Methods for preparing purified lipopeptides

Abstract

The present invention relates to crystalline and crystal-like forms of lipopeptides, including daptomycin, a lipopeptide antibiotic with potent bactericidal activity against gram-positive bacteria, including strains that are resistant to conventional antibiotics. The present invention relates to methods of purifying lipopeptides, including daptomycin, a lipopeptide antibiotic with potent bactericidal activity against gram-positive bacteria, including strains that are resistant to conventional antibiotics. The present invention also relates to pharmaceutical compositions comprising the purified form of the lipopeptide and methods of using these compositions.

1 - 56 . (canceled) 
     
     
         57 . A daptomycin calcium salt produced by a method comprising the steps of:
 a) combining daptomycin in a solution comprising an alcohol selected from the group consisting of isopropanol, tert-butanol, and methanol, and calcium acetate at a pH of about 5.5 to 7.5 and a temperature of about 0 to 30 degrees C.;   b) allowing the daptomycin to precipitate or crystallize from the solution; and   c) obtaining the daptomycin calcium salt from the precipitated or crystallized daptomycin.   
     
     
         58 . The daptomycin calcium salt of  claim 57 , wherein the solution comprises isopropanol and the temperature of the solution is about 20 to 30 degrees C. 
     
     
         59 . The daptomycin calcium salt of  claim 57 , wherein the solution comprises isopropanol and the daptomycin is precipitated or crystallized at a pH of about 5.9 to 6.3. 
     
     
         60 . The daptomycin calcium salt of  claim 57 , wherein the daptomycin of step a) is produced by a method comprising the steps of:
 i) fermenting  Streptomyces roseosporus  to obtain a fermentation culture containing daptomycin;   ii) clarifying the daptomycin from the fermentation culture to obtain a daptomycin preparation;   iii) subjecting the daptomycin preparation to size exclusion ultrafiltration to obtain a filtered daptomycin preparation;   iv) subjecting the filtered daptomycin preparation to hydrophobic interaction chromatography;   v) subjecting the daptomycin from the hydrophobic interaction chromatography to anionic exchange chromatography;   vi) adjusting the daptomycin obtained from the anion exchange chromatography to about pH 3.0-4.8 and about 2-15° C.;   vii) filtering the daptomycin of step vi) under conditions in which the daptomycin is retained on the filter; and   viii) obtaining the daptomycin of step a) from the daptomycin of step vii).   
     
     
         61 . The daptomycin calcium salt of  claim 57  having a daptomycin purity of at least 93%. 
     
     
         62 . The daptomycin calcium salt of  claim 57 , wherein the daptomycin combined in the solution of step a) has a daptomycin purity of less than about 93%. 
     
     
         63 . The daptomycin calcium salt of  claim 57  having a daptomycin purity of at least about 93%, wherein the daptomycin combined in the solution of step a) has a daptomycin purity of less than 93%. 
     
     
         64 . A daptomycin calcium salt produced by a method comprising the steps of:
 a) forming a daptomycin preparation comprising a solution of daptomycin at a pH of about 3.0-4.8 and a temperature of about 2-15° C.,   b) filtering the daptomycin preparation under conditions in which at least a portion of the daptomycin is retained on the filter;   c) forming a solution comprising the retained daptomycin of step b), isopropanol and calcium acetate;   d) maintaining the retained daptomycin from the solution in step c) at a temperature of about 0 to 30 degrees C. and a pH of about 5.5 to 7.5 to form a precipitated or crystalline daptomycin calcium salt; and   e) collecting the precipitated or crystalline daptomycin calcium salt.   
     
     
         65 . The daptomycin calcium salt of  claim 64 , wherein the daptomycin in the daptomycin preparation of step a) is obtained by a process comprising the step of
 i) fermenting  Streptomyces roseosporus  to obtain a fermentation culture containing daptomycin.   
     
     
         66 . The daptomycin calcium salt of  claim 65 , wherein the daptomycin in the daptomycin preparation of step a) is obtained by a process further comprising the steps of:
 ii) clarifying the daptomycin from the fermentation culture to obtain a daptomycin preparation; and   iii) subjecting the clarified daptomycin preparation from step i) to at least one of hydrophobic interaction chromatography and anion exchange chromatography to obtain the daptomycin for the daptomycin preparation of step a).   
     
     
         67 . The daptomycin calcium salt of  claim 64 , wherein the daptomycin from the solution in step c) is maintained at a temperature of about 20 to 30 degrees C. and a pH of about 5.9 to 6.3. 
     
     
         68 . A daptomycin calcium salt produced by a method comprising the steps of:
 a) fermenting  Streptomyces roseosporus  to obtain a fermentation culture containing daptomycin;   b) clarifying the daptomycin from the fermentation culture to obtain a daptomycin preparation;   c) subjecting the daptomycin preparation to size exclusion ultrafiltration to obtain a filtered daptomycin preparation;   d) subjecting the filtered daptomycin preparation to hydrophobic interaction chromatography;   e) subjecting the daptomycin obtained from the hydrophobic interaction chromatography to anion exchange chromatography;   f) altering at least one of the pH, temperature and salt concentration of the daptomycin obtained from the anion exchange chromatography;   g) filtering the daptomycin of step f) under conditions in which the daptomycin is retained on the filter; and   h) forming a solution containing the daptomycin of step g), isopropanol, and calcium acetate;   i) precipitating or crystallizing daptomycin from the solution at a temperature of about 0 to 30 degrees C. and a pH of about 5.5 to 7.5; and   j) collecting the precipitated or crystallized calcium salt of daptomycin from the solution.   
     
     
         69 . The daptomycin calcium salt of  claim 68 , wherein the daptomycin is precipitated or crystallized from the solution at a temperature of about 20 to 30 degrees C. and a pH of about 5.9 to 6.3. 
     
     
         70 . A composition comprising a daptomycin calcium salt having a purity of at least about 98%, the daptomycin calcium salt obtained by a process comprising the steps of:
 a) combining daptomycin in a solution comprising an alcohol selected from the group consisting of isopropanol, tert-butanol, and methanol, and a salt selected from the group consisting of calcium acetate and calcium chloride at a pH of about 5.5 to 7.5 and a temperature of about 0 to 30 degrees C.;   b) allowing the daptomycin to precipitate or crystallize from the solution;   c) obtaining daptomycin calcium salt from the precipitated or crystallized daptomycin.   
     
     
         71 . The composition of  claim 70 , wherein the composition comprises a lower level of anhydro-daptomycin and/or the β-isomer of daptomycin compared to the daptomycin combined in the solution of step a). 
     
     
         72 . The composition of  claim 70 , wherein the daptomycin of step a) is obtained by a process comprising the steps of:
 i) fermenting  Streptomyces roseosporus  to obtain a fermentation culture containing daptomycin;   ii) clarifying the fermentation culture to obtain a clarified daptomycin preparation; and   iii) subjecting the clarified daptomycin preparation to anion exchange chromatography.   
     
     
         73 . The composition of  claim 70 , wherein the salt is calcium acetate. 
     
     
         74 . The composition of  claim 70 , wherein the temperature of the solution is about 2-15 degrees C.