DESC:
FIELD OF THE INVENTION
The present invention relates to an improved process for preparing a daptomycin of formula I, which is a cyclic lipopeptide antibiotic derived from the organism Streptomyces roseporous. Daptomycin is used to treat various bacterial infections caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).

BACKGROUND OF THE INVENTION
PCT Application Publication No. WO0153330A3 describes a daptomycin purification method in which daptomycin is filtered under pressure on a 10, 000 dalton molecular weight cutoff (MWCO) ultrafilter at a pH of 7-8, under these conditions, daptomycin passes through the filter but pyrogens such as lipopolysaccharides do not pass into the filtrate. After the initial ultra-filtration, the pH of the filtrate is lowered to pH 2.5 to 4.5 and the filtrate is concentrated on a 10,000 MWCO ultra-filter to approximately 120 mg/mL and the daptomycin is retained on the filter. After concentration, the concentration of daptomycin is adjusted to 105 mg/mL and used to fill vials under aseptic conditions.

U.S. Patent No. 4,874,843 describes a daptomycin purification method in which the fermentation broth was filtered and passed through a column containing HP-20 resin. After elution, the semipurified daptomycin was passed through a column containing HP-20ss, and then separated again on HP-20 resin.

U.S. Patent No. 4,885,243 describes a method for preparing a highly pure form of daptomycin or a daptomycin-related lipopeptide.

The present invention provides an improved process for preparing daptomycin.

OBJECT OF THE INVENTION
Accordingly, it is an object of the present invention to overcome the drawbacks of prior art.

It is another object of the present invention to provide an improved process for preparing daptomycin.

SUMMARY OF THE INVENTION
1. In one aspect the present invention provides an improved process for the preparation of daptomycin comprising the steps of:
a) providing a fermentation broth obtained from the culture of daptomycin produced microorganism;
b) adjusting a pH of the fermentation broth in the range of 3.5 to 4.0;
c) precipitating the daptomycin;
d) subjecting the solution through microfiltration; and
e) obtaining the microfiltrate comprising precipitated

In another aspect the present invention provides a method for reducing the content of pH buffering salt content in the final daptomycin API comprising the steps of:
a) providing purified daptomycin, wherein content of pH buffering salt which used for crystallization of daptomycin is present in the range of 80,000 to 90,000 ppm;
b) dissolving daptomycin crystals in water and adjusted the pH in the range of 3.5 to 4.0;
c) adding n-butanol to the solution obtained in step a) and allow the layers to be separated;
d) extracting n-butanol layer containing daptomycin;
e) washing the n-butanol layer with EDTA solution followed by water;
f) subjecting washed n-butanol layer to crystallization at 15-25 ° C by slowly adding isopropanol; and
g) collecting the precipitated crystals and dried to obtain final daptomycin API, wherein the content of pH buffering salt reduced from 80,000-90,000 ppm to about 50-80 ppm.

SHORT DESCRIPTION OF THE INVENTION
A first embodiment of the invention relates to an improved process for preparing a daptomycin, the process comprising the steps of:
a) providing a fermentation broth obtained from the culture of daptomycin produced microorganism;
b) adjusting a pH between 3.5 to 4.0;
c) filtering in a micro filter membrane at room temperature;
d) removing the permeate and concentrating the resulting retentate;
e) extracting the precipitated daptomycin in the retentate with n-butanol;
f) treating the extracted butanol solution obtained in step e) with aqueous buffer at a pH 6.0-6.5;
g) back extracted daptomycin to aqueous buffer to clear light coloured solution comprising partly purified daptomycin with purity of 75-80 % ;
h) loading the solution of step g) into anion exchange chromatography and washed the column by 2 column volumes of washing buffer (30 mM Tris acetate, pH 6.5);
i) eluting the first eluate of anion exchange column with buffer 1, followed by buffer 2 then followed by buffer 3;
j) loading the first eluate of step j) onto a hydrophobic interaction chromatography (HIC) column and washed the column by 4 column volumes of washing buffer 1 (30 mM Tris acetate buffer: Acetonitrile at a ratio of 90:10, pH 6.5) followed by 6 column volumes of washing buffer 2 (30 mM Tris acetate buffer: Acetonitrile at a ratio of 80: 20);
k) eluting the second eluate of HIC column with 15 column volumes of buffer (30 mM Tris acetate buffer: Acetonitrile at ratio of 75:25;
l) concentrating the second eluate of step k) to remove residual solvent followed by increasing the concentration to 40-50 mg/g by Nano filtration.
m) precipitating or crystallizing daptomycin from the solution at a temperature of about 0 to 30 degrees Celsius and a pH of about 6.0 to 7.0; and
n) collecting the precipitated or crystallized daptomycin from the solution.

