNA plasmid Naked 0.04 0.05 0.08 0.57 0.23 0.67 A317 RNA RV01 (10) 0.07 0.10 0.13 1.30 0.59 1.32 RV01 (08) 0.02 0.04 0.06 0.46 0.30 0.51 RV01 (05) 0.08 0.12 0.15 1.90 0.68 1.94 RV01 (09) 0.06 0.08 0.09 1.62 0.67 1.71 RV01 (10) 0.03 0.08 DNA RV13 (02) 0.03 0.04 0.06 1.15 0.41 1.18 Thus the liposome formulations significantly enhanced immunogenicity relative to the naked RNA controls, as determined by increased F-specific IgG titers and T cell frequencies. Plasmid DNA formulated with liposomes, or delivered naked using electroporation, was significantly less immunogenic than liposome-formulated self-replicating RNA. The RV01 RNA vaccines were more immunogenic than the RV13 vaccine. RV01 has a tertiary amine in the headgroup with a pKa of about 5.8, and also include unsaturated alkyl tails. RV13 has unsaturated alkyl tails but its headgroup has a quaternary amine and is very strongly cationic. Liposomes—Requirement for Encapsulation To assess whether the effect seen in the liposome groups was due merely to the liposome components, or was linked to the encapsulation, the replicon was administered in encapsulated form (with two different purification protocols, 0.1 μg RNA), or mixed with the liposomes after their formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as naked RNA (1 μg). FIG. 10 shows that the lipoplex gave the lowest levels of expression, showing that shows encapsulation is essential for potent expression. Further experiments used three different RNAs: (i) ‘vA317’ replicon that expresses RSV-F i.e. the surface fusion glycoprotein of RSV; (ii) ‘vA17’ replicon that expresses GFP; and (iii) ‘vA336’ that is replication-defective and encodes GFP. RNAs were delivered either naked or with liposomes made by method (D). Empty liposomes were made by method (D) but without any RNA. Four liposome formulations had these characteristics: RNA Particle Size Zav (nm) Polydispersity RNA Encapsulation vA317 155.7 0.113 86.6% vA17 148.4 0.139  92% vA336 145.1 0.143 92.9% Empty 147.9 0.147 — BALB/c mice, 5 animals per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with: Group 1 naked self-replicating RSV-F RNA (vA317, 0.1 μg) Group 2 self-replicating RSV-F RNA (vA317, 0.1 μg) encapsulated in liposomes Group 3 self-replicating RSV-F RNA (vA317, 0.1 μg) added to empty liposomes Group 4 F subunit protein (5 μg) Serum was collected for antibody analysis on days 14, 35 and 51. F-specific specific serum IgG titers (GMT) were measured; if an individual animal had a titer of <25 (limit of detection), it was assigned a titer of 5. In addition, spleens were harvested from mice at day 51 for T cell analysis, to determine cells which were cytokine-positive and specific for RSV F51-66 peptide (CD4+) or for RSV F peptides F85-93 and F249-258 (CD8+). IgG titers were as follows in the 10 groups and in non-immunised control mice: Day 1 2 3 4 — 14 22 1819 5 5 5 35 290 32533 9 19877 5 51 463 30511 18 20853 5 RSV serum neutralization titers at day 51 were as follows: Day 1 2 3 4 51 35 50 24 38 Animals showing RSV F-specific CD4+ splenic T cells on day 51 were as follows, where a number (% positive cells) is given only if the stimulated response was statistically significantly above zero: Cytokine 1 2 3 4 IFN-γ 0.04 IL2 0.02 0.06 0.02 IL5 TNFα 0.03 0.05 Animals showing RSV F-specific CD8+ splenic T cells on day 51 were as follows, where a number is given only if the stimulated response was statistically significantly above zero: Cytokine 1 2 3 4 IFN-γ 0.37 0.87 IL2 0.11 0.40 0.04 IL5 TNFα 0.29 0.79 0.06 Thus encapsulation of RNA within the liposomes is necessary for high immunogenicity, as a simple admixture of RNA and the liposomes (group 3) was not immunogenic (in fact, less immunogenic than naked RNA). In other studies mice received various combinations of (i) self-replicating RNA replicon encoding full-length RSV F protein (ii) self-replicating GFP-encoding RNA replicon (iii) GFP-encoding RNA replicon with a knockout in nsP4 which eliminates self-replication (iv) full-length RSV F-protein. 13 groups in total received: A — — B 0.1 μg of (i), naked — C 0.1 μg of (i), encapsulated — in liposome D 0.1 μg of (i), with separate — liposomes E 0.1 μg of (i), naked 10 μg of (ii), naked F 0.1 μg of (i), naked 10 μg of (iii), naked G 0.1 μg of (i), encapsulated 10 μg of (ii), naked in liposome H 0.1 μg of (i), encapsulated 10 μg of (iii), naked in liposome I 0.1 μg of (i), encapsulated 1 μg of (ii), encapsulated in liposome in liposome J 0.1 μg of (i), encapsulated 1 μg of (iii), encapsulated in liposome in liposome K 5 μg F protein — L 5 μg F protein 1 μg of (ii), encapsulated in liposome M 5 μg F protein 1 μg of (iii), encapsulated in liposome Results in FIG. 16 show that F-specific IgG responses required encapsulation in the liposome rather than mere co-delivery (compare groups C & D). A comparison of groups K, L and M shows that the RNA provided an adjuvant effect against co-delivered protein, and this effect was seen with both replicating and non-replicating RNA. RSV Immunogenicity in Different Mouse Strains Replicon “vA142” encodes the full-length wild type surface fusion (F) glycoprotein of RSV but with the fusion peptide deleted, and the 3′ end is formed by ribozyme-mediated cleavage. It was tested in three different mouse strains. BALB/c mice were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 22. Animals were divided into 8 test groups (5 animals per group) and a naïve control (2 animals): Group 1 were given naked replicon (1 μg). Group 2 were given 1 μg replicon delivered in liposomes “RV01(37)” with 40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-conjugated DMG. Group 3 were given the same as group 2, but at 0.1 μg RNA. Group 4 were given 1 μg replicon in “RV17(10)” liposomes (40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG). Group 5 were 1 μg replicon in “RV05(11)” liposomes (40% RV07 lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG). Group 6 were given 0.1 μg replicon in “RV17(10)” liposomes. Group 7 were given 5 μg RSV-F subunit protein adjuvanted with aluminium hydroxide. Group 8 were a naïve control (2 animals) Sera were collected for antibody analysis on days 14, 35 and 49. F-specific serum IgG GMTs were: Day 1 2 3 4 5 6 7 8 14 82 2463 1789 2496 1171 1295 1293 5 35 1538 34181 25605 23579 13718 8887 73809 5 At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG 1 2 3 4 5 6 7 IgG1 94 6238 4836 7425 8288 1817 78604 IgG2a 5386 77064 59084 33749 14437 17624 24 RSV serum neutralizing antibody titers at days 35 and 49 were as follows (data are 60% plaque reduction neutralization titers of pools of 2-5 mice, 1 pool per group): Day 1 2 3 4 5 6 7 8 35 <20 143 20 101 32 30 111 <20 49 <20 139 <20 83 41 32 1009 <20 Spleens were harvested at day 49 for T cell analysis. Average net F-specific cytokine-positive T cell frequencies (CD4+ or CD8+) were as follows, showing only figures which were statistically significantly above zero (specific for RSV peptides F51-66, F164-178, F309-323 for CD4+, or for peptides F85-93 and F249-258 for CD8+): CD4+CD8− CD4−CD8+ Group IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα 1 0.03 0.06 0.08 0.47 0.29 0.48 2 0.05 0.10 0.08 1.35 0.52 1.11 3 0.03 0.07 0.06 0.64 0.31 0.61 4 0.05 0.09 0.07 1.17 0.65 1.09 5 0.03 0.08 0.07 0.65 0.28 0.58 6 0.05 0.07 0.07 0.74 0.36 0.66 7 0.02 0.04 0.04 8 C57BL/6 mice were immunised in the same way, but a 9th group received VRPs (1×106 IU) expressing the full-length wild-type surface fusion glycoprotein of RSV (fusion peptide deletion). Sera were collected for antibody analysis on days 14, 35 & 49. F-specific IgG titers (GMT) were: