WO2009144739A1 - Amorphous daptomycin and a method of purification thereof - Google Patents

Amorphous daptomycin and a method of purification thereof Download PDF

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Publication number
WO2009144739A1
WO2009144739A1 PCT/IN2009/000265 IN2009000265W WO2009144739A1 WO 2009144739 A1 WO2009144739 A1 WO 2009144739A1 IN 2009000265 W IN2009000265 W IN 2009000265W WO 2009144739 A1 WO2009144739 A1 WO 2009144739A1
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Prior art keywords
purification
daptomycin
reverse phase
chromatography
elute
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PCT/IN2009/000265
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French (fr)
Inventor
Nitin Sopanrao Patil
Shrivallabh Balwant Desai
Ganesh Ramachandran
Srinivasa Thimmashetty
Anuj Goel
Harish Iyer
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Biocon Limited
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Publication of WO2009144739A1 publication Critical patent/WO2009144739A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the invention relates to the purification of daptomycin. More specifically, the invention relates to a simplified and optimized purification process of daptomycin from a fermentation broth by chromatographic procedures enabling high yields, selectivity and purity of the desired end product.
  • the daptomycin is prepared in high purity of at least about 98%, and preferably at least about 96%.
  • the present invention also relates to a process for preparing daptomycin that is scalable for commercial production.
  • Daptomycin also known as LY 146032, is a cyclic lipopeptide antibiotic that can be derived from the fermentation of Streptomyces roseosporus. Daptomycin is a member of the factor A-21978C type antibiotics of S. roseosporus and is comprised of a decanoyl side chain linked to the N-terminaL tryptophan of a cyclic 13-amino acid peptide. Daptomycin has an excellent profile of activity because it is highly effective against most gram-positive bacteria; it is highly bactericidal and fast-acting; it has a low resistance rate and is effective against antibiotic-resistant organisms.
  • a number of United States Patents describe A-21978C antibiotics and derivatives thereof including daptomycin (LY 146032) as well as methods of producing and isolating the A-21978C antibiotics and derivatives thereof.
  • United States Patent No. RE32,333, RE32,455 and 4,800,157 describe a method of synthesizing daptomycin by cultivating Streptomyces roseosporus NRLl 5998 under submerged aerobic fermentation conditions.
  • United States Patent. No. 4,885,243 describes an improved method of synthesizing daptomycin by feeding a fermentation culture a decanoic fatty acid or ester or salt thereof.
  • United States Patent No. 4,874,843 describes a daptomycin purification method which is incorporated herein by reference.
  • US patent No. 6696412 describes commercially feasible methods to produce high levels of purified daptomycin at a purity level of 95-98%.
  • the process chromatography method comprises sequentially using anion exchange chromatography, hydrophobic interaction chromatography (HIC) and anion exchange chromatography to purify a preparation containing daptomycin.
  • HIC hydrophobic interaction chromatography
  • WO02056829 disclose the 92% pure amorphous form of daptomycin which is incorporated herein by reference
  • the present invention has steps novel with respect to any of the above known methods and uses RP-HPLC on commercial scale to prepare highly pure daptomycin at purity levels of 96-99%.
  • the invention further addresses the long process times as seen with anion exchange method of purification by replacing 2 rounds of ion exchange chromatography with single RP-HPLC purification.
  • the present purification process of daptomycin comprises sequentially carrying out hydrophobic interaction chromatography of microfiltered broth followed by RP-HPLC and subsequent liquid-liquid extraction, HIC or anion exchange chromatography. Finally, solvent wash for removal of endotoxin prior to ultrafiltration, nanofiltration or reverse osmosis followed by lyophilization is carried out to give formulated daptomycin.
  • Another important advantage of the purification process according to the present invention is that they may be scaled up in a reproducible and consistent manner. Further, the process of the present invention affords products which are superior to those obtained by purification methods hitherto known and give higher yields.
  • the main objective of the present invention is to obtain an amorphous daptomycin having at least 98% purity.
  • Another main objective of the present invention is to obtain an amorphous daptomycin having at least 97% purity.
  • Yet another main objective of the present invention is to obtain an amorphous daptomycin having at least 96% purity. Still another main objective of the present invention is to obtain an amorphous daptomycin having powder XRD pattern represented in fig 2.
  • Still another main objective of the present invention is to obtain a method of purification of daptomycin.
  • the present invention relates to an amorphous daptomycin having at least 98% purity; an amorphous daptomycin having at least 97% purity; an amorphous daptomycin having at least 96% purity; an amorphous daptomycin having powder XRD pattern represented in fig 2; and a method of purification of daptomycin comprising the steps of a) filtration of the fermentation broth, b) optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c) purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d) recovery of purified daptomycin from the elute of reverse phase chromatography, and e) depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product.
  • the present invention relates to an amorphous daptomycin having at least 98% purity.
  • the present invention also relates to an amorphous daptomycin having at least 97% purity.
  • the present invention also relates to an amorphous daptomycin having at least 96% purity.
  • the present invention also relates to an amorphous daptomycin having powder XRD pattern represented in fig 2.
  • the present invention also relates to a method of purification of daptomycin comprising the steps of: a. filtration of the fermentation broth, b. optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c. purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d. recovery of purified daptomycin from the elute of reverse phase chromatography, and e. depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product.
  • the recovery in step (d) is carried out using the steps of: a. optional extraction of the elute of reverse phase chromatography with a water immiscible solvent or treatment of the elute from reverse phase chromatography by ion exchange chromatography or hydrophobic interaction chromatography, b. optional concentration of the elute of reverse phase chromatography or the elute of hydrophobic interaction chromatography from step (a) by evaporation, c.
  • step (a) subjecting the elute of reverse phase chromatography, the daptomyc in- containing raffmate from step (a), the elute of ion exchange chromatography or hydrophobic interaction chromatography from step (a), or the concentrate from step (b) to endotoxin removal followed by ultrafiltration, nanofiltration or reverse osmosis.
  • the hydrophobic interaction chromatography is performed on resin selected from the group comprising HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD16, XAD1600,
  • the ultrafiltration or nanofiltration is performed using membrane selected from the group comprising polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE) and ceramic membranes.
  • membrane selected from the group comprising polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE) and ceramic membranes.
