PROCESS FOR THE PURIFICATION OF LIPOPOLYPEPTIDE ANTIBIOTICS

Abstract

Disclosed is a process for the purification of lipopolypeptide antibiotics from culture broths which comprises: a) removal of the mycelium from the broth; b) anion-exchange chromatography of the solution resulting from stage a), eluting with di- or trivalent ions; c) optional concentration of the purified fraction resulting from stage b); d) hydrophobic interaction chromatography of the fraction resulting from stage b) or c), eluting with C1-C4 alcohols; e) cation-exchange chromatography of the desired lipopolypeptide-enriched fraction resulting from stage d), eluting at a pH equal to or greater than the isoelectric point of the lipopolypeptide; and f) dialysis, concentration and freeze-drying or spray-drying of the purified lipopolypeptide.

Claims

1. A process for the purification of lipopolypeptide antibiotics from culture broths which comprises: a) removal of the mycelium from the broth to provide a solution; b) anion exchange chromatography of the solution from step a) eluting with di- or tri-valent ions to provide a purified fraction; c) optional concentration of the purified fraction from step b); d) hydrophobic interaction chromatography of the purified fraction from step b) or c) eluting with C1-C4 alcohols to provide a lipopolypeptide-enriched fraction; e) cation exchange chromatography of the lipopolypeptide-enriched fraction from step d) eluting at a pH equal to or higher than the lipopolypeptide isoelectric point to provide a purified lipopolypeptide antibiotic; and; f) dialysis, concentration and freeze-drying or spray-drying of the purified lipopolypeptide antibiotic.

2. The process according to claim 1 wherein the lipopolypeptide antibiotic is daptomycin or surotomycin.

3. The process according to claim 1 wherein elution of step b) is carried out with magnesium sulfate, of aluminum sulfate or a straight or cyclic diamine citrate.

4. The process according to claim 3 wherein elution of step b) is carried out with magnesium sulfate.

5. The process according to claim 1 wherein step b) is carried out using a resin functionalized with weak basic groups.

6. The process according to claim 1 wherein elution of hydrophobic interaction chromatography of step d) is carried out with isopropanol.

7. The process according to claim 1 wherein the cation exchange chromatography of step e) is carried out using a resin functionalized with strong acid groups.

8. The process according to claim 7 wherein the resin is eluted at pH ranging from 3 to 7.

9. The process according to claim 1 wherein a cation exchange resin is employed for removing water-miscible organic solvents from daptomycin or surotomycin aqueous solutions.

10. The process according to claim 1 wherein a cation exchange resin is employed for decolorizing daptomycin or surotomycin in water or water-miscible organic solvents solutions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0045] Using the process according to the invention, bivalent or trivalent ions, which may be metal ions such as Mg, Zn and Al or bivalent, straight organic bases such as ethylenediamine, dimethylethylenediamine and the like, or cyclic bases such as imidazole, piperazine and the like, can be successfully used in ion-exchange chromatography for the purification of daptomycin. The salt can be formed with an inorganic or organic monovalent, bivalent or trivalent counterion, such as acetates, formates, tartrates, citrates, sulphates, chlorides, phosphates, and polyphosphates of bivalent organic bases or of bivalent ions of metallic or metalloid elements.

[0046] When the bivalent ion system is selected, account should be taken of the solubility of the salt in water, whether it is able to buffer the pH of the solution, and whether it is liable to give oxidation-reduction reactions in the presence of dissolved oxygen. Different saline systems can also be used at the various stages of the process; in particular, when conditioning the resin before use and regenerating it after use, buffer systems and saline systems different from those selected as eluent at the chromatography stage can be used.

[0047] The preferred saline system used as eluent is magnesium sulphate, employed at concentrations ranging from zero to 1 M, and in particular from zero to 600 mM, with a lower concentration at the start of chromatography which is then gradually increased until the highest value indicated is reached, with an incremental profile that can be either the step type (discontinuous) or the gradient type (continuous). Preferably, the magnesium sulphate can be combined with a buffer system used at a low concentration but still able to control the pH, maintaining it at the desired value. One example of a buffer system is magnesium acetate and acetic acid.