Day 1 2 3 4 5 6 7 8 9 14 1140 2133 1026 2792 3045 1330 2975 5 1101 35 1721 5532 3184 3882 9525 2409 39251 5 12139 At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG 1 2 3 4 5 6 7 8 IgG1 66 247 14 328 468 92 56258 79 IgG2a 2170 7685 5055 6161 1573 2944 35 14229 RSV serum neutralizing antibody titers at days 35 and 49 were as follows (data are 60% plaque reduction neutralization titers of pools of 2-5 mice, 1 pool per group): Day 1 2 3 4 5 6 7 8 9 35 <20 27 29 22 36 <20 28 <20 <20 49 <20 44 30 23 36 <20 33 <20 37 Spleens were harvested at day 49 for T cell analysis. Average net F-specific cytokine-positive T cell frequencies (CD8+) were as follows, showing only figures which were statistically significantly above zero (specific for RSV peptides F85-93 and F249-258): CD4−CD8+ Group IFNγ IL2 IL5 TNFα 1 0.42 0.13 0.37 2 1.21 0.37 1.02 3 1.01 0.26 0.77 4 1.26 0.23 0.93 5 2.13 0.70 1.77 6 0.59 0.19 0.49 7 0.10 0.05 8 9 2.83 0.72 2.26 Nine groups of C3H/HeN mice were immunised in the same way. F-specific IgG titers (GMT) were: Day 1 2 3 4 5 6 7 8 9 14 5 2049 1666 1102 298 984 3519 5 806 35 152 27754 19008 17693 3424 6100 62297 5 17249 At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG 1 2 3 4 5 6 7 8 IgG1 5 1323 170 211 136 34 83114 189 IgG2a 302 136941 78424 67385 15667 27085 3800 72727 RSV serum neutralizing antibody titers at days 35 and 49 were as follows: Day 1 2 3 4 5 6 7 8 9 35 <20 539 260 65 101 95 443 <20 595 49 <20 456 296 35 82 125 1148 <20 387 Thus three different lipids (RV01, RV05, RV17; pKa 5.8, 5.85, 6.1) were tested in three different inbred mouse strains. For all 3 strains RV01 was more effective than RV17; for BALB/c and C3H strains RV05 was less effective than either RV01 or RV17, but it was more effective in B6 strain. In all cases, however, the liposomes were more effective than two cationic nanoemulsions which were tested in parallel. CMV Immunogenicity RV01 liposomes with DLinDMA as the cationic lipid were used to deliver RNA replicons encoding cytomegalovirus (CMV) glycoproteins. The “vA160” replicon encodes full-length glycoproteins H and L (gH/gL), whereas the “vA322” replicon encodes a soluble form (gHsol/gL). The two proteins are under the control of separate subgenomic promoters in a single replicon; co-administration of two separate vectors, one encoding gH and one encoding gL, did not give good results. BALB/c mice, 10 per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0, 21 and 42 with VRPs expressing gH/gL (1×106 IU), VRPs expressing gHsol/gL (1×106 IU) and PBS as the controls. Two test groups received 1 μg of the vA160 or vA322 replicon formulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG; made using method (D) but with 150 μg RNA batch size). The vA160 liposomes had a Zav diameter of 168 nm, a pdI of 0.144, and 87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162 nm, a pdI of 0.131, and 90% encapsulation. The replicons were able to express two proteins from a single vector. Sera were collected for immunological analysis on day 63 (3wp3). CMV neutralization titers (the reciprocal of the serum dilution producing a 50% reduction in number of positive virus foci per well, relative to controls) were as follows: gH/gL VRP gHsol/gL VRP gH/gL liposome gHsol/gL liposome 4576 2393 4240 10062 RNA expressing either a full-length or a soluble form of the CMV gH/gL complex thus elicited high titers of neutralizing antibodies, as assayed on epithelial cells. The average titers elicited by the liposome-encapsulated RNAs were at least as high as for the corresponding VRPs. Repeat experiments confirmed that the replicon was able to express two proteins from a single vector. The RNA replicon gave a 3wp3 titer of 11457, compared to 5516 with VRPs. Further experiments used different replicons in addition to vA160. The vA526 replicon expresses the CMV pentameric complex (gH-gL-UL128-UL130-UL-131) under the control of three subgenomic promoters: the first drives the expression of gH; the second drives expression of gL; the third drives the expression of the UL128-2A-UL130-2A-UL131 polyprotein, which contains two 2A cleavage sites between the three UL genes. The vA527 replicon expresses the CMV pentameric complex via three subgenomic promoters and two IRESs: the first subgenomic promoter drives the expression of gH; the second subgenomic promoter drives expression of gL; the third subgenomic promoter drives the expression of the UL128; UL130 is under the control of an EMCV IRES; UL131 is under control of an EV71 IRES. These three replicons were delivered by liposome (method (H), with 150 μg batch size) or by VRPs. BALB/c mice, 10 groups of 10 animals, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0, 21 and 42 with: Group 1 VRPs expressing gH FL/gL (1×106 IU) Group 2 pentameric, 2A VRP (1×105 IU) Group 3 pentameric, 2A VRP (1×106 IU) Group 4 pentameric, IRES VRP (1×105 IU) Group 5 self-replicating RNA vA160 (l μg) formulated in liposomes Group 6 self-replicating RNA vA526 (1 μg) formulated in liposomes Group 7 self-replicating RNA vA527 (1 μg) formulated in liposomes Group 8 self-replicating RNA vA160 (1 μg) formulated in a cationic nanoemulsion Group 9 self-replicating RNA vA526 (1 μg) formulated in a cationic nanoemulsion Group 10 self-replicating RNA vA527 (1 μg) formulated in a cationic nanoemulsion. Sera were collected for immunological analysis on days 21 (3wp1), 42 (3wp2) and 63 (3wp3). CMV serum neutralization titers on days 21, 42 and 63 were: Vaccine Group 3wp1 3wp2 3wp3 1 126 6296 26525 2 N/A N/A 6769 3 N/A 3442 7348 4 N/A N/A 2265 5 347 9848 42319 6 179 12210  80000 7 1510  51200  130000 8 N/A N/A 845 9 N/A N/A 228 10 N/A N/A 413 Thus self-replicating RNA can be used to express multiple antigens from a single vector and to raise a potent and specific immune response. The replicon can express five antigens (CMV pentamric complex (gH-gL-UL128-UL130-UL-131) and raise a potent immune response. Self-replicating RNA delivered in liposomes was able to elicit high titers of neutralizing antibody, as assayed on epithelial cells, at all time points assayed (3wp1, 3wp2, and 3wp3). These responses were superior to the corresponding VRPs and to cationic nanoemulsions. Delivery Volume Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution to overcome the physical barriers of cell membranes which prevent large and membrane-impermeable compounds from entering cells. This phenomenon has previously been shown to be useful for the intracellular delivery of DNA vaccines. A typical mouse delivery volume for intramuscular injection is 50 μl into the hind leg, which is a relatively high volume for a mouse leg muscle. In contrast, a human intramuscular dose of ˜0.5 ml is relatively small. If immunogenicity in mice would be volume-dependent then the replicon vaccines' efficacy might be due, at least in part, on hydrodynamic forces, which would not be encouraging for use of the same vaccines in humans and larger animals. The vA317 replicon was delivered to BALB/c mice, 10 per group, by bilateral intramuscular vaccinations (5 or 50 per leg) on day 0 and 21: Group 1 received naked replicon, 0.2 μg in 50 μL per leg Group 2 received naked replicon, 0.2 μg in 5 μL per leg Group 3 received liposome-formulated replicon (0.2 μg, 50 μL per leg) Group 4 received liposome-formulated replicon (0.2 μg, 5 μL per leg) Serum was collected for antibody analysis on days 14 and 35. F-specific serum IgG GMTs were: Day 1 2 3 4 14 42 21 2669 2610 35 241 154 17655 18516 Thus immunogenicity of the formulated replicon did not vary according to the delivered volume, thus indicating that these RNA vaccines