  • the reverse phase resin is selected from C4, C8 and Cl 8 reverse phase resin.
  • the elution from the reverse phase resin is carried out with mixture of organic solvent and water or mixture of organic solvent and buffer at pH 2.5 to 8.
  • the organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof.
  • the reverse phase chromatography elution is carried out in isocratic or gradient manner.
  • the gradient elution is achieved by changing the content of organic solvent, pH and/or buffer molarity during elution.
  • the water-immiscible solvent is selected from ethyl acetate, propyl acetate, butyl acetate, amyl acetate, chloroform, dichloromethane, methyl-t-butyl ether, diethyl ether, butanol, hexane, heptane, cyclohexane or mixture thereof.
  • the ion exchange chromatography is anion exchange chromatography or cation exchange chromatography. In still another embodiment of the present invention, the ion exchange chromatography is anion exchange chromatography.
  • the ion exchange chromatography is performed on resin selected from the group comprising diethyl aminoethyl, quaternary aminoethyl, quaternary ammonium, polyethyleneimine and quaternized polyethyleneimine type resins.
  • the ion exchange chromatography elution is carried out using a salt solution.
  • the salt is a halogenide.
  • the salt is an alkali or alkaline earth halogenide.
  • the evaporation is done using a thin or wiped film evaporator, falling film evaporator, forced circulation evaporator, short tube evaporator or long tube evaporator.
  • the ultrafiltration or nanofiltration is performed at pH 2.5 to 8.
  • chromatography refers to the process by which a solute of interest in a mixture is separated from other solutes in a mixture as a result of differences in rates at which the individual solutes of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.
  • High Performance liquid chromatography refers to that chromatographic procedure in which the particles (stationary phase) used in the column packing are small (between 3 and 50 microns) and regular with little variation from the selected size. Such chromatography typically employs relatively high (around 500-3500 psi) inlet pressures.
  • ion-exchange and ion-exchange chromatography refers to the chromatographic process in which a solute of interest (such as a protein) in a mixture interacts with a charged compound linked (such as by covalent attachment) to a solid phase ion exchange material such that the solute of interest interacts non-specifically with the charged compound more or less than solute impurities or contaminants in the mixture.
  • the contaminating solutes in the mixture elute from a column of the ion exchange material faster or slower than the solute of interest or are bound to or excluded from the resin relative to the solute of interest.
  • Ion-exchange chromatography specifically includes cation exchange, anion exchange, and mixed mode chromatography.
  • the object of the present invention is provided in a specifically delineated process for purification of daptomycin from fermentation broth by using chromatographic process.
  • the present invention relates to a chromatography process for purifying a daptomycin from fermentation broth comprising the steps of: a) filtration of the fermentation broth, b) optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c) purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d) recovery of purified daptomycin from the elute of reverse phase chromatography, and e) Lyophilization of the recovered daptomycin to give highly pure formulated product.
  • the present invention also relates to the above process, wherein the recovery of purified daptomycin from the elute of reverse phase chromatography is carried out using the following steps:
  • the final purified product daptomycin is amorphous in nature and 98% pure.
  • variables which can be adjusted during the chromatographic procedures of the present invention. Such variables include resin selection; loading, wash and eluting conditions, such as ionic strength, buffer composition, pH, temperature, addition of one or more organic solvents, etc. However, such variables are routinely adjusted in this field and those skilled in the art can readily establish optimum conditions.
  • the present invention relates to a chromatography process for purifying a daptomycin from fermentation broth comprising the steps of: a) filtration of the fermentation broth, b) optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c) purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d) recovery of purified daptomycin from the elute of reverse phase chromatography, and e) depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product.
  • the present invention also relates to the above process, wherein the recovery of purified daptomycin from the elute of reverse phase chromatography is carried out using the following steps:
  • the final purified product daptomycin is amorphous in nature and 98% pure .
  • the daptomycin of the present invention can be produced by fermentation. After fermentation, the daptomycin-containing extracellular solution is clarified by removing the mycelia from the fermentation broth. This clarification is performed by any standard solid-liquid separation technique, such as centrifugation or filtration. Preferably, the fermentation broth clarification is carried out using a microfiltration system, filter press, rotary drum filter, depth filter or industrial centrifuge.
  • the clarified solution may be directly subjected to a reverse phase chromatography.
  • the clarified solution is concentrated and the concentrate is subjected to reverse phase chromatography.
  • the concentration of clarified solution can be carried out by ultrafiltration, nanofiltration, or hydrophobic interaction chromatography or combinations thereof.
  • the ultrafiltration and nanofiltration membrane can be selected from polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE), or ceramic membranes.
  • the pH during ultrafiltration or nanofiltration is selected in the range 2.5 to 8.
  • the retentate of ultrafiltration or nanofiltration gives concentrated clarified solution.
  • the feed for the reverse phase chromatography may be treated with activated charcoal, alumina, silica gel or may be passed through a guard column before passing it through the reverse phase chromatography column.
  • the resin can be selected from HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD16, XAD1600, XAD 18, XAD761, XAD7HP, etc.
  • the clarified solution is contacted with the hydrophobic interaction chromatography resin under conditions such that daptomycin binds to the resin. This contact is carried out at pH in the range from 2.5 to 8. Preferably, the contact is carried out at pH in the range from 3.5 to 5.0.
  • additives including salts and/or surfactants are added to the clarified solution. Surfactants are added at small concentrations to prevent precipitation during pH adjustment and column blockage in hydrophobic interaction chromatography.
  • the surfactant can be selected from Triton X, Tween, SDS, etc.
  • the surfactant concentration is selected in the range from 0.001% to 1.0%.
  • Additive salts are selected from sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, di-ammonium hydrogen phosphate, sodium acetate, ammonium acetate, ammonium chloride, sodium carbonate, sodium bicarbonate and sodium citrate.
  • the resin is washed and eluted with water, buffer, mixture of organic solvent and water or mixture of organic solvent and buffer.
  • the organic solvent is selected from methanol, ethanol, n- propanol, isopropanol, n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof.
  • the pH during elution is selected in the range from 2.5 to 8.
  • the product-containing elute from hydrophobic interaction chromatography gives concentrated clarified solution.
  • the clarified solution or the concentrated clarified solution is subjected to a reverse phase chromatography resin.