[0048] The same type of chromatography on anion-exchange resins can be obtained using buffers based on bivalent non-metallic ions, such as straight or cyclic diamines, also salified with a monovalent, bivalent or trivalent acid counterpart.

[0049] According to the invention, the use of bivalent ions as eluents for ion-exchange chromatography in the purification of daptomycin offers the advantages described below. The procedure is particularly useful to obtain good separation of some correlated impurities which are difficult to separate in known hydrophobic interaction chromatography processes, while simultaneously eliminating some aspecific impurities deriving from fermentation. In particular, the technique is directly applicable to fermentation broths, preferably after separating the mycelium by centrifugation or microfiltration, a good degree of purity already being obtained after the first chromatography step.

[0050] Various aqueous solutions can be used for this purpose, comprising a) a buffer system, which can be of any type, organic or inorganic, provided that it can buffer at a pH ranging from 2 to 7, and b) a bivalent salt, which is used at increasing concentrations and has the task of selectively causing the daptomycin and the correlated impurities to detach from the resin, which are divided into different fractions. A variation on the process described herein involves the use of the same salt to control both the pH and the ionic strength, for example by using it at a low concentration for pH control only, and then increasing the concentration to obtain the elution of the product.

[0051] Various types of ion-exchange resins can be used for this purpose, based on natural polymers like dextran and agarose, and on synthetic polymers like polymethacrylates and polystyrenes; weak bases like diethylamines and strong bases like quaternary ammonium ions can both be used as functional groups. Polymethacrylic resins with a diethylaminoethyl function, such as Diaion FPDA13 resin, are particularly suitable for this purpose, due to their low cost and the absence of aspecific interactions; said resins bond to daptomycin in the pH range wherein the product is most stable, and then release it with good yields.

[0052] Unlike calcium, some bivalent ions do not interfere with the process of bonding to the resins, so that the fractions obtained by purification with anionic resin can be used directly in HIC chromatography, with no need for dialysis or concentration steps.

[0053] A second field of application of chromatography on ion-exchange resin using bivalent ions is desolvation of daptomycin solutions, such as the fractions obtained by HIC chromatography. As already stated, HIC chromatography uses increasing quantities of water-miscible organic solvents to elute the product adsorbed on the resin; acetonitrile, isopropanol, ethanol or other similar solvents can be used, at variable concentrations

[0054] The invention is illustrated in greater detail in the examples below.

[0055] “Purity” here means the percentage ratio between the peak area of daptomycin and the sum total of the peak areas of daptomycin and the impurities, determined by HPLC analysis with a UV detector at 214 nm, as described in U.S. Pat. No. 8,129,342 (column 22). Where indicated, the individual impurity contents relate to the ratio between the peak area of the substance indicated and the total of the areas, determined by HPLC as above.

EXAMPLE 1

[0056] A culture of Streptomyces roseosporus is grown in submerged aerobic fermentation as described in patent EP0178152B1, administering decanoic acid during the final stages of fermentation and taking the necessary precautions to prevent its accumulation, as described in patent U.S. Pat. No. 4,208,403.

[0057] 40 liters of a suspension containing about 2.5 grams of daptomycin per gram of fermentation broth is obtained, and purified in the following steps:

[0058] a) The whole broth undergoes microfiltration using titanium dioxide-based membranes with suitable porosity (0.2 μm). An almost clear filtrate is obtained, and conveyed to the subsequent nanofiltration stages; the mycelium in the retentate is resuspended in water and microfiltered again, and the second filtrate is combined with the first to improve recovery of the product. Finally, a dark aqueous solution is obtained, with a daptomycin concentration of about 1.5 g/l, corresponding to a process yield of over 90%. The solution is partly concentrated by nanofiltration, eliminating the permeate (which is devoid of product) and retaining the retentate; to shorten the processing time and limit degradation of the product, nanofiltration is conducted at low temperature, and commenced simultaneously with microfiltration. A reddish-brown concentrated solution of crude daptomycin is obtained, with a purity of about 50-55% in the HPLC area (determined as described above), which is called the microfiltered broth;