do not rely on hydrodynamic delivery for their efficacy. Expression Kinetics A self-replicating RNA replicon (“vA311”) that expresses a luciferase reporter gene (luc) was used for studying the kinetics of protein expression after injection. BALB/c mice, 5 animals per group, received bilateral intramuscular vaccinations (50 μL per leg) on day 0 with: Group 1 DNA expressing luciferase, delivered using electroporation (10 μg) Group 2 self-replicating RNA (1 μg) formulated in liposomes Group 3 self-replicating RNA (1 μg) formulated with a cationic nanoemulsion Group 4 self-replicating RNA (1 μg) formulated with a cationic nanoemulsion Group 5 VRP (1×106 IU) expressing luciferase Prior to vaccination mice were depilated. Mice were anesthetized (2% isoflurane in oxygen), hair was first removed with an electric razor and then chemical Nair. Bioluminescence data was then acquired using a Xenogen IVIS 200 imaging system (Caliper Life Sciences) on days 3, 7, 14, 21, 28, 35, 42, 49, 63 and 70. Five minutes prior to imaging mice were injected intraperitoneally with 8 mg/kg of luciferin solution. Animals were then anesthetized and transferred to the imaging system. Image acquisition times were kept constant as bioluminescence signal was measured with a cooled CCD camera. In visual terms, luciferase-expressing cells were seen to remain primarily at the site of RNA injection, and animals imaged after removal of quads showed no signal. In quantitative terms, luciferase expression was measured as average radiance over a period of 70 days (p/s/cm2/sr), and results were as follows for the 5 groups: Days 1 2 3 4 5 3 8.69E+07 3.33E+06 2.11E+06 9.71E+06 1.46E+07 7 1.04E+08 8.14E+06 1.83E+07 5.94E+07 1.64E+07 14 8.16E+07 2.91E+06 9.22E+06 3.48E+07 8.49E+05 21 1.27E+07 3.13E+05 6.79E+04 5.07E+05 6.79E+05 28 1.42E+07 6.37E+05 2.36E+04 4.06E+03 2.00E+03 35 1.21E+07 6.12E+05 2.08E+03 42 1.49E+07 8.70E+05 49 1.17E+07 2.04E+05 63 9.69E+06 1.72E+03 70 9.29E+06 The self-replicating RNA formulated with cationic nanoemulsions showed measurable bioluminescence at day 3, which peaked at day 7 and then reduced to background levels by days 28 to 35. When formulated in liposomes the RNA showed measurable bioluminescence at day 3, which peaked at day 7 and reduced to background levels by day 63. RNA delivered using VRPs showed enhanced bioluminescence at day 21 when compared to the formulated RNA, but expression had reduced to background levels by day 28. Electroporated DNA showed the highest level of bioluminescence at all time points measured and levels of bioluminescence did not reduce to background levels within the 70 days of the experiment. Delivery Route Liposome-encapsulated RNA encoding HIV gp140 was delivered to mice intramuscularly, intradermally, or subcutaneously. All three routes led to high serum IgG levels of HIV-specific antibodies (FIG. 15), exceeding titers seen in response to electroporated intramuscular DNA. Cotton Rats A study was performed in cotton rats (Sigmodon hispidis) instead of mice. At a 1 μg dose liposome encapsulation increased F-specific IgG titers by 8.3-fold compared to naked RNA and increased PRNT titers by 9.5-fold. The magnitude of the antibody response was equivalent to that induced by 5×106 IU VRP. Both naked and liposome-encapsulated RNA were able to protect the cotton rats from RSV challenge (1×105 plaque forming units), reducing lung viral load by at least 3.5 logs. Encapsulation increased the reduction by about 2-fold. Further work in cotton rats used four different replicons: vA317 expresses full-length RSV-F; vA318 expresses truncated (transmembrane and cytoplasmic tail removed) RSV-F; vA142 expresses RSV-F with its fusion peptide deleted; vA140 expresses the truncated RSV-F also without its peptide. Cotton rats, 4 to 8 animals per group, were given intramuscular vaccinations (100 μL in one leg) on days 0 and 21 with the four different replicons at two doses (1.0 and 0.1 μg) formulated in liposomes made by method (D), but with a 150 μg RNA batch size. Control groups received a RSV-F subunit protein vaccine (5 μg) adjuvanted with alum (8 animals/group), VRPs expressing full-length RSV-F (1×106 IU, 8 animals/group), or naïve control (4 animals/group). Serum was collected for antibody analysis on days 0, 21 and 34. F-specific serum IgG titers and RSV serum neutralizing antibody titers on day 21 and 34 were: IgG, IgG, NT, NT, Group day 21 day 34 day 21 day 34 1 μg vA317 915 2249 115 459 0.1 μg vA317 343 734 87 95 1 μg vA318 335 1861 50 277 0.1 μg vA318 129 926 66 239 1 μg vA142 778 4819 92 211 0.1 μg vA142 554 2549 78 141 1 μg vA140 182 919 96 194 0.1 μg vA140 61 332 29 72 5 μg F trimer subunit/alum 13765 86506 930 4744 1 × 106 IU VRP-F full 1877 19179 104 4528 Naïve 5 5 10 15 All four replicons evaluated in this study (vA317, vA318, vA142, vA140) were immunogenic in cotton rats when delivered by liposome, although serum neutralization titers were at least ten-fold lower than those induced by adjuvanted protein vaccines or by VRPs. The liposome/RNA vaccines elicited serum F-specific IgG and RSV neutralizing antibodies after the first vaccination, and a second vaccination boosted the response effectively. F-specific IgG titers after the second vaccination with 1 μg replicon were 2- to 3-fold higher than after the second vaccination with 0.1 μg replicon. The four replicons elicited comparable antibody titers, suggesting that full length and truncated RSV-F, each with or without the fusion peptide, are similarly immunogenic in cotton rats. Further work in cotton rats again used the vA317, vA318 and vA142 replicons. Cotton rats, 2-8 animals per group, were given intramuscular vaccinations (100 μL in one leg) on days 0 and 21 with the replicons (0.1 or 1 μg) encapsulated in RV01 liposomes made by method (D) but with a 150 μg RNA batch size. Control groups received the RSV-F subunit protein vaccine (5 μg) adjuvanted with alum or VRPs expressing full-length RSV-F (1×106 IU, 8 animals/group). All these animals received a third vaccination (day 56) with RSV-F subunit protein vaccine (5 μg) adjuvanted with alum. In addition there was a naïve control (4 animals/group). In addition, an extra group was given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 56 with 1 μg vA317 RNA in liposomes but did not receive a third vaccination with the subunit protein vaccine. Serum was collected for antibody analysis on days 0, 21, 35, 56, 70, plus days 14, 28 & 42 for the extra group. F-specific serum IgG titers (GMT) were as follows: Day 21 Day 35 Day 56 Day 70 1 μg vA318 260 1027 332 14263 0.1 μg vA318 95 274 144 2017 1 μg vA142 483 1847 1124 11168 0.1 μg vA142 314 871 418 11023 1 μg vA317 841 4032 1452 13852 1 × 106 VRP (F-full) 2075 3938 1596 14574 5 μg F trimer subunit/alum 12685 54526 25846 48864 Naïve 5 5 5 5 Serum neutralisation titers were as follows (60% RSV neutralization titers for 2 pools of 3-4 animals per group, GMT of these 2 pools per group): Day 21 Day 35 Day 56 Day 70 1 μg vA318 58 134 111 6344 0.1 μg vA318 41 102 63 6647 1 μg vA142 77 340 202 5427 0.1 μg vA142 35 65 56 2223 1 μg vA317 19 290 200 4189 1 × 106 VRP (F-full) 104 1539 558 2876 5 μg F trimer subunit/alum 448 4457 1630 3631 Naïve 10 10 10 Serum titers and neutralising titers for the extra group were as follows: Day 14 21 28 35 42 56 70 IgG 397 561 535 501 405 295 3589 NT 52 82 90 106 80 101 1348 Thus the replicons are confirmed as immunogenic in cotton rats, eliciting serum F-specific IgG and RSV neutralizing antibodies after the first vaccination. A second vaccination boosted the responses effectively. F-specific IgG titers after the second