  • the feed for the reverse phase chromatography column is diluted with water.
  • the feed is loaded onto the column at pH in the range from 2.5 to 8.
  • the feed pH is in the range from 3.5 to 5.0.
  • the reverse phase resin is selected from C4, C8, C18 silica or polystyrene-divinylbenzene. Daptomycin from the column is eluted with mixture of organic solvent and water or mixture of organic solvent and buffer.
  • the organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof.
  • the pH during elution is selected in the range from 2.5 to 8.
  • the elution is carried out in isocratic or gradient manner. In gradient elution, the content of organic solvent, pH and/or buffer molarity is changed during elution. During product elution, fractions are collected. Fractions containing product with desired purity are combined to obtain elute from reverse phase chromatography.
  • the buffer can be prepared using phosphates of sodium, potassium or ammonium salts, acetates of sodium, potassium or ammonium salts, citrates of sodium, potassium or ammonium salts, oxalates sodium, potassium or ammonium salts, acetic acid, phosphoric acid, citric acid, oxalic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide and ammonium hydroxide or mixtures thereof.
  • the elute from reverse phase chromatography is optionally processed by extraction, ion exchange chromatography or hydrophobic interaction chromatography.
  • extraction the elute from reverse phase chromatography is extracted with a water-immiscible organic solvent.
  • the water-immiscible solvent is selected from ethyl acetate, propyl acetate, butyl acetate, amyl acetate, chloroform, dichloromethane, methyl-t-butyl ether, diethyl ether, butanol, hexane, heptane, cyclohexane or mixture thereof.
  • the extraction is carried out in batch manner in single or multiple stages. Alternately, the extraction is carried out in a co-current or counter-current continuous manner. Daptomycin remains in the aqueous layer to give raffinate.
  • the elute from reverse phase chromatography is passed through ion exchange resin column.
  • the ion exchange resin is selected from diethyl aminoethyl, quaternary aminoethyl, quaternary ammonium, polyethyleneimine and quaternized polyethyleneimine type resins.
  • the elute from reverse phase chromatography is diluted with water before passing through the ion exchange column.
  • the resin is washed and eluted with water and salt solution in water.
  • the elution is carried out in isocratic or gradient manner.
  • the gradient elution is carried out using step or continuous gradient.
  • the salt concentration is selected in the range from 0 to 1000 mM. Preferably, this concentration is selected in the range from 0 to 500 mM.
  • the salt is selected from NaCl or KCl.
  • hydrophobic interaction chromatography the elute from reverse phase chromatography is passed through a hydrophobic interaction resin column.
  • the hydrophobic interaction resin is selected from HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD16, XAD1600, XADl 8, XAD761, XAD7HP, etc.
  • the elute from reverse phase chromatography is diluted with water before passing through the hydrophobic interaction resin column.
  • the loading is carried out at pH in the range from 2.5 to 8.
  • the loading is carried out at pH in the range from 3.5 to 5.0.
  • the resin is washed and eluted with water, buffer, mixture of organic solvent and water or mixture of organic solvent and buffer.
  • the organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, * n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof.
  • the pH during elution is selected in the range from 2.5 to 8.
  • the elute from reverse phase chromatography or the elute from hydrophobic interaction chromatography is optionally concentrated by evaporation.
  • Various evaporators including thin or wiped film evaporator, falling film evaporator, forced circulation evaporator, short tube evaporator or long tube evaporator can be used for this concentration.
  • the elute of reverse phase chromatography, the daptomycin-containing raff ⁇ nate from extraction, the elute of ion exchange chromatography, the elute hydrophobic interaction chromatography or the concentrate after evaporation is then subjected to depyrogenation by employing solvent wash.
  • the organic solvent is selected from n- butanol, ethyl acetate, butyl acetate, hexane, heptane, petroleum ether or mixture thereof.
  • the pH during solvent wash is normally selected in the range from 5 to 8. Daptomycin remains in the aqueous layer to give raff ⁇ nate which is subjected to ultrafiltration or nanofiltration.
  • the feed to the ultrafiltration or nanofiltration is diluted with water.
  • the filtration is carried out at a pH in the range from 2.5 to 8.
  • the ultrafiltration and nanofiltration membrane is selected from polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE), or ceramic membranes.
  • the daptomycin containing retentate is diafiltered against water.
  • the daptomycin solution is sterile filtered and lyophilized to obtain amorphous pure daptomycin.
  • the purity of final product has at least 98% as measured by HPLC.
  • the column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 30:70. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 6.5 and passed over weak anion exchange resin to remove organic solvent. The resin was pre-equilibrated with 30 mM Tris HCl buffer at pH 6.5. The column was eluted with 0.5 M NaCl solution. The product containing elute was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized. The purity of daptomycin was 97.5%.
  • the column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 30:70. During elution, fractions were collected. The fractions with desired purity were combined, and subjected to liquid-liquid extraction using n-butyl acetate. Post-extraction, the aqueous layer containing daptomycin was concentrated and diafiltered by ultrafiltration. The concentrate was sterile filtered, filled in vials and lyophilized to give daptomycin of purity more than 96%.
  • the column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 30:70. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 4.5. This solution was passed through an HP20ss • column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The resin was washed with water and eluted with 70% methanol in buffer. The product-containing elute of purity 97.2% was diluted with water and pH of this solution was adjusted to 4.5. The pH-adjusted solution was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized.
  • the product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column.
  • the column was eluted using a linear gradient from 30 to 50% acetonitrile over 90 minutes.
  • the fractions containing product with purities ⁇ 98% were combined, pH was adjusted to 6.5 and extracted with n-butyl acetate in multiple stages.
  • Post-extraction, the aqueous layer containing daptomycin was concentrated and diafiltered at pH 6.5 using a nanof ⁇ ltration membrane.
  • the concentrate was sterile filtered, filled in vials and lyophilized.
  • the fractions containing product with desired purity were combined and diluted with water. pH of the diluted solution was adjusted to 4.5 and passed through an HP20ss column. The column was washed with water and eluted with 70% acetonitrile. The elute was sterile filtered and lyophilized to give the daptomycin. The purity of daptomycin obtained was 98.8%.