[0059] b) The microfiltered broth is loaded, corrected to pH=6, and loaded onto a Diaion FPDA13 anionic resin column, pre-balanced with a buffer solution of 50 mM magnesium acetate at pH 6. The daptomycin bonds entirely to the resin, while a clear, colored solution is eliminated in the effluent. The resin is washed with demineralized water, then with a buffer solution of 50 mM magnesium acetate at pH=6; the effluent obtained from the column mainly contains impurities, and is eliminated. The product is eluted from the resin with a solution of 50 mM magnesium acetate and magnesium sulphate ranging from zero to 500 mM at pH 6, dividing the effluent into various fractions, followed by HPLC analysis of each fraction as described above. The fractions with adequate purity are combined, then concentrated by nanofiltration, using polymer membranes with a cut-off of about 500 Da; no micelle formation is observed;

[0060] b) The daptomycin solution is loaded onto a Diaion HP20ss resin column, pre-conditioned in 50 mM ammonium acetate buffer at a pH of about 6.3, and packed under pressure in a fixed-bed container. The solution leaving the column during loading is discarded, and a volume of demineralized water equal to the volume of resin is loaded, discarding the leaving solution. The product is eluted with a 50 mM pH 6 ammonium acetate buffer solution with increasing quantities of isopropanol, increasing the solvent concentration in a gradual linear progression from 5% to 40% (by volume); the leaving solution is fractionated in portions amounting to half the volume of resin. The fractions are analysed by HPLC and combined or discarded on the basis of the daptomycin purity data in area % by the method indicated above;

[0061] d) The purified solution of daptomycin is diluted with an equal volume of demineralized water, then loaded onto a Relisorb SP400 (Resindion) resin column pre-conditioned to pH 3 with dilute formic acid. The resin is washed with an 0.1% formic acid solution diluted in water for injection (WFI), using two volumes of solution per volume of resin; at this stage, the loss of product in the effluent is almost nil. The daptomycin is eluted from the resin with an aqueous solution of 100 mM ammonium acetate at pH 5, then concentrated by nanofiltration until the volume is reduced to ⅕th of the initial volume. The concentrate is dialyzed with WFI, adding it continuously to the retentate in quantities equal to the permeate flow.

[0062] The resulting daptomycin solution is further concentrated until a concentration of 130 g/l is reached, and then freeze-dried. Powdered daptomycin with 96% purity and a residual magnesium content of less than 10 ppm is obtained.

EXAMPLE 2

[0063] The fermentation and microfiltration of S. roseosporus are conducted as described in example 1:

[0064] a) Microfiltered broth 1 is corrected to pH=6 and loaded onto a column of Diaion FPDA13 anionic resin, pre-balanced with a buffer solution of 50 mM magnesium acetate at pH 6; the daptomycin bonds entirely to the resin, while a clear, colored solution is eliminated in the effluent. The resin is washed with demineralized water, then with a buffer solution of 50 mM magnesium acetate at pH=6; the effluent obtained from the column mainly contains impurities, and is eliminated. The product is eluted from the resin with a solution of 50 mM magnesium acetate and aluminium sulphate ranging from zero to 300 mM at pH 6, dividing the effluent into various fractions. HPLC analysis of each fraction is then conducted as described above; the fractions with adequate purity are combined and concentrated by nanofiltration, without observing micelle formation;