vaccination with 1.0 μg replicon were 1.5 to 4-fold higher than after the second vaccination with 0.1 μg replicon. The third vaccination (protein at day 56) did not boost titers in cotton rats previously vaccinated with F trimer subunit+alum, but it did provide a large boost to titers in cotton rats previously vaccinated with replicon. In most cases the RSV serum neutralization titers after two replicon vaccinations followed by protein boost were equal to or greater than titers induced by two or three sequential protein vaccinations. This study also evaluated the kinetics of the antibody response to 1.0 μg vA317. F-specific serum IgG and RSV neutralization titers induced by a single vaccination reached their peak around day 21 and were maintained through at least day 56 (50-70% drop in F-specific IgG titer, little change in RSV neutralization titer). A homologous second vaccination was given to these animals on day 56, and boosted antibody titers to a level at least equal to that achieved when the second vaccination was administered on day 21. Further experiments involved a viral challenge. The vA368 replicon encodes the full-length wild type surface fusion glycoprotein of RSV with the fusion peptide deleted, with expression driven by the EV71 IRES. Cotton rats, 7 per group, were given intramuscular vaccinations (100 μL per leg) on days 0 and 21 with vA368 in liposomes prepared by method (H), 175 μg RNA batch size, or with VRPs having the same replicon. A control group received 5 μg alum-adjuvanted protein, and a naïve control group was also included. All groups received an intranasal challenge (i.n.) with 1×106 PFU RSV four weeks after the final immunization. Serum was collected for antibody analysis on days 0, 21, 35. Viral lung titers were measured 5 days post challenge. Results were as follows: Liposome VRP Protein Naïve F-specific Serum IgG titers (GMT) Day 21 370 1017 28988 5 Day 35 2636 2002 113843 5 Neutralising titers (GMT) Day 21 47 65 336 10 Day 35 308 271 5188 10 Lung viral load (pfu per gram of lung) Day 54 422 225 124 694110 Thus the RNA vaccine reduced the lung viral load by over three logs, from approximately 106 PFU/g in unvaccinated control cotton rats to less than 103 PFU/g in vaccinated cotton rats. Large Mammal Study A large-animal study was performed in cattle. Calves (4-6 weeks old, ˜60-80 kg, 5 per group) were immunised with 66 μg of replicon vA317 encoding full-length RSV F protein at days 0, 21, 86 and 146. The replicons were formulated inside liposomes. PBS alone was used as a negative control, and a licensed vaccine was used as a positive control (“Triangle 4” from Fort Dodge, containing killed virus). All calves received 15 μg F protein adjuvanted with the MF59 emulsion on day 146. One cow was mistakenly vaccinated with the wrong vaccine on day 86 instead of Triangle 4 and so its data were excluded from day 100 onwards. The RNA vaccines encoded human RSV F whereas the “Triangle 4” vaccine contains bovine RSV F, but the RSV F protein is highly conserved between BRSV and HRSV. The liposomes were made by method (E), except a 1.5 mg RNA batch size was used. Calves received 2 ml of each experimental vaccine, administered intramuscularly as 2×1 ml on each side of the neck. In contrast, the “Triangle 4” vaccine was given as a single 2 ml dose in the neck. Serum was collected for antibody analysis on days 0, 14, 21, 35, 42, 56, 63, 86, 100, 107, 114, 121, 128, 135, 146, 160, 167, 174, 181, 188, 195, and 202. If an individual animal had a titer below the limit of detection it was assigned a titer of 5 FIG. 14A shows F-specific IgG titers over the first 63 days. The RNA replicon was immunogenic in the cows via liposomes, although it gave lower titers than the licensed vaccine. All vaccinated cows showed F-specific antibodies after the second dose, and titers were very stable from the period of 2 to 6 weeks after the second dose (and were particularly stable for the RNA vaccines). FIG. 14B shows F-specific serum IgG titers (GMT) over 210 days, and measured values up to day 202 were as follows: 3wp1 2wp2 5wp2 ~9wp2 2wp3 5wp3 8wp3 2wp4 5wp4 8wp4 D 0 D 21 D 35 D 56 D 86 D 100 D 121 D 146 D 160 D 181 D 202 PBS 5 5 5 5 5 5 5 5 46 98 150 Liposome 5 5 12 11 20 768 428 74 20774 7022 2353 Triangle 4 5 5 1784 721 514 3406 2786 336 13376 4775 2133 RSV serum neutralizing antibody titers were as follows: 2wp2 5wp2 2wp3 3wp3 4wp3 8wp3 2wp4 3wp4 4wp4 D 0 D 35 D 56 D 100 D 107 D 114 D 146 D 160 D 167 D 174 PBS 12 10 10 14 18 20 14 10 10 10 Liposome 13 10 10 20 13 17 13 47 26 21 Triangle 4 12 15 13 39 38 41 13 24 26 15 The material used for the second liposome dose was not freshly prepared, and the same lot of RNA showed a decrease in potency in a mouse immunogenicity study. Therefore it is possible that the vaccine would have been more immunogenic if fresh material had been used for all vaccinations. When assayed with complement, neutralizing antibodies were detected in all vaccinated cows. In this assay, all vaccinated calves had good neutralizing antibody titers after the second RNA vaccination Furthermore, the RNA vaccine elicited F-specific serum IgG titers that were detected in a few calves after the second vaccination and in all calves after the third. MF59-adjuvanted RSV-F was able to boost the IgG response in all previously vaccinated calves, and to boost complement-independent neutralization titers of calves previously vaccinated with RNA. Proof of concept for RNA vaccines in large animals is particularly important in light of the loss in potency observed previously with DNA-based vaccines when moving from small animal models to larger animals and humans. A typical dose for a cow DNA vaccine would be 0.5-1 mg [47,48] and so it is very encouraging that immune responses were induced with only 66 μg of RNA. It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. TABLE 1 useful phospholipids DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA 1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC 1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE 1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanol- amine DEPG 1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA 1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC 1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE 1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanol- amine DLPG 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DLPS 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA 1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanol- amine DMPG 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DMPS 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA 1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanol- amine DOPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DOPS 1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA 1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanol- amine DPPG 1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DPPS 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA 1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC 1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE 1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanol- amine DSPG 1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol . . .) DSPS 1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPC Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPC Hydrogenated Soy PC LYSOPC 1-Myristoyl-sn-Glycero-3-phosphatidylcholine MYRISTIC LYSOPC 1-Palmitoyl-sn-Glycero-3-phosphatidylcholine PALMITIC LYSOPC 1-Stearoyl-sn-Glycero-3-phosphatidylcholine STEARIC Milk 