  • EXAMPLE 7 10 L fermentation broth containing daptomycin was microfiltered through 0.1 ⁇ m filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column.
  • the column was eluted using a linear gradient from 30 to 50% acetonitrile over 90 minutes. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 4.5. This solution was passed through an HP20ss column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The resin was washed with water and eluted with aqueous acetonitrile. The product-containing elute of -98% purity was concentrated by solvent evaporation. The concentrated solution was diluted with water and diafiltered using an ultrafiltration membrane. The diafiltered solution was sterile filtered and lyophilized.
  • EXAMPLE 8 10 L fermentation broth containing daptomycin was microfiltered through 0.1 ⁇ m filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column.
  • the column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 35:65. During elution, the fractions with desired purity were combined, diluted with water, pH-adjusted to 6.5 and passed over weak anion exchange resin to remove organic solvent. The resin was pre-equilibrated with 30 mM Tris HCl buffer at pH 6.5. The column was eluted with 0.5 M NaCl solution. The product containing elute was depyrogenated by contacting with n-butanol in the ratio of 1: 0.2 for 30 mins. The aqueous layer containing the product was further contacted with n-heptane in the ratio of 1: 0.4 for 30 mins. Finally the aqueous 'product containing layer' was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized. The purity of daptomycin obtained was 96.6%.
  • fractions were collected. The fractions with 97% purity were combined, diluted with water, pH-adjusted to 4.5. This solution was passed through an HP20ss column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The resin was washed with water and eluted with 70% methanol in buffer. The product-containing elute was diluted with water and pH of this solution was adjusted to 4.5. The pH-adjusted solution was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized.
  • fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 6.5 and passed over weak anion exchange resin to remove organic solvent.
  • the resin was pre- equilibrated with 30 mM Tris HCl buffer at pH 6.5.
  • the column was eluted with 0.5 M NaCl solution.
  • the product containing elute was depyrogenated by contacting with n- butanol in the ratio of 1: 0.2 for 30 mins.
  • the aqueous layer containing the product was further contacted with n-heptane in the ratio of 1: 0.4 for 30 mins.
  • the aqueous 'product containing layer' was concentrated and diafiltered using an ultrafiltration membrane.
  • the concentrate was sterile filtered, filled in vials and lyophilized to give daptomycin of 96%.

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Abstract

The invention relates to the purification of daptomycin. More specifically, the invention relates to a simplified and optimized purification process of daptomycin from a fermentation broth by chromatographic procedures enabling high yields, selectivity and purity of the desired end product. The daptomycin is prepared in high purity of at least about 98%, and preferably at least about 96%. The present invention also relates to a process for preparing daptomycin that is scalable for commercial production.

Description

AMORPHOUS DAPTOMYCIN AND A METHOD OF PURIFICATION THEREOF
FIELD OF THE INVENTION The invention relates to the purification of daptomycin. More specifically, the invention relates to a simplified and optimized purification process of daptomycin from a fermentation broth by chromatographic procedures enabling high yields, selectivity and purity of the desired end product. The daptomycin is prepared in high purity of at least about 98%, and preferably at least about 96%. The present invention also relates to a process for preparing daptomycin that is scalable for commercial production.
BACKGROUND AND PRIOR ART OF THE INVENTION
Daptomycin, also known as LY 146032, is a cyclic lipopeptide antibiotic that can be derived from the fermentation of Streptomyces roseosporus. Daptomycin is a member of the factor A-21978C type antibiotics of S. roseosporus and is comprised of a decanoyl side chain linked to the N-terminaL tryptophan of a cyclic 13-amino acid peptide. Daptomycin has an excellent profile of activity because it is highly effective against most gram-positive bacteria; it is highly bactericidal and fast-acting; it has a low resistance rate and is effective against antibiotic-resistant organisms.
A number of United States Patents describe A-21978C antibiotics and derivatives thereof including daptomycin (LY 146032) as well as methods of producing and isolating the A-21978C antibiotics and derivatives thereof.
United States Patent No. RE32,333, RE32,455 and 4,800,157 describe a method of synthesizing daptomycin by cultivating Streptomyces roseosporus NRLl 5998 under submerged aerobic fermentation conditions.
United States Patent. No. 4,885,243 describes an improved method of synthesizing daptomycin by feeding a fermentation culture a decanoic fatty acid or ester or salt thereof. United States Patent No. 4,874,843 describes a daptomycin purification method which is incorporated herein by reference.
U.S. Patent. No. 5,912,226 describes the identification and isolation of two impurities produced during the manufacture of daptomycin which is incorporated herein by reference.
US patent No. 6696412 describes commercially feasible methods to produce high levels of purified daptomycin at a purity level of 95-98%. The process chromatography method comprises sequentially using anion exchange chromatography, hydrophobic interaction chromatography (HIC) and anion exchange chromatography to purify a preparation containing daptomycin. WO02056829 disclose the 92% pure amorphous form of daptomycin which is incorporated herein by reference
A number of different chromatographic procedures are applied to obtain the desired end result with respect to purity and yield as discussed above. There is a need in the art to develop a simple chromatographic purification procedure that may be operational on a large scale with minimal steps to isolate the purified daptomycin from a fermentation broth.
The present invention has steps novel with respect to any of the above known methods and uses RP-HPLC on commercial scale to prepare highly pure daptomycin at purity levels of 96-99%. The invention further addresses the long process times as seen with anion exchange method of purification by replacing 2 rounds of ion exchange chromatography with single RP-HPLC purification. The present purification process of daptomycin comprises sequentially carrying out hydrophobic interaction chromatography of microfiltered broth followed by RP-HPLC and subsequent liquid-liquid extraction, HIC or anion exchange chromatography. Finally, solvent wash for removal of endotoxin prior to ultrafiltration, nanofiltration or reverse osmosis followed by lyophilization is carried out to give formulated daptomycin. Another important advantage of the purification process according to the present invention is that they may be scaled up in a reproducible and consistent manner. Further, the process of the present invention affords products which are superior to those obtained by purification methods hitherto known and give higher yields.
OBJECTIVES QF THE INVENTION
The main objective of the present invention is to obtain an amorphous daptomycin having at least 98% purity.
Another main objective of the present invention is to obtain an amorphous daptomycin having at least 97% purity.