In a first aspect of the first embodiment, step b) comprises, adjusting the pH of end of fermentation broth before microfiltration between 3.5 and 4.0, where daptomycin is precipitated and bound to cell mass which retained in the retentate side of microfiltration.

The precipitated daptomycin broth subjected to microfiltration step in the initial clarification steps and handling volume reduced by 5 times with less than 5-7% loss of product, for example 100 kL reduced to 20 kL.

In a second aspect of the first embodiment, step f) comprises, extracting the daptomycin in the concentrated retentate with butanol at a ratio of 1:1 to 1.2. The extraction is performed four to five times and the handling clarified solution becomes 20 - 24kL.

At pH 3.5 to 4.0 during microfiltration, the daptomycin net charge becomes Zero (PI), this helps in selective extraction of daptomycin and solvent soluble impurities to the butanol layer leaving behind the water-soluble junk and cell mass.

The water-soluble metabolites and unused media components of end of fermentation broth removed along with permeate of microfiltration, which helps in the better clarification of product and avoiding the emulsion formation in the later butanol and aqueous back extraction steps of product.

In a third aspect of the first embodiment, step g) comprises back extraction of daptomycin from the butanol solution with the aqueous buffer at a ratio of 1: 0.25 two times followed by third time at a ratio of 1:0.1 two times. The final handling clarified solution becomes 14 - 17kL.

In a fourth aspect of the first embodiment, aqueous extraction of step g) is performed at pH 6.0 to 6.5. At pH 6.0 to 6.5, the daptomycin becomes charged and solubilized in the aqueous solution, this helps in selective back extraction of daptomycin and related impurities to the aqueous layer leaving behind organic soluble impurities in spent n-butanol layer.

The aqueous layer extracted finally appears in clear light coloured in nature with purity of 75%-80%.

In a fifth aspect of the first embodiment, the reduced handling volume of partly purified daptomycin solution of step h) increased the chromatographic loading capacity for at least 3-5 times, for examples from 5 g/L to 15-25 g/L in the subsequent anion exchange chromatography purification step.

In a sixth aspect of the first embodiment, because of the loading solution of partly purified daptomycin in clear light coloured nature, there is no binding of coloured junk on to the resin used in anion exchange chromatography. This helps life of resin, so resin can be used for many cycles and also consistency in run expected batch to batch because of clear clarified load.

In a seventh aspect of the first embodiment, step j) comprises eluting first eluate of anion exchange column with 5 column volumes of buffer 1 (75 mM TRIS acetate and 10 mM CaCl2, pH 6.5) followed by 10 column volumes of buffer 2 (75 mM TRIS acetate and 30 mM CaCl2) then followed by 5 column volumes of buffer 3 (75 mM TRIS acetate buffer and 40 mM CaCl2).

The loading solution of partly purified daptomycin in clear light colored nature reduced number of buffers used in the column to neutralize the column.

In a preferred embodiment, regeneration of resin used 2-3 column volumes of 4N sodium chloride solution followed by 2-3 column volumes of water. In general, if load is not clean and dark colored in nature, it requires sodium hydroxide and sodium chloride solution buffer washing in mixture or separate, followed by water wash to neutralise the resin and regeneration. This generally requires multiple column volumes of liquid to regenerate and neutralise. In spite complete cleaning of resin is not expected and resin deteriorate fast. The present invention provides loading of clean and clear clarified load into anion exchange chromatography, the regeneration of column required mild treatment to clean the resin and required minimal water consumption.

In an eight aspect of first embodiment, the fractionated pools from anion exchange column were combined and loaded into hydrophobic interaction chromatography (HIC). The HP-20 SS resin was equilibrated with an equilibration buffer. After the loading was finished, the column was washed with Tris buffer and isopropanol followed by elution with elution buffer comprising Tris acetate and isopropanol. In a preferred embodiment, the buffer is a mixture of Tris buffer and isopropanol or a mixture of Tris buffer and acetonitrile.