1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidyl- Sphingomyelin choline MPPC MSPC 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidyl- choline PMPC 1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidyl- choline POPC 1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidyl- choline POPE 1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidyl- ethanolamine POPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac- (1-glycerol) . . .] PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidyl- choline SMPC 1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidyl- choline SOPC 1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidyl- choline SPPC 1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidyl- choline REFERENCES [1] Johanning et al. (1995) Nucleic Acids Res 23:1495-1501. [2] Heyes et al. (2005) J Controlled Release 107:276-87. [3] WO2005/121348. [4] Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers: Methods and Protocols. (ed. Weissig). Humana Press, 2009. ISBN 160327359X. [5] Liposome Technology, volumes I, II & III. (ed. Gregoriadis). Informa Healthcare, 2006. [6] Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes). (eds. Arshady & Guyot). Citus Books, 2002. [7] Jeffs et al. (2005) Pharmaceutical Research 22 (3):362-372. [8] Polymers in Drug Delivery. (eds. Uchegbu & Schatzlein). CRC Press, 2006. [9] Microparticulate Systems for the Delivery of Proteins and Vaccines. (eds. Cohen & Bernstein). CRC Press, 1996. [10] O'Hagan et al. (2001) J Virology 75:9037-9043. [11] Singh et al. (2003) Pharmaceutical Research 20: 247-251. [12] WO2009/132206. [13] Martinon et al. (1993) Eur J Immunol 23:1719-22. [14] WO2005/113782. [15] WO2011/005799. [16] El Ouahabi et al. (1996) FEBS Letts 380:108-12. [17] Giuliani et al. (2006) Proc Natl Acad Sci USA 103(29): 10834-9. [18] WO2009/016515. [19] WO02/34771. [20] WO2005/032582. [21] WO2010/119343. [22] WO2006/110413. [23] WO2005/111066. [24] WO2005/002619. [25] WO2006/138004. [26] WO2009/109860. [27] WO02/02606. [28] WO03/018054. [29] WO2006/091517. [30] WO2008/020330. [31] WO2006/089264. [32] WO2009/104092. [33] WO2009/031043. [34] WO2007/049155. [35] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [36] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [37] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications) [38] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press). [39] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed., CRC Press, 1997) [40] Ausubel et al. (eds) (2002) Short protocols in molecular biology, 5th edition (Current Protocols). [41] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press) [42] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag) [43] Yoneyama & Fujita (2007) Cytokine & Growth Factor Reviews 18:545-51. [44] Maurer et al. (2001) Biophysical Journal, 80: 2310-2326. [45] Perri et al. (2003) J Virol 77:10394-10403. [46] Iavarone et al. (2011) J Immunol 186; 4213-22. [47] Boxus et al. (2007) J Virol 81:6879-89. [48] Taylor et al. (2005) Vaccine 23:1242-50. 13808089 glaxosmithkline biological sa USA B2 Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001. Open Apr 5th, 2022 05:12PM Apr 5th, 2022 05:12PM GlaxoSmithKline Health Care Pharmaceuticals & Biotechnology
nyse:gsk GlaxoSmithKline Dec 1st, 2009 12:00AM Jun 25th, 2007 12:00AM https://www.uspto.gov?id=US07626041-20091201 Carvedilol phosphate salts and/or solvates thereof, corresponding compositions, and/or methods of treatment The present invention relates to carvedilol phosphate salts, which include novel crystalline forms of carvedilol dihydrogen phosphate (i.e., dihydrogen phosphate salt of 1-(carbazol-4-yloxy-3-[[2-(o-methoxyphenoxy) ethyl]amino]-2-propanol) and/or carvedilol hydrogen phosphate, etc.), and/or solvates thereof, compositions containing the aforementioned salts and/or solvates, and methods of using the aforementioned salts and/or solvates to treat hypertension, congestive heart failure and angina, etc. 7626041 1. A compound which is carvedilol dihydrogen phosphate dihydrate having an x-ray diffraction pattern which comprises characteristic peaks in degrees two-theta (2θ) as shown in FIG. 9. 2. A compound which is carvedilol dihydrogen phosphate dihydrate having characteristic peaks from 0° degrees 2-theta (2θ) to 35° degrees 2-theta (2θ) at about 6.5±0.2 (2θ), 7.1±0.2 (2θ), 13.5 ±0.2 (2θ), 14.0±0.2 (2θ), 17.8±0.2 (2θ), 18.9±0.2 (2θ), and 21.0±0.2 (2θ). 3. A compound which is carvedilol dihydrogen phosphate dihydrate having an x-ray diffraction pattern which comprises characteristic peaks in degrees two-theta (2θ) as shown in FIG. 25. 4. A compound which is carvedilol dihydrogen phosphate dihydrate having characteristic peaks from 0° degrees 2-theta (2θ) to 35° degrees 2-theta (2θ) at about 6.4±0.2 (2θ), 9.6±0.2 (2θ), 16.0 ±0.2 (2θ), 18.4±0.2 (2θ), 20.7±0.2 (2θ), and 24.5±0.2 (2θ). 4 This application is a divisional application of U.S. application Ser. No. 10/518,654, now U.S. Pat. No. 7,268,156, Filed Dec. 16, 2004, which is a 371 application of PCT/US03/20408, Filed: Jun. 27, 2003, which derives priority to U.S. Prov. Appln. Ser. No. 60/392,175, now abandoned, Filed Jun. 27, 2002. FIELD OF THE INVENTION The present invention relates to a salt of carvedilol and/or corresponding solvates thereof, compositions containing such a salt of carvedilol and/or corresponding solvates thereof, and/or methods of using the aforementioned compound(s) in the treatment of certain disease states in mammals, in particular man. The present invention further relates to carvedilol phosphate salts, which include novel crystalline forms of carvedilol dihydrogen phosphate (i.e., such as dihydrogen phosphate salt of 1-(carbazol-4-yloxy-3-[[2-(o-methoxyphenoxy) ethyl]amino]-2-propanol), carvedilol hydrogen phosphate, etc.), and/or other corresponding solvates thereof, compositions containing such salts and/or solvates, and methods of using the aformentioned compounds to treat hypertension, congestive heart failure and angina, etc. BACKGROUND OF THE INVENTION The compound, 1-(carbazol-4-yloxy-3-[[2-(o-methoxyphenoxy) ethyl]-amino]-2-propanol is known as Carvedilol. Carvedilol is depicted by the following chemical structure: Carvedilol is disclosed in U.S. Pat. No. 4,503,067 to Wiedemann et al. (i.e., assigned to Boehringer Mannheim, GmbH, Mannheim-Waldhof, Fed. Rep. of Germany), which was issued on Mar. 5, 1985. Currently, carvedilol is synthesized as free base for incorporation in medication that is available commercially. The aforementioned free base form of Carvedilol is a racemic mixture of R(+) and S(−) enantiomers, where nonselective β-adrenoreceptor blocking activity is exhibited by the S(−) enantiomer and α-adrenergic blocking activity is exhibited by both R(+) and S(−) enantiomers. Those unique features or characteristics associated with such a racemic Carvedilol mixture contributes to two complementary pharmacologic actions: i.e., mixed venous and arterial vasodilation and non-cardioselective, beta-adrenergic blockade. Carvedilol is used for treatment of hypertension, congestive heart failure and angina. The currently commercially available carvedilol product is a conventional, tablet prescribed as a twice-a-day (BID) medication in the United States. Carvedilol contains an α-hydroxyl secondary amine functional group, which has a pKa of 7.8. Carvedilol exhibits predictable solubility behaviour in neutral or alkaline media, i.e. above a pH of 9.0, the solubility of carvedilol is relatively low (<1 μg/mL). The solubility of carvedilol increases with decreasing pH and reaches a plateau near pH=5, i.e. where saturation solubility is about 23 μg/mL