Yet another main objective of the present invention is to obtain an amorphous daptomycin having at least 96% purity. Still another main objective of the present invention is to obtain an amorphous daptomycin having powder XRD pattern represented in fig 2.
Still another main objective of the present invention is to obtain a method of purification of daptomycin.
STATEMENT OF THE INVENTION
Accordingly, the present invention relates to an amorphous daptomycin having at least 98% purity; an amorphous daptomycin having at least 97% purity; an amorphous daptomycin having at least 96% purity; an amorphous daptomycin having powder XRD pattern represented in fig 2; and a method of purification of daptomycin comprising the steps of a) filtration of the fermentation broth, b) optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c) purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d) recovery of purified daptomycin from the elute of reverse phase chromatography, and e) depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product. BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Figurel: XRD of Amorphous Daptoraycin Figure 2: Chromatogram of Daptomycin
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an amorphous daptomycin having at least 98% purity.
The present invention also relates to an amorphous daptomycin having at least 97% purity.
The present invention also relates to an amorphous daptomycin having at least 96% purity.
The present invention also relates to an amorphous daptomycin having powder XRD pattern represented in fig 2.
The present invention also relates to a method of purification of daptomycin comprising the steps of: a. filtration of the fermentation broth, b. optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c. purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d. recovery of purified daptomycin from the elute of reverse phase chromatography, and e. depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product.
In another embodiment of the present invention, the recovery in step (d) is carried out using the steps of: a. optional extraction of the elute of reverse phase chromatography with a water immiscible solvent or treatment of the elute from reverse phase chromatography by ion exchange chromatography or hydrophobic interaction chromatography, b. optional concentration of the elute of reverse phase chromatography or the elute of hydrophobic interaction chromatography from step (a) by evaporation, c. subjecting the elute of reverse phase chromatography, the daptomyc in- containing raffmate from step (a), the elute of ion exchange chromatography or hydrophobic interaction chromatography from step (a), or the concentrate from step (b) to endotoxin removal followed by ultrafiltration, nanofiltration or reverse osmosis.
In yet another embodiment of the present invention, the hydrophobic interaction chromatography is performed on resin selected from the group comprising HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD16, XAD1600,
XAD 18, XAD761 and XAD7HP .
In still another embodiment of the present invention, the ultrafiltration or nanofiltration is performed using membrane selected from the group comprising polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE) and ceramic membranes.
In still another embodiment of the present invention, the reverse phase resin is selected from C4, C8 and Cl 8 reverse phase resin.
In still another embodiment of the present invention, the elution from the reverse phase resin is carried out with mixture of organic solvent and water or mixture of organic solvent and buffer at pH 2.5 to 8.
In still another embodiment of the present invention, the organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof. In still another embodiment of the present invention, the reverse phase chromatography elution is carried out in isocratic or gradient manner.
In still another embodiment of the present invention, the gradient elution is achieved by changing the content of organic solvent, pH and/or buffer molarity during elution.
In still another embodiment of the present invention, the water-immiscible solvent is selected from ethyl acetate, propyl acetate, butyl acetate, amyl acetate, chloroform, dichloromethane, methyl-t-butyl ether, diethyl ether, butanol, hexane, heptane, cyclohexane or mixture thereof.
In still another embodiment of the present invention, the ion exchange chromatography is anion exchange chromatography or cation exchange chromatography. In still another embodiment of the present invention, the ion exchange chromatography is anion exchange chromatography.
In still another embodiment of the present invention, the ion exchange chromatography is performed on resin selected from the group comprising diethyl aminoethyl, quaternary aminoethyl, quaternary ammonium, polyethyleneimine and quaternized polyethyleneimine type resins.
In still another embodiment of the present invention, the ion exchange chromatography elution is carried out using a salt solution. In still another embodiment of the present invention, the salt is a halogenide.
In still another embodiment of the present invention, the salt is an alkali or alkaline earth halogenide.
In still another embodiment of the present invention, the evaporation is done using a thin or wiped film evaporator, falling film evaporator, forced circulation evaporator, short tube evaporator or long tube evaporator.
In still another embodiment of the present invention, the ultrafiltration or nanofiltration is performed at pH 2.5 to 8.
The term "chromatography" refers to the process by which a solute of interest in a mixture is separated from other solutes in a mixture as a result of differences in rates at which the individual solutes of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.
The term "High Performance liquid chromatography", as used herein, refers to that chromatographic procedure in which the particles (stationary phase) used in the column packing are small (between 3 and 50 microns) and regular with little variation from the selected size. Such chromatography typically employs relatively high (around 500-3500 psi) inlet pressures.
The term "ion-exchange" and "ion-exchange chromatography" refers to the chromatographic process in which a solute of interest (such as a protein) in a mixture interacts with a charged compound linked (such as by covalent attachment) to a solid phase ion exchange material such that the solute of interest interacts non-specifically with the charged compound more or less than solute impurities or contaminants in the mixture. The contaminating solutes in the mixture elute from a column of the ion exchange material faster or slower than the solute of interest or are bound to or excluded from the resin relative to the solute of interest. "Ion-exchange chromatography" specifically includes cation exchange, anion exchange, and mixed mode chromatography. The object of the present invention is provided in a specifically delineated process for purification of daptomycin from fermentation broth by using chromatographic process.
In a broad aspect, the present invention relates to a chromatography process for purifying a daptomycin from fermentation broth comprising the steps of: a) filtration of the fermentation broth, b) optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c) purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d) recovery of purified daptomycin from the elute of reverse phase chromatography, and e) Lyophilization of the recovered daptomycin to give highly pure formulated product.
The present invention also relates to the above process, wherein the recovery of purified daptomycin from the elute of reverse phase chromatography is carried out using the following steps:
a) optional extraction of the elute of reverse phase chromatography with a water immiscible solvent or treatment of the elute from reverse phase chromatography by ion exchange chromatography or hydrophobic interaction chromatography, b) optional concentration of the elute of reverse phase chromatography or the elute of hydrophobic interaction chromatography from step (a) by evaporation, c) subjecting the elute of reverse phase chromatography, the daptomycin- containing raffϊnate from step (a), the elute of ion exchange chromatography from step (a), the elute of hydrophobic interaction chromatography from step
(a), or the concentrate from step (b) to endotoxin removal by solvent wash followed by ultrafiltration, nanofiltration or reverse osmosis.