The fractionated pools from HIC concentrated to remove residual solvent and increasing the concentration by nano filtration. After filtration, concentrated daptomycin subjected to crystallization in the presence of calcium chloride or calcium acetate to obtain precipated daptomycin.

In a preferred embodiment, the purified daptomycin after crystallization comprises daptomycin with purity of 96-98% as measured by HPLC.

The purified daptomycin obtained after crystallization by using salts like calcium chloride, calcium acetate as a pH buffering agent may contain residual amount of these salts as an impurity in the final product. The trace amount of salt must be removed to obtain final daptomycin API which may be assayed by purity method of HPLC to ensure its meet API specification.

In ninth aspect of first embodiment, the purified daptomycin collected after crystallization is subjected to ultrafiltration followed by lyophilization to obtain final daptomycin API.

In another aspect, the purified daptomycin crystals is dissolved in water and extracted with n-butanol. The n-butanol extract of daptomycin is subsequently washed with EDTA and water, this helps in removal of residual salt present along with the product. The n-butanol extract of daptomycin after EDTA and water washing is subjected to recrystallization and isolating daptomycin in solid form.

A method for reducing the content of pH buffering salt content in the final daptomycin API comprising the steps of:
a) providing purified daptomycin, wherein content of pH buffering salt which used for crystallization of daptomycin is present in the range of 80,000 to 90,000 ppm;
b) dissolving daptomycin crystals in water and adjusted the pH in the range of 3.5 to 4.0;
c) adding n-butanol to the solution obtained in step a) and allow the layers to be separated;
d) extracting n-butanol layer containing daptomycin;
e) washing the n-butanol layer with EDTA solution followed by water;
f) subjecting washed n-butanol layer to crystallization at 15-25 ° C by slowly adding isopropanol; and
g) collecting the precipitated crystals and dried to obtain final daptomycin API, wherein the content of pH buffering salt reduced from 80,000-90,000 ppm to about 50-80 ppm

A second embodiment of the invention relates to an improved process for preparing daptomycin comprising the steps of:
a) providing a fermentation broth obtained from the culture of daptomycin produced microorganism;
b) adjusting a pH between 3.5 to 4.0;
c) precipitating the daptomycin;
d) subjecting the solution through microfiltration; and
e) obtaining the microfilter retentate comprising precipitated daptomycin

In first aspect of the second embodiment, the microfiltration of step d) is performed at room temperature.

In second aspect of the second embodiment, wherein the microfiltration process of step d) is performed using a microfiltration membrane formed from a material selected from the group consisting of natural polymers, synthetic polymers, and ceramics.

In a preferred embodiment, 0.2? or 0.1µ ceramic membrane is used.

In third aspect of the second embodiment, wherein the microfiltration process is followed by at least one chromatographic purification step.

A third embodiment of the invention relates to an improved process for preparing daptomycin comprising the steps of:
a) providing a fermentation broth obtained from the culture of daptomycin produced microorganism;
b) adjusting a pH between 3.5 to 4.0;
c) precipitating the daptomycin;
d) subjecting the solution through microfiltration;
e) obtaining the microfilter retentate comprising precipitated daptomycin;
f) extracting the concentrated daptomycin with n-butanol;
g) back extracting with the aqueous buffer at pH 6.0 to 6.5;
h) removing colored impurities to obtain light colored aqueous solution comprising partly purified daptomycin having purity of 75 to 80%; and
i) optionally further purification by atleast one chromatographic step.

In general, colour is a process stream can be derived from a variety of different sources but there are two major sources. The first is the fermentation from which the product, or bulk intermediate is derived. Those colours that are derived from the carbon source used in the fermentation and are often similar to those encountered during the processing of sugar tend to be large molecules. The second source is derived from degradation products.

One of the biggest challenges in the purification of fermentation products is the reduction of costs. The bacterial cultures can be very darkly coloured, and this coloured junk interfere in the purification process and effects the consistency of the process and also overall recovery of the process as requires additional purification steps to clean the junk. In addition resins used in the process life cycle depend on the nature of junk.

The present invention provides an improved process for purifying daptomycin, wherein all coloured impurities are removed in the clarified fermentation broth before loading into the chromatographic column for purification.

