Fidaxomicin purification method

Patent metadata

Field	Value
Publication number	US-10316052-B2
Application number	US-201515324555-A
Country	US
Kind code	B2
Filing date	Jul 7, 2015
Priority date	Jul 9, 2014
Publication date	Jun 11, 2019
Grant date	Jun 11, 2019

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.

First claim

What is claimed is:

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).

Assignees

• Zhejiang Hisun Pharm Co Ltd

Inventors

• Ren Huijun
• Li Daochao
• Ying Xuexiao
• Chen Feng
• Zheng Linghui
• Wang Lingping
• Bai Hua

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• A61P1/12

  Antidiarrhoeals · CPC title

• C07H17/08

  Hetero rings containing eight or more ring members, e.g. erythromycins · CPC title

• C07H1/06 Primary

  Separation; Purification · CPC title

• C12P19/62

  the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin · CPC title