In another aspect of the invention the final purified product daptomycin is amorphous in nature and 98% pure. Those skilled in the art will recognize that there are various variables which can be adjusted during the chromatographic procedures of the present invention. Such variables include resin selection; loading, wash and eluting conditions, such as ionic strength, buffer composition, pH, temperature, addition of one or more organic solvents, etc. However, such variables are routinely adjusted in this field and those skilled in the art can readily establish optimum conditions.
The present invention relates to a chromatography process for purifying a daptomycin from fermentation broth comprising the steps of: a) filtration of the fermentation broth, b) optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c) purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d) recovery of purified daptomycin from the elute of reverse phase chromatography, and e) depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product.
The present invention also relates to the above process, wherein the recovery of purified daptomycin from the elute of reverse phase chromatography is carried out using the following steps:
a) optional extraction of the elute of reverse phase chromatography with a water immiscible solvent or treatment of the elute from reverse phase chromatography by ion exchange chromatography or hydrophobic interaction chromatography, b) optional concentration of the elute of reverse phase chromatography or the elute of hydrophobic interaction chromatography from step (a) by evaporation, c) subjecting the elute of reverse phase chromatography, the daptomycin- containing raffinate from step (a), the elute of ion exchange chromatography from step (a), the elute of hydrophobic interaction chromatography from step (a), or the concentrate from step (b) to endotoxin removal followed by ultrafiltration, nanofiltration or reverse osmosis. In another aspect of the invention the final purified product daptomycin is amorphous in nature and 98% pure .The daptomycin of the present invention can be produced by fermentation. After fermentation, the daptomycin-containing extracellular solution is clarified by removing the mycelia from the fermentation broth. This clarification is performed by any standard solid-liquid separation technique, such as centrifugation or filtration. Preferably, the fermentation broth clarification is carried out using a microfiltration system, filter press, rotary drum filter, depth filter or industrial centrifuge.
The clarified solution may be directly subjected to a reverse phase chromatography. Alternately, the clarified solution is concentrated and the concentrate is subjected to reverse phase chromatography. The concentration of clarified solution can be carried out by ultrafiltration, nanofiltration, or hydrophobic interaction chromatography or combinations thereof. The ultrafiltration and nanofiltration membrane can be selected from polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE), or ceramic membranes. The pH during ultrafiltration or nanofiltration is selected in the range 2.5 to 8. The retentate of ultrafiltration or nanofiltration gives concentrated clarified solution. Optionally, the feed for the reverse phase chromatography may be treated with activated charcoal, alumina, silica gel or may be passed through a guard column before passing it through the reverse phase chromatography column.
In hydrophobic interaction chromatography, the resin can be selected from HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD16, XAD1600, XAD 18, XAD761, XAD7HP, etc. The clarified solution is contacted with the hydrophobic interaction chromatography resin under conditions such that daptomycin binds to the resin. This contact is carried out at pH in the range from 2.5 to 8. Preferably, the contact is carried out at pH in the range from 3.5 to 5.0. Optionally, additives including salts and/or surfactants are added to the clarified solution. Surfactants are added at small concentrations to prevent precipitation during pH adjustment and column blockage in hydrophobic interaction chromatography. The surfactant can be selected from Triton X, Tween, SDS, etc. The surfactant concentration is selected in the range from 0.001% to 1.0%. Additive salts are selected from sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, di-ammonium hydrogen phosphate, sodium acetate, ammonium acetate, ammonium chloride, sodium carbonate, sodium bicarbonate and sodium citrate. The resin is washed and eluted with water, buffer, mixture of organic solvent and water or mixture of organic solvent and buffer. The organic solvent is selected from methanol, ethanol, n- propanol, isopropanol, n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof. The pH during elution is selected in the range from 2.5 to 8. The product-containing elute from hydrophobic interaction chromatography gives concentrated clarified solution.
The clarified solution or the concentrated clarified solution is subjected to a reverse phase chromatography resin. Optionally, the feed for the reverse phase chromatography column is diluted with water. The feed is loaded onto the column at pH in the range from 2.5 to 8. Preferably, the feed pH is in the range from 3.5 to 5.0. The reverse phase resin is selected from C4, C8, C18 silica or polystyrene-divinylbenzene. Daptomycin from the column is eluted with mixture of organic solvent and water or mixture of organic solvent and buffer. The organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof. The pH during elution is selected in the range from 2.5 to 8. The elution is carried out in isocratic or gradient manner. In gradient elution, the content of organic solvent, pH and/or buffer molarity is changed during elution. During product elution, fractions are collected. Fractions containing product with desired purity are combined to obtain elute from reverse phase chromatography. The buffer can be prepared using phosphates of sodium, potassium or ammonium salts, acetates of sodium, potassium or ammonium salts, citrates of sodium, potassium or ammonium salts, oxalates sodium, potassium or ammonium salts, acetic acid, phosphoric acid, citric acid, oxalic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide and ammonium hydroxide or mixtures thereof.
Purified daptomycin in the elute from reverse phase chromatography is then recovered. The elute from reverse phase chromatography is optionally processed by extraction, ion exchange chromatography or hydrophobic interaction chromatography. In extraction, the elute from reverse phase chromatography is extracted with a water-immiscible organic solvent. The water-immiscible solvent is selected from ethyl acetate, propyl acetate, butyl acetate, amyl acetate, chloroform, dichloromethane, methyl-t-butyl ether, diethyl ether, butanol, hexane, heptane, cyclohexane or mixture thereof. The extraction is carried out in batch manner in single or multiple stages. Alternately, the extraction is carried out in a co-current or counter-current continuous manner. Daptomycin remains in the aqueous layer to give raffinate.
In ion exchange chromatography, the elute from reverse phase chromatography is passed through ion exchange resin column. The ion exchange resin is selected from diethyl aminoethyl, quaternary aminoethyl, quaternary ammonium, polyethyleneimine and quaternized polyethyleneimine type resins. Optionally, the elute from reverse phase chromatography is diluted with water before passing through the ion exchange column. The resin is washed and eluted with water and salt solution in water. The elution is carried out in isocratic or gradient manner. The gradient elution is carried out using step or continuous gradient. During washing and elution, the salt concentration is selected in the range from 0 to 1000 mM. Preferably, this concentration is selected in the range from 0 to 500 mM. The salt is selected from NaCl or KCl.