DETAILED DESCRIPTION OF THE INVENTION
The best mode of carrying out the present invention is illustrated by the below mentioned examples. These examples are provided as illustration only and hence should not be construed as limitation to the scope of the invention.

Example: 1
The end of fermentation broth obtained from the culture of daptomycin produced microorganism was subject to microfiltration as follows,
The daptomycin end of fermentation broth (100 kL) was provided, the pH was adjusted to 3.5 to 4.0, mixed the broth for 30-60 minutes at room temperature, and at pH 3.5 – 4.0 the daptomycin was precipitated and bound to cell mass which retained in the retentate side of microfiltration. The microfiltration was carried through 0.2? or 0.1µ ceramic membrane at room temperature to give microfilter retentate concentration of 75 -80%, diafilter the retentate with a ratio of 1:1 50 mM sodium acetate buffer at pH 4.5 * 2 times.

Example: 2
The microfilter retentate (20 kL) comprising daptomycin was extracted with n-butanol at a ratio of 1:0.25 at room temperature, allow the layers to separated, and the extraction was performed four times. Combined the organic layers (20 kL) and washed with water at a ratio of 1: 0.25.

After the water washing, back extracting the daptomycin containing n-butanol extract with aqueous buffer (50 mM Tris acetate buffer, pH 6.5 to 7.0) 2 times at ratio of 1:0.25, followed by two times at a ratio of 1:0.1, allow the layers to be separated. Combined the aqueous layers (14 kL) to obtain clear light colored aqueous solution containing partly purified daptomycin having purity of 75-80%.

Example: 3
The clear light colored aqueous solution containing partly purified daptomycin having purity of 75-80% obtained in Example: 1 was diluted with purified water at a ratio of 1:1, the pH was adjusted to 6.3-6.5 and obtained conditioned harvest, this conditioned harvest was purified by anion exchange chromatography as follows:

The FPDA-13 resin was equilibrated with 4 column volumes of equilibration buffer (30 mM Tris acetate, pH 6.5) followed by the loading of conditioned harvest at a linear flow rate of 150 cm/hr, after loading was finished the column was washed with 2 column volumes of wash buffer (30 mM Tris acetate, pH 6.5). Bound product was eluted with buffer 1 (75 mM tris acetate and 10 mM calcium chloride, pH 6.5) followed by elution with buffer 2 (75 mM tris acetate and 30 mM calcium chloride, pH 6.5) and finally eluted with buffer 3 (75 mM tris acetate and 40 mM calcium chloride). The pooled fractions from the anion exchange column were collected based on purity.

Example: 4
The fractionation pool from the anion exchange column was further loaded into hydrophobic interaction chromatographic column as follows,

The HP-20 SS resin was equilibrated with 4 column volumes of equilibration buffer (30 mM Tris acetate buffer and isopropanol at a ratio of 90: 10, pH 6.5) followed by the loading of collected fraction from the anion exchange column at a linear flow rate of 250 cm/hr, after loading was finished the column was washed 4 column volumes of wash buffer 1 (30 mM TRIS acetate buffer and isopropanol at ratio of 90:10, pH 6.5) and 6 column volumes of wash buffer 2 (30 mM TRIS acetate buffer and isopropanol at ratio of 80:20, pH 6.5) . Bound product was eluted with eluate buffer (30 mM TRIS acetate buffer and isopropanol at ratio of 75: 25, pH 6.5). The pooled fractions from the hydrophobic interaction chromatography (HIC) were collected.

Example: 5
The fractionation pool from the HIC column was concentrated to remove the isopropanol to less than 5-7% in the pool at 30-35 degree celcius. The solution was then processed through a nanofiltation step with 180-300 Daltons molecular weight PES or PS membrane. Diafiltration was performed using water till the conductivity of permeate less than 100-150µS.

Example: 6
After nanofiltration, the daptomycin solution containing 40-50 mg/mL of daptomycin was subject to crystallization as follows,

Adjust the pH of the nanofiltrated daptomycin solution to pH 4.5 to 6.5, calcium acetate was added at a mole ratio of 1:4 with respect to daptomycin solution at room temperature and allowed the solution to stand for 45-60 minutes at room temperature. Then isopropanol was added to a concentration of 78%. The solution was mixed, then slowly added another aliquots of isopropanol atleast for 3 hrs till the final concentration of 84%. A precipitated material was formed after incubation of mixture for 12-18 hrs, filtered the precipitate and suck dried for 3-6 hrs to obtain purified daptomycin has a HPLC purity of 96-98%. The content of calcium acetate is 80,000- 90,000 ppm.