at pH=7 and about 100 μg/mL at pH=5 at room temperature. At lower pH values (i.e., at a pH of 1 to 4 in various buffer systems), solubility of carvedilol is limited by the solubility of its protonated form or its corresponding salt formed in-situ. The hydrochloride salt of carvedilol generated in situ in acidic medium, which simulates gastric fluid, is less soluble in such medium. In light of the foregoing, a salt, and/or novel crystalline form of carvedilol with greater aqueous solubility, chemical stability, etc. would offer many potential benefits for provision of medicinal products containing the drug carvedilol. Such benefits would include products with the ability to achieve desired or prolonged drug levels in a systemic system by sustaining absorption along the gastro-intestinal tract of mammals (i.e., such as humans), particularly in regions of neutral pH, where a drug, such as carvedilol, has minimal solubility. Surprisingly, it has now been shown that a novel crystalline form of carvedilol phosphate salt (i.e., such as carvedilol dihydrogen phosphate and/or carvedilol hydrogen phosphate, etc.) can be isolated as a pure, crystalline solid, which exhibits much higher aqueous solubility than the corresponding free base or other prepared crystalline salts of carvedilol, such as the hydrochloride salt. This novel crystalline form also has potential to improve the stability of carvedilol in formulations due to the fact that the secondary amine functional group attached to the carvedilol core structure, a moiety pivotal to degradation processes, is protonated as a salt. In light of the above, a need exists to develop different carvedilol forms and/or different compositions, respectively, which have greater aqueous solubility, chemical stability, sustained or prolonged drug or absorption levels (i.e., such as in neutral gastrointestinal tract pH regions, etc.). There also exists a need to develop methods of treatment for hypertension, congestive heart failure or angina, etc. which comprises administration of the aforementioned carvedilol phosphate salts and/or solvates thereof or corresponding pharmaceutical compositions, which contain such salts, and/or solvates. The present invention is directed to overcoming these and other problems encountered in the art. SUMMARY OF THE INVENTION The present invention relates to a salt of carvedilol and/or corresponding solvates thereof, compositions containing such carvedilol and/or corresponding solvates thereof, and/or methods of using the aforementioned compound(s) in the treatment of certain disease states in mammals, in particular man. The present invention further relates to carvedilol phosphate salts, which include novel crystalline forms of carvedilol phosphate (i.e., such as dihydrogen phosphate salt of 1-(carbazol-4-yloxy-3-[[2-(o-methoxyphenoxy) ethyl]amino]-2-propanol), carvedilol hydrogen phosphate, etc.), and/or other corresponding solvates thereof. The present invention relates to a pharmaceutical composition, which contains carvedilol phosphate salts and/or solvates thereof. The present invention further relates to a method of treating hypertension, congestive heart failure and angina, which comprises administering to a subject in need thereof an effective amount of a carvedilol phosphate salt (which include novel crystalline forms) and/or solvates thereof or a pharmaceutical composition (i.e., which contains such salts and/or solvates of carvedilol phosphate), etc. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is an x-ray powder diffractogram for carvedilol dihydrogen phosphate hemihydrate (Form I). FIG. 2 shows the thermal analysis results for carvedilol dihydrogen phosphate hemihydrate (Form I). FIG. 3 is an FT-Raman spectrum for carvedilol dihydrogen phosphate hemihydrate (Form I). FIG. 4 is an FT-Raman spectrum for carvedilol dihydrogen phosphate hemihydrate in the 4000-2000 cm−1 region of the spectrum (Form I). FIG. 5 is an FT-Raman spectrum for carvedilol dihydrogen phosphate hemihydrate in the 2000-400 cm−1 region of the spectrum (Form I). FIG. 6 is an FT-IR spectrum for carvedilol dihydrogen phosphate hemihydrate (Form I). FIG. 7 is an FT-IR spectrum for carvedilol dihydrogen phosphate hemihydrate in the 4000-2000 cm−1 region of the spectrum (Form I). FIG. 8 is an FT-IR spectrum for carvedilol dihydrogen phosphate hemihydrate in the 2000-500 cm−1 region of the spectrum (Form I). FIG. 9 is an x-ray powder diffractogram for carvedilol dihydrogen phosphate dihydrate (Form II). FIG. 10 shows the thermal analysis results for carvedilol dihydrogen phosphate dihydrate (Form II). FIG. 11 is an FT-Raman spectrum for carvedilol dihydrogen phosphate dihydrate (Form II). FIG. 12 is an FT-Raman spectrum for carvedilol dihydrogen phosphate dihydrate in the 4000-2000 cm−1 region of the spectrum (Form II). FIG. 13 is an FT-Raman spectrum for carvedilol dihydrogen phosphate dihydrate in the 2000-400 cm−1 region of the spectrum (Form II). FIG. 14 is an FT-IR spectrum for carvedilol dihydrogen phosphate dihydrate (Form II). FIG. 15 is an FT-IR spectrum for carvedilol dihydrogen phosphate dihydrate in the 4000-2000 cm−1 region of the spectrum (Form II). FIG. 16 is an FT-IR spectrum for carvedilol dihydrogen phosphate dihydrate in the 2000-500 cm−1 region of the spectrum (Form II). FIG. 17 shows the thermal analysis results for carvedilol dihydrogen phosphate methanol solvate (Form III). FIG. 18 is an FT-Raman spectrum for carvedilol dihydrogen phosphate methanol solvate (Form III). FIG. 19 is an FT-Raman spectrum for carvedilol dihydrogen phosphate methanol solvate in the 4000-2000 cm−1 region of the spectrum (Form III). FIG. 20 is an FT-Raman spectrum for carvedilol dihydrogen phosphate methanol solvate in the 2000-400 cm−1 region of the spectrum (Form III). FIG. 21 is an FT-IR spectrum for carvedilol dihydrogen phosphate methanol solvate (Form III). FIG. 22 is an FT-IR spectrum for carvedilol dihydrogen phosphate methanol solvate in the 4000-2000 cm−1 region of the spectrum (Form III). FIG. 23 is an FT-IR spectrum for carvedilol dihydrogen phosphate methanol solvate in the 2000-500 cm−1 region of the spectrum (Form III). FIG. 24 is an x-ray powder diffractogram for carvedilol dihydrogen phosphate methanol solvate (Form III). FIG. 25 is an x-ray powder diffractogram for carvedilol dihydrogen phosphate dihydrate (Form IV). FIG. 26 is a solid state 13C NMR for carvedilol dihydrogen phosphate dihydrate (Form I). FIG. 27 is a solid state 31P NMR for carvedilol dihydrogen phosphate dihydrate (Form I). FIG. 28 is an x-ray powder diffractogram for carvedilol dihydrogen phosphate (Form V). FIG. 29 is an x-ray powder diffractogram for carvedilol hydrogen phosphate (Form VI). DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a salt of carvedilol and/or corresponding solvates thereof, compositions containing such carvedilol salts and/or corresponding solvates thereof, and/or methods of using the aforementioned compound(s) in the treatment of certain disease states in mammals, in particular man. The present invention further relates to carvedilol phosphate salts, which include novel crystalline forms of carvedilol dihydrogen phosphate (i.e., such as dihydrogen phosphate salt of 1-(carbazol-4-yloxy-3-[[2-(o-methoxyphenoxy) ethyl]amino]-2-propanol), carvedilol hydrogen phosphate, etc.) and/or other carvedilol phosphate solvates thereof. The present invention relates to a pharmaceutical composition, which contains carvedilol phosphate salts and/or solvates thereof. The present invention further relates to a method of treating hypertension, congestive heart failure