In hydrophobic interaction chromatography, the elute from reverse phase chromatography is passed through a hydrophobic interaction resin column. The hydrophobic interaction resin is selected from HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD16, XAD1600, XADl 8, XAD761, XAD7HP, etc. Optionally, the elute from reverse phase chromatography is diluted with water before passing through the hydrophobic interaction resin column. The loading is carried out at pH in the range from 2.5 to 8. Preferably, the loading is carried out at pH in the range from 3.5 to 5.0. The resin is washed and eluted with water, buffer, mixture of organic solvent and water or mixture of organic solvent and buffer. The organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, *n-butanol, t-butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof. The pH during elution is selected in the range from 2.5 to 8.
The elute from reverse phase chromatography or the elute from hydrophobic interaction chromatography is optionally concentrated by evaporation. Various evaporators including thin or wiped film evaporator, falling film evaporator, forced circulation evaporator, short tube evaporator or long tube evaporator can be used for this concentration.
The elute of reverse phase chromatography, the daptomycin-containing raffϊnate from extraction, the elute of ion exchange chromatography, the elute hydrophobic interaction chromatography or the concentrate after evaporation is then subjected to depyrogenation by employing solvent wash. The organic solvent is selected from n- butanol, ethyl acetate, butyl acetate, hexane, heptane, petroleum ether or mixture thereof. The pH during solvent wash is normally selected in the range from 5 to 8. Daptomycin remains in the aqueous layer to give raffϊnate which is subjected to ultrafiltration or nanofiltration. Optionally, the feed to the ultrafiltration or nanofiltration is diluted with water. The filtration is carried out at a pH in the range from 2.5 to 8. The ultrafiltration and nanofiltration membrane is selected from polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE), or ceramic membranes. The daptomycin containing retentate is diafiltered against water.
Finally, the daptomycin solution is sterile filtered and lyophilized to obtain amorphous pure daptomycin. The purity of final product has at least 98% as measured by HPLC.
The disclosures of the prior art references referred to in this patent application are incorporated herein by reference. The invention is further defined by reference to the following examples describing in detail the process and compositions of the invention. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention.
EXAMPLE 1
10 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 30:70. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 6.5 and passed over weak anion exchange resin to remove organic solvent. The resin was pre-equilibrated with 30 mM Tris HCl buffer at pH 6.5. The column was eluted with 0.5 M NaCl solution. The product containing elute was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized. The purity of daptomycin was 97.5%.
EXAMPLE 2
10 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 30:70. During elution, fractions were collected. The fractions with desired purity were combined, and subjected to liquid-liquid extraction using n-butyl acetate. Post-extraction, the aqueous layer containing daptomycin was concentrated and diafiltered by ultrafiltration. The concentrate was sterile filtered, filled in vials and lyophilized to give daptomycin of purity more than 96%.
EXAMPLE 3
10 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 30:70. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 4.5. This solution was passed through an HP20ss • column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The resin was washed with water and eluted with 70% methanol in buffer. The product-containing elute of purity 97.2% was diluted with water and pH of this solution was adjusted to 4.5. The pH-adjusted solution was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized.
EXAMPLE 4
50 L broth was microfiltered through 0.1 μm and the permeate was concentrated 10 fold on a 0.6KDa nanofiltration membrane. The concentrate was mixed with activated charcoal and filtered to remove the charcoal. The filtrate was loaded on a C8 reverse phase chromatography column. The product was eluted using a linear gradient of 30-50% acetonitrile over 90 minutes. The fractions containing product with desired purity were combined and extracted with n-butyl acetate. Post-extraction, the aqueous layer containing daptomycin was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized.
EXAMPLE 5
50 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The product containing fractions were combined, diluted with water and purified on a C8 reverse phase chromatography column. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted using a linear gradient from 30 to 50% acetonitrile over 90 minutes. The fractions containing product with purities ~98% were combined, pH was adjusted to 6.5 and extracted with n-butyl acetate in multiple stages. Post-extraction, the aqueous layer containing daptomycin was concentrated and diafiltered at pH 6.5 using a nanofϊltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized.
EXAMPLE 6 pH of 50 L fermentation broth was adjusted to 4.5. The pH-adjusted broth was extracted with n-butanol. The butanol extract was concentrated partially and extracted with sodium phosphate buffer at pH 6.5. pH of this extract was adjusted to 4.5 and loaded onto an HP20ss column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10% isopropanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer. The product-containing elute was diluted with water and loaded on reverse phase C-8 resin, elution was carried as described in example 1. The fractions containing product with desired purity were combined and diluted with water. pH of the diluted solution was adjusted to 4.5 and passed through an HP20ss column. The column was washed with water and eluted with 70% acetonitrile. The elute was sterile filtered and lyophilized to give the daptomycin. The purity of daptomycin obtained was 98.8%.
EXAMPLE 7 10 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted using a linear gradient from 30 to 50% acetonitrile over 90 minutes. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 4.5. This solution was passed through an HP20ss column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The resin was washed with water and eluted with aqueous acetonitrile. The product-containing elute of -98% purity was concentrated by solvent evaporation. The concentrated solution was diluted with water and diafiltered using an ultrafiltration membrane. The diafiltered solution was sterile filtered and lyophilized.
EXAMPLE 8 10 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted under isocratic conditions with acetonitrile and buffer in the ratio of 35:65. During elution, the fractions with desired purity were combined, diluted with water, pH-adjusted to 6.5 and passed over weak anion exchange resin to remove organic solvent. The resin was pre-equilibrated with 30 mM Tris HCl buffer at pH 6.5. The column was eluted with 0.5 M NaCl solution. The product containing elute was depyrogenated by contacting with n-butanol in the ratio of 1: 0.2 for 30 mins. The aqueous layer containing the product was further contacted with n-heptane in the ratio of 1: 0.4 for 30 mins. Finally the aqueous 'product containing layer' was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized. The purity of daptomycin obtained was 96.6%.