[0065] b) The partly purified solution is loaded onto a column of Purolite PCG1200M resin, pre-conditioned in 50 mM ammonium acetate buffer at a pH of about 6.3, and packed under pressure in a fixed-bed container. The solution leaving the column during loading is discarded, and a volume of demineralized water equal to the volume of resin is loaded, discarding the leaving solution. The product is eluted with a 50 mM pH 6 ammonium acetate buffer solution with increasing quantities of ethanol, increasing the solvent concentration in a gradual linear progression from 10% to 60% (by volume); the leaving solution is fractionated in portions amounting to half the volume of resin. The fractions are analysed by HPLC and combined or discarded on the basis of the daptomycin purity data in area % by the method indicated above. An aqueous solution containing ethanol is obtained, wherein daptomycin is present with a purity of about 96%;

[0066] c) The purified solution of daptomycin is diluted with an equal volume of demineralized water, then loaded onto a Relisorb SP400 (Resindion) resin column pre-conditioned to pH 3 with dilute formic acid. The resin is washed with an 0.1% formic acid solution diluted in water for injection (WFI), using two volumes of solution per volume of resin; at this stage, the loss of product in the effluent is almost nil. The daptomycin is eluted from the resin with an aqueous solution of 500 mM magnesium sulphate at pH 3, then concentrated by nanofiltration, dialyzed and freeze-dried as described in example 1.

[0067] Powdered daptomycin with a purity exceeding 95% is obtained.

EXAMPLE 3

[0068] a) The microfiltered broth obtained as described in example 1 is corrected to pH 6.0-6.5 with acetic acid and loaded onto a Diaion FPDA13 anionic resin column, pre-balanced with a buffer solution of 50 mM piperazine citrate at pH 6; the daptomycin bonds entirely to the resin, while a clear, colored solution is eliminated in the effluent. The resin is washed with demineralized water, and then with a buffer solution of 50 mM piperazine citrate at pH 6; the effluent obtained from the column mainly contains impurities, and is eliminated;

[0069] The product is eluted from the resin with a solution of piperazine citrate ranging from 50 mM to 200 mM at pH 6, dividing the effluent into various fractions, followed by HPLC analysis of each fraction as described above; the fractions with adequate purity are combined and concentrated by nanofiltration, without observing micelle formation;

[0070] b) The solution of the concentrated product is acidified to pH 3.8, and then subjected to liquid/liquid extraction, adding an equal volume of n-butanol; the daptomycin is again extracted from the butanol solution with a small volume (½ the solvent volume) of aqueous buffer at pH 6.3. The solution is distilled under vacuum to reduce the residual quantity of solvent;

[0071] c) The solution is loaded onto HP20ss resin as described in example 1, paragraph c), but using solutions with an increasing isopropanol concentration. The fractions with purity exceeding 95% in HPLC area are selected and combined;

[0072] d) The purified solution of daptomycin is diluted with an equal volume of demineralized water, then loaded onto a Relisorb SP400 (Resindion) resin column pre-conditioned to pH 3 with dilute formic acid. The resin is washed with an 0.1% formic acid solution diluted in water for injection (WFI), using two volumes of solution per volume of resin; at this stage, the loss of product in the effluent is almost nil. The daptomycin is eluted from the resin with an aqueous solution of 500 mM sodium chloride in 20% ethanol at pH 3, then concentrated by nanofiltration, dialyzed and freeze-dried as described in example 1.

EXAMPLE 4

[0073] a) The microfiltered broth obtained as described in example 1 is corrected to pH 6.0-6.5 and loaded onto a Diaion FPDA13 anionic resin column, pre-balanced with a buffer solution of 50 mM ethylenediamine acetate at pH 6; the daptomycin bonds entirely to the resin, while a clear, colored solution is eliminated in the effluent. The resin is washed with demineralized water, and then with a buffer solution of 50 mM ethylenediamine acetate at pH 6; the effluent obtained from the column mainly contains impurities, and is eliminated. The product is eluted from the resin with a solution of 50 mM to 300 mM ethylenediamine acetate at pH 6, dividing the effluent into various fractions, followed by HPLC analysis of each fraction as described above; the fractions with adequate purity are combined;

