High purity lipopeptides

Abstract

The invention discloses highly purified daptomycin and to pharmaceutical compositions comprising this compound. The invention discloses a method of purifying daptomycin comprising the sequential steps of anion exchange chromatography, hydrophobic interaction chromatography and anion exchange chromatography. The invention also discloses a method of purifying daptomycin by modified buffer enhanced anion exchange chromatography. The invention also discloses an improved method for producing daptomycin by fermentation of Streptomyces roseosporus. The invention also discloses high pressure liquid chromatography methods for analysis of daptomycin purity. The invention also discloses lipopeptide micelles and methods of making the micelles. The invention also discloses methods of using lipopeptide micelles for purifying lipopeptide antibiotics, such as daptomycin. The invention also discloses using lipopeptide micelles therapeutically.

1 . A composition comprising (a) essentially pure daptomycin, (b) daptomycin that is substantially free of anhydro-daptomycin and substantially free of β-isomer of daptomycin, (c) daptomycin that is essentially free of anhydro-daptomycin and substantially free of β-isomer of daptomycin, (d) daptomycin that is free of anhydro-daptomycin and substantially free of β-isomer of daptomycin, (e) daptomycin that is substantially free of impurities 1 to 14 or (f) daptomycin that is essentially free of impurities 1 to 14.  
     
     
         2 . The composition of  claim 1  comprising essentially pure daptomycin.  
     
     
         3 . The composition of  claim 1  compromising daptomycin that is substantially free of anhydro-daptomycin and substantially free of β-isomer of daptomycin.  
     
     
         4 . The composition according to  claim 3  that is essentially free of anhydro-daptomycin.  
     
     
         5 . The composition according to  claim 3  that is free of anhydro-daptomycin.  
     
     
         6 . The composition of  claim 1  that is substantially free of each of impurities 1 to 14.  
     
     
         7 . The composition according to  claim 6  that is essentially free of each of impurities 1 to 14.  
     
     
         8 . The composition of  claim 1 , wherein daptomycin purity is measured by HPLC.  
     
     
         9 . A pharmaceutical composition comprising the composition of  claim 1 .  
     
     
         10 . A pharmaceutical composition according to  claim 9 , further comprising one or more antibiotics, one or more antifungal agents, or both an antibiotic and an antifungal agent.  
     
     
         11 . The composition according to  claim 1  wherein the daptomycin is purified by a process comprising the steps of: 
 b) clarifying the fermentation broth to obtain a clarified solution;    c) subjecting the clarified solution to anion exchange chromatography to obtain an enriched daptomycin preparation;    d) subjecting the enriched daptomycin preparation to hydrophobic interaction chromatography to obtain a semi-purified daptomycin preparation; and    e) subjecting the semi-purified daptomycin preparation to anion exchange chromatography to obtain purified daptomycin.    
     
     
         12 . The composition according to  claim 11 , wherein the feed of n-decanoic acid in step a) is regulated to achieve a residual concentration of n-decanoic acid of no more than 50 parts per million (ppm) during fermentation.  
     
     
         13 . The composition according to  claim 11 , wherein said clarifying in step b) comprises filtration or centrifugation and depth filtration.  
     
     
         14 . The composition according to  claim 11 , wherein the anion exchange chromatography in step c) is performed using a resin comprising a copolymer of 2-methacrylic acid and ethyleneglycol dimethacrylate (EDGM).  
     
     
         15 . The composition according to  claim 11 , wherein the hydrophobia interaction chromatography in step d) is performed using a resin comprising a co-polymer of cross-linked divinylbenzene/stryene.  
     
     
         16 . The composition according to  claim 15 , wherein the hydrophobic interaction chromatography is performed at neutral pH and a solvent concentration that is reduced compared to the solvent concentration used when performing the hydrophobic interaction chromatography at acidic pH.  
     
     
         17 . The composition according to  claim 16 , wherein the resin is recycled by loading the column at an acidic pH and eluting the column at a neutral pH.  
     
     
         18 . The composition according to  claim 11 , wherein the anion exchange chromatography in step e) is performed using a resin comprising a copolymer of 2-methacrylic acid and ethyleneglycol dimethacrylate (EDGM).  
     
     
         19 . The composition according to  claim 1   1 , wherein the anion exchange chromatography in step e) is used to reduce the level of solvent from step b).  
     
     
         20 . The composition according to  claim 11 , wherein the anion exchange chromatography is performed via continuous flow chromatography.  
     
     
         21 . The composition according to  claim 11 , wherein the process further comprises the step of filtering daptomycin.  
     
     
         22 . The composition according to  claim 11 , wherein the process further comprises the step of depyrogenating daptomycin using ultrafiltration.  
     
     
         23 . The composition according to  claim 22  wherein said depyrogenating comprises the steps of: 
 i) providing a daptomycin solution under conditions in which the daptomycin is in a monomeric and nonmicellar state;    ii) filtering the daptomycin solution under conditions in which the daptomycin passes through the filter but pyrogens do not pass through the filter;    iii) subjecting the daptomycin solution to conditions forming a daptomycin aggregate;    iv) filtering the daptomycin aggregate under conditions in which the daptomycin aggregate is retained on the filter; and    v) collecting the daptomycin aggregate.    
     
     
         24 . The composition according to  claim 22 , wherein the process further comprises the step of lyophilizing daptomycin.  
     
     
         25 . The composition according to  claim 22 , wherein the anion exchange chromatography is performed via radial flow chromatography.  
     
     
         26 . The composition according to any one of  claim 11 , wherein said clarifying in step b) comprises microfiltration or centrifugation.  
     
     
         27 . The composition according to  claim 11 , wherein the process further comprises the steps of filtering and concentrating daptomycin.  
     
     
         28 . The composition according to  claim 11 , wherein the process further comprises the step of separating the enriched daptomycin obtained in step c) from low molecular weight material by ultrafiltration.  
     
     
         29 . The composition according to  claim 28 , wherein the process further comprises the step of depyrogenating the daptomycin.