Fidaxomicin purification method

Abstract

A fidaxomicin purification method, comprising: fermenting Actinoplanes sp. HS-16-20 to generate fermented liquid; conducting solid/liquid separation on the fermented liquid, soaking mycelium in an organic solvent, and filtering to obtain a solution containing fidaxomicin; treating the solution with nanofiltration concentrate, and separating to obtain fidaxomicin crude product; conducting preparative column chromatography on the fidaxomicin crude product, eluting with an acid aqueous solution containing an organic solvent, and separating to obtain the refined fidaxomicin product.

Claims

1. A fidaxomicin purification process, comprising: (1) conducting preparative column chromatography with a preparative column on a crude sample comprising fidaxomicin, eluting the fidaxomicin from the crude sample with an acid aqueous solution containing an organic solvent to generate eluates containing the fidaxomicin, and collecting and combining the eluates containing the fidaxomicin to form an eluate containing the fidaxomicin; and (2) separating the fidaxomicin from the acid aqueous solution of the eluate to yield refined fidaxomicin; wherein the fidaxomicin is generated by Actinoplanes sp. HS-16-20, accession number is CGMCC No. 7294, and the acid aqueous solution containing an organic solvent is a solution comprising a methanol:water:formic acid ratio of 55-75:45-25:0.1, or an acetonitrile:water:formic acid ratio of 45-65:55-35:0.1 (v:v:v).

2. The process according to claim 1, wherein the crude sample comprising fidaxomicin in step (1) is prepared according to the following process: (a) pre-treating fidaxomicin fermentation broth to obtain a solution containing fidaxomicin; (b) conducting nanofiltration and concentrating on the solution containing fidaxomicin to generate a concentrated solution containing the fidaxomicin; and (c) separating the concentrated solution to yield the crude sample comprising fidaxomicin.

3. The process according to claim 2, wherein the pre-treating in step (a) comprises conducting a solid/liquid separation on the fidaxomicin fermentation broth to obtain mycelium, soaking the mycelium in an organic solvent, and filtering to obtain a solution containing fidaxomicin.

4. The process according to claim 3, wherein the organic solvent is methanol or ethanol.

5. The process according to claim 2, wherein the process for preparing fidaxomicin fermentation broth in step (a) comprises: {circle around (1)} incubating a producer strain of fidaxomicin in plate medium, culturing the producer strain to make mature mycelium, and obtaining a colony that produces fidaxomicin; {circle around (2)} incubating the colony in a shaking flask of seed medium, culturing the seed medium, and obtaining a seed culture; {circle around (3)} incubating the seed culture in seed tank medium, culturing the seed tank medium, and obtaining a seed culture of fermenter; and {circle around (4)} incubating the seed culture of fermenter in fermentation medium, culturing the fermentation medium, and obtaining the fidaxomicin fermentation broth.

6. The process according to claim 2, wherein the conducting nanofiltration and concentrating is performed with a DK or a DL nanofiltration membrane.

7. The process according to claim 2, wherein the concentrated solution containing the fidaxomicin has a fidaxomicin concentration of 10000mg/L.

8. The process according to claim 2, wherein the separating of step (c) comprises adding an anti-solvent to the concentrated solution containing the fidaxomicin and separating the fidaxomicin from the anti-solvent to yield the crude sample comprising fidaxomicin.

9. The process according to claim 8, wherein the anti-solvent is water, and the addition of the water makes the concentration of the organic solvent in the concentrated solution less than 35%, wherein the percentage refers to the volume of the organic solvent in the concentrated solution accounting for the total volume of the concentrated solution obtained after the addition of the water.

10. The process according to claim 1, wherein the preparative column in step (1) contains C8 filler.

11. The process according to claim 1, wherein the acid aqueous solution containing the organic solvent used in step (1) is a solution comprising a methanol:water:formic acid ration of 60-70:40-30:0.1, or an acetonitrile:water:formic acid ratio of 50-60:50-40:0.1 (v:v:v).

12. The process according to claim 1, wherein the eluate containing the fidaxomicin has a purity of 99.5% as determined by high-performance liquid chromatography (HPLC).

13. The process according to claim 1, wherein separating comprises adding an anti-solvent into the eluate containing the fidaxomicin, and separating the refined fidaxomicin from the eluate.

14. The process according to claim 13, wherein the anti-solvent is water and the water is added to generate a water:eluate ratio of 1-2:1 (v:v).

