High purity lipopeptides
View Patent ↗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 method to purify daptomycin, comprising the steps of:
a) fermenting Streptomyces roseosporus with a feed of n-decanoic acid to produce daptomycin in a fermentation broth;
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) adjusting the enriched daptomycin preparation to a pH of about 2.5-5 and a temperature of about 2-15 degrees C.; and
e) subjecting the enriched daptomycin preparation from step d) to filtration to obtain purified daptomycin.
2. The method of claim 1 , further comprising the step of subjecting the clarified solution of step b) to hydrophobic interaction chromatography.
3. The method of claim 2 , wherein the hydrophobic interaction chromatography utilizes an HP-20ss resin.
4. The method of claim 2 , wherein the hydrophobic interaction chromatography utilizes an eluent comprising isopropyl alcohol.
5. The method of claim 1 , wherein the anion exchange chromatography of step c) utilizes an FP-DA 13 resin.
6. The method of claim 1 , wherein the anion exchange chromatography of step c) utilizes 100-500 mM NaCl.
7. The method of claim 1 , wherein the filtration of step e) utilizes a 10,000 nominal molecular weight (NMW) filter.
8. The method of claim 1 , wherein the fermentation broth of step a) further comprises a compound having the structure:
9. The method of claim 1 , wherein the purified daptomycin of step e) comprises a compound having the structure:
10. The method of claim 1 , further comprising step f): freezing the purified daptomycin.
11. A method to purify daptomycin, comprising the steps of:
a) fermenting Streptomyces roseosporus with a feed of n-decanoic acid to produce daptomycin in a fermentation broth;
b) clarifying the fermentation broth to obtain a clarified solution;
c) subjecting the clarified solution of step b) to hydrophobic interaction chromatography to produce a daptomycin solution, wherein the hydrophobic interaction chromatography utilizes an HP-20ss resin and an eluent comprising isopropanol;
d) subjecting the daptomycin solution from step c) to anion exchange chromatography to obtain an enriched daptomycin preparation, wherein the anion exchange chromatography utilizes an FP-DA 13 resin and an eluent comprising 100-500 mM NaCl;
e) adjusting the enriched daptomycin preparation from step d) to a pH of about 2.5-5 and a temperature of about 2-15 degrees C.;
f) subjecting the enriched daptomycin preparation from step e) to filtration to obtain purified daptomycin, wherein the filtration utilizes a 10,000 nominal molecular weight (NMW) filter; and
g) freezing the purified daptomycin.