EXAMPLE 9
10 L fermentation broth containing daptomycin was microfiltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopronanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted under isocratic conditions with acetone and buffer in the ratio of 37:63. During elution, fractions were collected. The fractions with 97% purity were combined, diluted with water, pH-adjusted to 4.5. This solution was passed through an HP20ss column, which was pre-equilibrated with 5% isopropanol in sodium acetate buffer at pH 4.5. The resin was washed with water and eluted with 70% methanol in buffer. The product-containing elute was diluted with water and pH of this solution was adjusted to 4.5. The pH-adjusted solution was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized.
EXAMPLE 10
5 L fermentation broth containing daptomycin was microfϊltered through 0.1 μm filter. The filtrate was mixed with 0.05% triton XlOO and pH of the solution was adjusted to 4.5. The solution was passed through an HP20ss column, which was pre-equilibrated with 5 % isopropanol in sodium acetate buffer at pH 4.5. The column was washed with 10 % isopropanol in sodium acetate buffer and eluted with 25% isopropanol in sodium acetate buffer at pH 6.5. The product containing fractions were combined, diluted with water and loaded on a C8 reverse phase chromatography column. The column was eluted using 40% acetone over 300 minutes. During elution, fractions were collected. The fractions with desired purity were combined, diluted with water, pH-adjusted to 6.5 and passed over weak anion exchange resin to remove organic solvent. The resin was pre- equilibrated with 30 mM Tris HCl buffer at pH 6.5. The column was eluted with 0.5 M NaCl solution. The product containing elute was depyrogenated by contacting with n- butanol in the ratio of 1: 0.2 for 30 mins. The aqueous layer containing the product was further contacted with n-heptane in the ratio of 1: 0.4 for 30 mins. Finally the aqueous 'product containing layer' was concentrated and diafiltered using an ultrafiltration membrane. The concentrate was sterile filtered, filled in vials and lyophilized to give daptomycin of 96%.

Claims

We Claim
1. Amorphous daptomycin having at least 98% purity.
2. Amorphous daptomycin having at least 97% purity.
3. Amorphous daptomycin having at least 96% purity.
4. Amorphous daptomycin having powder XRD pattern represented in fig 2
5. A method of purification of daptomycin comprising the steps of: a. filtration of the fermentation broth, b. optional concentration of the filtrate from step (a) by hydrophobic interaction chromatography, ultrafiltration or nanofiltration, c. purification of daptomycin by loading the filtrate from step (a) or the concentrate from step (b) on reverse phase resin followed by elution, d. recovery of purified daptomycin from the elute of reverse phase chromatography, and e. depyrogenation and lyophilization of the recovered daptomycin to give highly pure formulated product.
6. The method of purification as claimed in claim 5, wherein the recovery in step (d) is carried out using the steps of:
a. optional extraction of the elute of reverse phase chromatography with a water immiscible solvent or treatment of the elute from reverse phase chromatography by ion exchange chromatography or hydrophobic interaction chromatography, b. optional concentration of the elute of reverse phase chromatography or the elute of hydrophobic interaction chromatography from step (a) by evaporation, c. subjecting the elute of reverse phase chromatography, the daptomycin- containing raffinate from step (a), the elute of ion exchange chromatography or hydrophobic interaction chromatography from step (a), or the concentrate from step (b) to endotoxin removal followed by ultrafiltration, nanofiltration or reverse osmosis.
7. The method of purification as claimed in claim 5 or 6, wherein the hydrophobic interaction chromatography is performed on resin selected from the group comprising HP2MG, HP20, HP21, HP20SS, SP20, SP20SS, SP825, SP850, SP207, XAD 16, XAD 1600, XAD 18, XAD761 and XAD7HP .
8. The method of purification as claimed in claim 5 or 6, wherein ultrafiltration or nanofiltration is performed using membrane selected from the group comprising polysulfone, polyether sulfone, polypropylene, polyacrylonitrile, cellulose esters, mixed cellulose esters, regenerated cellulose, polyvinylidene difluoride, nylon, teflon (PTFE) and ceramic membranes.
9. The method of purification as claimed in claim 5, wherein the reverse phase resin is selected from C4, C8 and Cl 8 reverse phase resin.
10. The method of purification according to claim 5, wherein elution from the reverse phase resin is carried out with mixture of organic solvent and water or mixture of organic solvent and buffer at pH 2.5 to 8.
11. The method of purification as claimed in claim 10, wherein the organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, t- butanol, acetonitrile, acetone, tetrahydrofuran or mixture thereof.
12. The method of purification as claimed in claim 5, wherein the reverse phase chromatography elution is carried out in isocratic or gradient manner.
13. The method of purification as claimed in claim 12, wherein the gradient elution is achieved by changing the content of organic solvent, pH and/or buffer molarity during elution.
14. The method of purification as claimed in claim 6, wherein the water-immiscible solvent is selected from ethyl acetate, propyl acetate, butyl acetate, amyl acetate, chloroform, dichloromethane, methyl-t-butyl ether, diethyl ether, butanol, hexane, heptane, cyclohexane or mixture thereof.
15. The method of purification as claimed in claim 6, wherein the ion exchange chromatography is anion exchange chromatography or cation exchange chromatography.
16. The method of purification as claimed in claim 15, wherein the ion exchange chromatography is anion exchange chromatography.
17. The method of purification as claimed in claim 6, wherein the ion exchange chromatography is performed on resin selected from the group comprising diethyl aminoethyl, quaternary aminoethyl, quaternary ammonium, polyethyleneimine and quaternized polyethyleneimine type resins.
18. The method of purification as claimed in claim 6, wherein the ion exchange chromatography elution is carried out using a salt solution.
19. The method of purification as claimed in claim 18, wherein the salt is a halogenide.
20. The method of purification as claimed in 18, wherein the salt is an alkali or alkaline earth halogenide.
21. The method of purification as claimed in claim 6, wherein the evaporation is done using a thin or wiped film evaporator, falling film evaporator, forced circulation evaporator, short tube evaporator or long tube evaporator.
22. The method of purification as claimed in claim 5 or 6, wherein the ultrafiltration or nanofϊltration is performed at pH 2.5 to 8.
PCT/IN2009/000265 2008-05-26 2009-05-04 Amorphous daptomycin and a method of purification thereof WO2009144739A1 (en)

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