15. A fidaxomicin purification process, comprising: incubating a producer strain of fidaxomicin in plate medium, culturing the producer strain to make mature mycelium, and obtaining a colony that produces fidaxomicin; incubating the colony in a shaking flask of seed medium, culturing the seed medium, and obtaining a seed culture; incubating the seed culture in seed tank medium, culturing the seed tank medium, and obtaining a seed culture of fermenter; incubating the seed culture of fermenter in fermentation medium, culturing the fermentation medium, and obtaining a crude sample comprising fidaxomicin; conducting preparative column chromatography with a preparative column on the crude sample comprising fidaxomicin, eluting the fidaxomicin from the crude sample with an acid aqueous solution containing an organic solvent to generate eluates containing the fidaxomicin, and collecting and combining the eluates containing the fidaxomicin to form an eluate containing the fidaxomicin; and separating the fidaxomicin from the acid aqueous solution of the eluate to yield refined fidaxomicin; wherein the fidaxomicin is generated by Actinoplanes sp. HS-16-20, accession number is CGMCC No. 7294, and the acid aqueous solution containing an organic solvent is a solution comprising a methanol:water:formic acid ratio of 55-75:45-25:0.1, or an acetonitrile:water:formic acid ratio of 45-65:55-35:0.1 (v:v:v).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is HPLC graph of the fidaxomicin fermentation broth in embodiment 1.

(2) FIG. 2 is HPLC graph of the precipitated crude fidaxomicin prepared according to embodiment 1.

(3) FIG. 3 is HPLC graph of the refined fidaxomicin prepared according to embodiment 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(4) The Actinoplanes sp. used for fermentation culture in the present invention has been deposited in China General Microbiological Culture Collection Center on Mar. 11, 2013, whose accession number is CGMCC No. 7294; more details is recited in Chinese patent application CN201310501389.3 filed on Oct. 16, 2013, the contents of which are incorporated herein by reference in their entireties. The nanofiltration membrane is supplied by General Electric Company (GE); the preparation column is supplied by Beijing Tong Heng Innovation Technology Co., Ltd.; the C8 filler is supplied by Acchorom Technology and Akzo Nobel Company; the high performance liquid chromatograph is LC2010HT supplied by Shimadzu; the ethanol and methanol added into the fidaxomicin mycelium are commercially available industrial grade; the methanol and acetonitrile used for preparation are commercially available chromatographic grade; the formic acid is commercially available reagent grade. The chromatographic purity in the following embodiments refers to HPLC purity, and ventilation means introducing the air.

(5) The process for incubating the fermentation broth of the present invention is as below:

(6) (1) Preparing and Incubating Plate Colony

(7) ISP2 medium was employed as the plate medium, the formulation of ISP2 medium comprised (g/L): glucose 4, yeast extract 4, malt extract 10, agar 15, and distilled water was added to reach a constant volume of 1000 mL, pH 7.3, wherein g/L referred to the mass of each component in 1 L ISP2 medium. The ISP2 medium underwent sterilization for 20 min at a pressure of 1.05 kg/cm.sup.2 before use, and then was poured into the plate after cooled to 50-60 C., Actinoplanes sp. (CGMCC No. 7294) mycelium was inoculated, incubated at 281 C. for 8 days, the mycelium was mature, and a Actinoplanes sp. colony was obtained.

(8) (2) Preparing and Incubating the Shaking Flask Seed Liquid

(9) The formulation of the seed medium included (g/L): sucrose 2, sorbitol 3, cottonseed meal 3, peanut meal 1.5, CaCO.sub.3 0.6, MgSO.sub.4.7H.sub.2O 0.3 and water was added to reach a constant volume of 1000 mL, pH 7.2, wherein g/L referred to the mass of each component in 1 L seed medium. The liquid volume for the shaking flask was 25 mL/250 mL, i.e. 25 mL seed medium in 250 mL shaking flask. The seed medium underwent sterilization at 121 C. for 30 min before use. Then the Actinoplanes sp. colony obtained according to step (1) was inoculated into the seed medium with a inoculum rate of 10.sup.5-10.sup.6 c.f.u/mL, incubated in a shaking bed at 281 C. and 250 rpm for 28 hours, pH value of the culture liquid usually 6.8-7.0, the (Actinoplanes sp.) mycelium concentration was 25-30% (volume percent).

(10) (3) Preparing the Seed Tank Culture Liquid

(11) 10 L seed medium was inoculated into a 15 L seed tank (the formulation of the seed tank medium included (g/L): sucrose 10, sorbitol 2, soluble starch 3, (NH.sub.4).sub.2SO.sub.4 0.5, beef extract 2, peanut meal 1, KH.sub.2PO.sub.4 0.04, and water was added to reach a constant volume of 1000 mL, wherein g/L referred to the mass of each component in 1 L seed tank medium), and steam sterilization was conducted at 121 C. for 30 min. After cooling, 100 mL shaking flask seed liquid was inoculated into the seed tank, incubated at 281 C. under a rotational speed of 200 rpm and 1 vvm (ventilation volume) for 24 hours, pH value of the culture liquid usually 6.8-7.0, the (Actinoplanes sp.) mycelium concentration was 25-30% (v/v).