[0074] b) The resulting daptomycin solution is adjusted to pH 3 with hydrochloric acid, then further purified with Purolite PCG1200M resin. The product is eluted with a 50 mM pH 6 ammonium acetate buffer solution with increasing quantities of isopropanol, increasing the solvent concentration in a gradual linear progression from zero to 40% (by volume). The fractions are analysed by HPLC and combined on the basis of purity;

[0075] c) The pure daptomycin solution is desolvated, correcting to pH 3 and capturing the product on Relite SP400 resin. The resulting product is eluted with a sodium acetate solution at pH 6, obtaining a quantitative yield. The solution is then dialyzed with water by nanofiltration on polysulphone membranes with a cut-off of 500 Da, concentrated to 100 g/l and freeze-dried.

EXAMPLE 5

[0076] a) The fermentation and microfiltration are conducted as described in example 1, with the difference that the microfiltered broth is loaded directly onto the FPDA13 resin pre-conditioned with acetate buffer at pH 6. Demineralized water equal to two volumes of resin is loaded, then eluted with an ammonium acetate buffer containing ammonium sulphate in increasing quantities from 50 mM to 500 mM, at pH 6;

[0077] b) The resulting solution is loaded directly (at the same concentration) onto a Purolite PCG1200M resin column, pre-conditioned with formic acid at pH 3 and packed under pressure in a fixed-bed container. The solution leaving the column during loading is discarded, and a volume of demineralized water equal to the volume of resin is loaded, discarding the leaving solution. The product is eluted with a solution containing increasing quantities of isopropanol with the addition of formic acid to pH 3, increasing the solvent concentration in a gradual linear progression from zero to 50% (by volume); the leaving solution is fractionated in portions amounting to half the volume of resin. The fractions are analysed by HPLC and combined on the basis of the daptomycin purity data;

[0078] c) The pure daptomycin solution is desolvated with Relite SP400, loading at pH 3 and eluting with ammonium acetate buffer at pH 7. The yield obtained is quantitative.

EXAMPLE 6

[0079] a) The microfiltered solution obtained in example 1 is loaded onto a column containing Amberlite 1200H cationic resin pre-balanced with 50 mM sodium acetate buffer at pH 6; a yellow solution containing about the same concentration of daptomycin leaves the column. The resin is further eluted with the same buffer, using a quantity by volume equal to twice the volume of resin; the solutions eluted are concentrated by ultrafiltration without observing micelle formation. A bleached solution with a 95% daptomycin yield is obtained;

[0080] b) The solution is concentrated by nanofiltration, then acidified to pH 3 with HCl and extracted with an equal volume of n-butanol; after separation, the aqueous phase is discarded. The organic phase is extracted with a 50 mM buffer solution of ammonium acetate, adding ammonia to correct the pH to 6;

[0081] c) The resulting solution is purified by chromatography on Relite Diaion HP20 resin, eluting with a linear gradient of ethanol from zero to 60%, at pH 6. The fractions with a purity exceeding 85% are selected, and desolvated as described in example 5, point c). The solution is dialyzed and concentrated by nanofiltration on 500 Da membranes.

EXAMPLE 7

[0082] The purification of the microfiltered broth proceeds as described in example 1, up to point c), with the difference that the desolvation is conducted with anionic resin. The aqueous solution of daptomycin originating from HIC chromatography, containing isopropanol, is loaded onto an FPDA13 resin column pre-conditioned to pH 6 with magnesium acetate buffer.

[0083] The resin is washed with water, used in quantities equal to twice the volume of resin. The product is eluted with a solution of 500 mM magnesium sulphate and 50 mM magnesium acetate at pH 6.

[0084] The resulting solution is concentrated with a nanofilter and dialyzed with water; the solution is corrected with HCL to pH 3, and finally, further concentrated to 130 g/l. The solution is frozen and freeze-dried under high vacuum, to obtain a pale yellow powder consisting of daptomycin with 96% purity containing less than 10 ppm of magnesium.