Example: 7
The filtered daptomycin was further passed through ultrafiltration membrane of 10,000 kilo daltons molecular weight cutoff at pH 3.5 to 4.5. The ultrafiltration was first performed with diafiltration of EDTA to remove calcium ions, followed by acetic acid to remove sodium and other monovalent cations and then finally followed by diafiltration with purified water.

After ultrafiltration, the daptomycin solution was lyophilized using standard conditions to get final daptomycin API.

Example: 8
The purified daptomycin with HPLC purity of 98% obtained in Example: 7 was dissolved in water and adjusted the pH in the range of 3.5 to 5 with 3M HCl/ortho phosphoric acid solution. The n-butanol was added to the solution obtained and allow the layers to be separated. The extracted n-butanol layer containing daptomycin was washed with EDTA solution followed by water. After washing, n-butanol layer was subjected to crystallization by adding isopropanol at 15-25° C. The precipitated crystals collected and vacuum dried at 22-28°C for 24-48 hrs to final daptomycin API. The HPLC Purity is 97.5%. Calcium acetate content is 55 ppm.
,CLAIMS:
1. An improved process for the preparation of daptomycin comprising the steps of:
a) providing a fermentation broth obtained from the culture of daptomycin produced microorganism;
b) adjusting a pH of the fermentation broth in the range of 3.5 to 4.0;
c) precipitating the daptomycin;
d) subjecting the solution through microfiltration; and
e) obtaining the microfiltrate comprising precipitated daptomycin.

2. The process as claimed in claim 1, wherein daptomycin net charge becomes Zero (PI) at pH in the range of 3.5 to 4.

3. The process as claimed in claim 1, wherein daptomycin is precipated after pH adjustment and bound to cell mass which retained in the retentate side of microfiltrate during microfiltration

4. The process as claimed in claim 1, wherein the microfiltration is carried out by using 0.2? or 0.1? ceramic membrane.

5. The process as claimed in claim 1, wherein the handling volume of daptomycin after pH adjustment and microfiltration reduced to 20 kL from 100 kL.

6. The process as claimed in claim 1, further comprising the steps of:
a) extracting the precipated daptomycin with n-butanol;
b) back extracting with the aqueous buffer at pH 6.0 to 6.5;
c) removing colored impurities to obtain light colored aqueous solution comprising partly purified daptomycin having HPLC purity of 75 to 80%;
d) subjecting the solution of step c) to anion exchange chromatography followed by hydrophobic interaction chromatography (HIC);
e) concentrating the HIC eluate obtained in step d) to remove residual solvent and increasing the concentration by Nano filtration;
f) crystallizing daptomycin from the solution at a temperature of about 0 to 30° C at pH 6.0 to 7.0;
g) collecting the crystallized daptomycin from the solution; and
h) purifying optionally by recrystallization.

7. The process as claim in claim 6, wherein the loading solution of step c) into chromatographic purification results in no binding of colored junk onto the resin used in chromatography.

8. A method for reducing the content of pH buffering salt content in the final daptomycin API comprising the steps of:
a) providing purified daptomycin, wherein content of pH buffering salt which used for crystallization of daptomycin is present in the range of 80,000 to 90,000 ppm;
b) dissolving daptomycin crystals in water and adjusted the pH in the range of 3.5 to 4.0;
c) adding n-butanol to the solution obtained in step a) and allow the layers to be separated;
d) extracting n-butanol layer containing daptomycin;
e) washing the n-butanol layer with EDTA solution followed by water;
f) subjecting washed n-butanol layer to crystallization at 15-25 ° C by slowly adding isopropanol; and
g) collecting the precipitated crystals and dried to obtain final daptomycin API, wherein the content of pH buffering salt reduced from 80,000-90,000 ppm to about 50-80 ppm.

9. The process as claimed in claim 9, wherein pH buffering salt in step a) selected from calcium chloride, calcium acetate.

10. The process according to claim 8, wherein the final daptomycin API has purity of 96-98% by HPLC and pH buffering salt of about 50-80 ppm