(12) (4) Preparing and Incubating the Fermentation Tank Medium

(13) The formulation of the fermentation medium included (g/L): sucrose 10, sorbitol 2, soluble starch 3, (NH.sub.4).sub.2SO.sub.4 0.5, beef extract 2, peanut meal 1, KH.sub.2PO.sub.4 0.04 and water was added to reach a constant volume of 1000 mL, wherein g/L referred to the mass of each component in 1 L fermentation medium. 1% PPG (polypropylene glycol) was added into the fermentation medium as a defoamer. The feeding volume was 35 L (i.e. 35 L fermentation medium), pH was at 7.0, and steam sterilization was conducted at 121 C. for 20 min. After cooling, about 3.5 L seed tank culture liquid was inoculated, incubated at 281 C. under a rotational speed of 200-300 rpm and 0.8-1.0 vvm (ventilation volume) for 8 days.

(14) Embodiment 1

(15) 30 L fermentation broth containing a fidaxomicin fermenting unit of 3026 mg/L (liquid chromatogram referring to FIG. 1) was filtered to obtain 7.4 kg mycelium, and the mycelium was soaked in 22 L ethanol and then filtered, the soaked filtrate was collected, nanofiltrated and concentrated via a DK nanofiltration membrane until fidaxomicin unit was more than 10000 mg/L, followed by adding purified water to the concentrated solution until the ethanol concentration reached 30%, continuing stirring for 30 min and filtering to give a crude fidaxomicin (liquid chromatogram referring to FIG. 2) with chromatographic purity of 72.6%.

(16) The crude fidaxomicin was purified by DAC200 preparation column packed with C8 filler (supplied by HuaPu) and mobile phase was acetonitrile:water:formic acid=55:45:0.1 (v:v:v), fractions of HPLC purity99.5% were collected and combined, 1.5 times volume of purified water was added under stirring, filtered and dried to give 47.7 g fidaxomicin dry powder with chromatographic purity of 99.67% (liquid chromatogram referring to FIG. 3) and the total extraction yield was 52.5%.

(17) Embodiment 2

(18) 30 L fermentation broth containing fidaxomicin fermenting unit of 2963 mg/L was filtered to obtain 7.1 kg mycelium, and the mycelium was soaked in 21 L methanol and then filtered, the soaked filtrate was collected, nanofiltrated and concentrated via a DK nanofiltration membrane until fidaxomicin unit was more than 10000 mg/L, followed by adding purified water to the concentrated solution until the methanol concentration reached 30%, continuing stirring for 30 min and filtering to give a crude fidaxomicin with chromatographic purity of 70.9%.

(19) The crude fidaxomicin was purified by DAC200 preparation column packed with C8 filler (supplied by HuaPu) and mobile phase was methanol:water:formic acid=65:35:0.1 (v:v:v), fractions of HPLC purity99.5% were collected and combined, 1.5 times volume of purified water was added under stirring, filtered and dried to give 45.6 g fidaxomicin dry powder with chromatographic purity of 99.58% and the total extraction yield was 51.3%.

(20) Embodiment 3

(21) 30 L fermentation broth containing fidaxomicin fermenting unit of 3079 mg/L was filtered to obtain 8.1 kg mycelium, and the mycelium was soaked in 24 L ethanol and then filtered, the soaked filtrate was collected, nanofiltrated and concentrated via a DL nanofiltration membrane until fidaxomicin unit was more than 10000 mg/L, followed by adding purified water to the concentrated solution until the ethanol concentration reached 30%, continuing stirring for 30 min and filtering to give a crude fidaxomicin with chromatographic purity of 72.0%.

(22) The crude fidaxomicin was purified by DAC200 preparation column packed with kromasil C8 filler and mobile phase was acetonitrile:water:formic acid=55:45:0.1 (v:v:v), fractions of HPLC purity99.5% were collected and combined, 1.5 times volume of purified water was added under stirring, filtered and dried to give 48.1 g fidaxomicin dry powder with chromatographic purity of 99.62% and the total extraction yield was 52.1%.

(23) Embodiment 4

(24) 30 L fermentation broth containing a fidaxomicin fermenting unit of 3102 mg/L was filtered to obtain 7.8 kg mycelium, and the mycelium was soaked in 23 L ethanol and then filtered, the soaked filtrate was collected, nanofiltrated and concentrated via a DL nanofiltration membrane until fidaxomicin unit was more than 10000 mg/L, followed by adding purified water to the concentrated solution until the ethanol concentration reached 30%, continuing stirring for 30 min and filtering to give a crude fidaxomicin with chromatographic purity of 73.5%.

(25) The crude fidaxomicin was purified by DAC200 preparation column packed with kromasil C8 filler and mobile phase was methanol:water:formic acid=65:35:0.1 (v:v:v), fractions of HPLC purity99.5% were collected and combined, 1.5 times volume of purified water was added under stirring, filtered and dried to give 49.0 g fidaxomicin dry powder with chromatographic purity of 99.68% and the total extraction yield was 52.7%.

(26) It is to be understood that the foregoing description of the preferred embodiments is intended to be purely illustrative of the principles of the invention, rather than exhaustive thereof, and that changes and variations will be apparent to those skilled in the art, and that the present invention is not intended to be limited other than expressly set forth in the following claims.