and angina, which comprises administering to a subject in need thereof an effective amount of a carvedilol phosphate salt (which include novel crystalline forms), and/or solvates thereof or a pharmaceutical composition (i.e., which contains such salts and/or solvates of carvedilol phosphate), etc. Carvedilol is disclosed and claimed in U.S. Pat. No. 4,503,067 to Wiedemann et al. (“U.S. '067 Patent”). Reference should be made to U.S. '067 Patent for its full disclosure, which include methods of preparing and/or using the carvedilol compound, etc. The entire disclosure of the U.S. '067 Patent is incorporated hereby by reference in its entirety. The present invention relates to a compound, which is a salt and/or novel crystalline forms of carvedilol phosphate (i.e., which include crystalline forms of carvedilol dihydrogen phosphate, carvedilol hydrogen phosphate, etc.) and/or solvates of carvedilol phosphate (i.e., which include carvedilol dihydrogen phosphate hemihydrate, carvedilol dihydrogen phosphate dihydrate (i.e., such as Forms II and IV, respectively, etc.), and/or carvedilol dihydrogen phosphate methanol solvate, etc.) In accordance with the present invention, it has been unexpectedly found that carvedilol dihydrogen phosphate can be isolated readily as novel crystalline forms, which displays much higher solubility when compared to the free base of carvedilol. An example in the present invention of a novel carvedilol phosphate salt is a novel crystalline form of carvedilol dihydrogen phosphate (i.e., identified as the dihydrogen phosphate salt of 1-(carbazol-4-yloxy-3-[[2-(o-methoxyphenoxy) ethyl]amino]-2-propanol). In accordance with the present invention, other carvedilol phosphate salts, and/or solvates of the present invention may be isolated as different solid and/or crystalline forms. Moreover, a specific identified species of a carvedilol phosphate salt (or a specific identified corresponding solvate species) also may also be isolated in various different crystalline or solid forms. For example, carvedilol dihydrogen phosphate, may be isolated in two different and distinct crystalline forms, Forms II and IV (see, Examples 2 and 4), respectively represented and substantially shown FIGS. 9 to 6 (for Form II) and FIG. 25 (for Form IV), which are represent spectroscopic and/or other characterizing data. It is recognized that the compounds of the present invention may exist in forms as stereoisomers, regioisomers, or diastereiomers, etc. These compounds may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. For example, carvedilol may exist as as racemic mixture of R(+) and S(−) enantiomers, or in separate respectively optically forms, i.e., existing separately as either the R(+) enantiomer form or in the S(+) enantiomer form. All of these individual compounds, isomers, and mixtures thereof are included within the scope of the present invention. Suitable solvates of carvedilol phosphate as defined in the present invention, include, but are not limited to carvedilol dihydrogen phosphate hemihydrate, carvedilol dihydrogen phosphate dihydrate (i.e., which include Forms II and IV, respectively), carvedilol dihydrogen phosphate methanol solvate, and carvedilol hydrogen phosphate, etc. In particular, crystalline carvedilol dihydrogen phosphate hemihydrate of the instant invention can be prepared by crystallization from an acetone-water solvent system containing carvedilol and H3PO4. In accordance with the present invention suitable, solvates of the present invention may be prepared by preparing a slurrying a carvedilol phosphate salt, such as a carvedilol dihydrogen salt, in a solvent, such as methanol. According to the instant invention, the various forms of carvedilol dihydrogen phosphate (i.e. which include salts and/or solvates thereof) are distinguished from each other using different characterization or identification techniques. Such techniques, include solid state 13C Nuclear Magnetic Resonance (NMR), 31P Nuclear Magnetic Resonance (NMR), Infrared (IR), Raman, X-ray powder diffraction, etc. and/or other techniques, such as Differential Scanning Calorimetry (DSC) (i.e., which measures the amount of energy (heat) absorbed or released by a sample as it is heated, cooled or held at constant temperature). In general, the aforementioned solid state NMR techniques are non-destructive techniques to yield spectra, which depict an NMR peak for each magnetically non-equivalent carbon site the solid-state For example, in identification of compounds of the present invention, 13C NMR spectrum of a powdered microcrystalline organic molecules reflect that the number of peaks observed for a given sample will depend on the number of chemically unique carbons per molecule and the number of non-equivalent molecules per unit cell. Peak positions (chemical shifts) of carbon atoms reflect the chemical environment of the carbon in much the same manner as in solution-state 13C NMR. Although peaks can overlap, each peak is in principle assignable to a single type of carbon. Therefore, an approximate count of the number of carbon sites observed yields useful information about the crystalline phase of a small organic molecule. Based upon the foregoing, the same principles apply to phosphorus, which has additional advantages due to high sensitivity of the 31P nucleus. Polymorphism also can be studied by comparison of 13C and 31P spectra. In the case of amorphous material, broadened peak shapes are usually observed, reflecting the range of environments experienced by the 13C or 31P sites in amorphous material types. Specifically, carvedilol dihydrogen phosphate salts, hydrates, and/or solvates thereof, substantially shown by the data described in FIGS. 1-29. For example, crystalline carvedilol dihydrogen phosphate hemihydrate (see, Example 1: Form I) is identified by an x-ray diffraction pattern as shown substantially in FIG. 1, which depicts characteristic peaks in degrees two-theta (2θ): i.e., 7.0±0.2 (2θ), 11.4±0.2 (2θ), 15.9±0.2 (2θ), 18.8±0.2 (2θ), 20.6±0.2 (2θ), 22.8±0.2 (2θ), and 25.4±0.2 (2θ). Crystalline carvedilol dihydrogen phosphate dihydrate (see, Example 2: Form II) is identified by an x-ray diffraction pattern as shown substantially in FIG. 9, which depicts characteristic peaks in degrees two-theta (2θ): i.e., 6.5±0.2 (2θ), 7.1±0.2 (2θ), 13.5±0.2 (2θ), 14.0±0.2 (2θ), 17.8±0.2 (2θ), 18.9±0.2 (2θ), and 21.0±0.2 (2θ). Crystalline carvedilol dihydrogen phosphate methanol solvate (see, Example 3: Form III) is identified by an x-ray diffraction pattern as shown substantially in FIG. 24, which depicts characteristic peaks in degrees two-theta (2θ): i.e., 6.9±0.2 (2θ), 7.2±0.2 (2θ), 13.5±0.2 (2θ), 14.1±0.2 (2θ), 17.8±0.2 (2θ), and 34.0±0.2 (2θ). Crystalline carvedilol dihydrogen phosphate dihydrate (see, Example 4: Form IV) is identified by an x-ray diffraction pattern as shown substantially in FIG. 25, which depicts characteristic peaks in degrees two-theta (2θ): i.e., 6.4±0.2 (2θ), 9.6±0.2 (2θ), 16.0±0.2 (2θ), 18.4±0.2 (2θ), 20.7±0.2 (2θ), and 24.5±0.2 (2θ). Crystalline carvedilol dihydrogen phosphate (see, Example 5: Form V) is identified by an x-ray diffraction pattern as shown substantially in FIG. 28, which depicts characteristic peaks in degrees two-theta (2θ): i.e., 13.2±0.2 (2θ), 15.8±0.2 (2θ), 16.3±0.2 (2θ), 21.2±0.2 (2θ), 23.7±0.2 (2θ), and 26.0±0.2 (2θ). Crystalline carvedilol hydrogen phosphate (see, Example 6: Form VI) is identified by an x-ray diffraction pattern as shown substantially in FIG. 29, which depicts characteristic peaks in degrees two-theta (2θ): i.e., 5.5±0.2 (2θ), 12.3±0.2 (2θ), 15.3±0.2 (2θ), 19.5±0.2 (2θ), 21.6±0.2 (2θ), and 24.9±0.2 (2θ). The present invention also relates to a pharmaceutical composition, which contains a salt of carvedilol phosphate and/or corresponding solvates thereof. Importantly, the chemical and/or physical properties of carvedilol forms described herein, which include salts of carvedilol dihydrogen phosphates, such as novel crystalline forms, and/or solvates thereof indicate that those forms may be particularly suitable for inclusion in medicinal agents, pharmaceutical compositions, etc. For example, solubility of various carvedilol salts, and/or solvates as those described herein may facilitate provision or development of a dosage form from which the drug substance becomes available for bioabsorption throughout the gastrointestinal tract (i.e., in particular the lower small intestine and colon). In light of the foregoing, it may be possible to develop stable controlled release dosage forms containing such carvedilol phosphate salts and/or solvates of the present invention, etc., for once-per-day dosage, delayed release or pulsatile release to optimize therapy by matching pharmacokinetic performance with pharmacodynamic requirements. Compounds or compositions within the scope of this invention include all compounds or compositions, wherein the compound of the present invention is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Thus, this invention also relates to a pharmaceutical composition comprising an effective amount of carvedilol dihydrogen phosphate salts and/or solvates thereof, with any of the characteristics noted herein, in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents thereof, and if desired, other active ingredients. Moreover, the quantity of the compound or composition of the present invention administered will vary depending on the patient and the mode of administration and can be any effective amount. Treatment regimen for the administration of the compounds and/or compositions of the present invention can also be determined readily by those with ordinary skill in art. The quantity of the compound and/or composition of the present invention administered may vary over a wide range to provide in a unit dosage an effective amount based upon the body weight of the patient per day to achieve the desired effect. In particular, a composition of the present invention is presented as a unit dose and taken preferably from 1 to 2 times daily, most preferably once daily to achieve the desired effect. Depending upon the treatment being effected, the compounds, and/or or compositions of the present invention can be administered orally, intravascularly, intraperitoneally, subcutaneously, intramuscularly or topically. Preferably, the composition is adapted for oral administration. In general, pharmaceutical compositions of the present invention are prepared using conventional materials and techniques, such as mixing, blending and the like. In accordance with the present invention, compounds and/or pharmaceutical composition can also include, but are not limited to, suitable adjuvants, carriers, excipients, or stabilizers, etc. and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, etc. Typically, the composition will contain a compound of the present invention, such as a salt of carvedilol or active compound(s), together with the adjuvants, carriers and/or excipients. In particular, a pharmaceutical composition of the present invention comprises an effective amount of a salt of carvedilol (i.e., such as carvedilol dihydrogen phosphate salts) and/or corresponding solvates (i.e., as identified herein) thereof, with any of the characteristics noted herein, in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents thereof, and if desired, other active ingredients. In accordance with the present invention, solid unit dosage forms can be conventional types known in the art. The solid form can be a capsule and the like, such as an ordinary gelatin type containing the compounds of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch, etc. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate, etc. The tablets, capsules, and the like can also contain a binder, such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose, or saccharin, etc. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets can be coated with shellac, sugar, or both, etc. A syrup can contain, in addition to active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor, etc. For oral therapeutic administration, these active compounds can be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. The percentage of the compound in compositions can, of course, be varied as the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Typically in accordance with the present invention, the oral maintenance dose is between about 25 mg and about 50 mg, preferably given once daily. In accordance with the present invention, the preferred unit dosage forms include tablets or capsules. The active compounds of the present invention may be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they can be enclosed in hard or soft shell capsules, or they can be compressed into tablets, or they can be incorporated directly with the food of the diet, etc. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils, etc. The compounds or pharmaceutical compositions of the present invention may also be administered in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical adjuvant, carrier or excipients. Such adjuvants, carriers and/or excipients, include, but are not limited to sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers, etc. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil, etc. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions, etc. These active compounds may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil, etc. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, etc., are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compounds and/or compositions prepared according to the present invention can be used to treat warm blooded animals, such as mammals, which include humans. Conventional administration methods may be suitable for use in the present invention. The present invention further relates to a method of treating hypertension, congestive heart failure and angina, which comprises administering to a subject in need thereof an effective amount of a carvedilol phosphate salt (i.e., which include novel crystalline forms) and/or solvates thereof or a pharmaceutical composition (i.e., which contains such salts and/or solvates of carvedilol phosphate), etc. The Examples set forth below are illustrative of the present invention and are not intended to limit, in any way, the scope of the present invention. EXAMPLES Example 1 Form I Carvedilol Dihydrogen Phosphate Hemihydrate Preparation A suitable reactor is charged with acetone. The acetone solution is sequentially charged with carvedilol and water. Upon addition of the water, the slurry dissolves quicky. To the solution is added aqueous H3PO4. The reaction mixture is stirred at room temperature and carvedilol dihydrogen phosphate seeds are added in one portion. The solid precipitate formed is stirred, then filtered and the collected cake is washed with aqueous acetone. The cake is dried under vacuum to a constant weight. The cake is weighed and stored in a polyethylene container. Example 2 Form II Carvedilol Dihydrogen Phosphate Dihydrate Preparation Form I is slurried in acetone/water mixture between 10 and 30° C. for several days. Example 3 Form III Carvedilol Dihydrogen phosphate Methanol Solvate Preparation Form I i