Preparation method of high-purity fidaxomicin

A fidaxomicin, high-purity technology, applied in the preparation of sugar derivatives, chemical instruments and methods, sugar derivatives, etc., can solve the problems of large damage to the downstream purification medium, less refined powder, and more impurities. The effect of improving quality and product yield, eliminating interference and simple process

Active Publication Date: 2013-09-04
NCPC NEW DRUG RES & DEV
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Problems solved by technology

This method has the following disadvantages: (1) After the fermentation broth is filtered, the resin and mycelium coexist, and the screening is difficult, the resin is difficult to reuse, and the environmental protection treatment is difficult; (2) The eluate is directly obtained without decolorization and impurity removal. , contains more impurities, and causes great damage to the downstream purification medium; (3) reversed-phase silica column chromatography uses MeOH / H 2 O / AcOH three-phase elution system, in which acetic acid will not only destroy the bonded phase of the column itself and reduce the utilization rate of the reversed-phase column, but also has a higher boiling point of 118°C. When recovering solvent, water and methanol can be recovered completely, but acetic acid can only recover a part, so that the proportion of the recovered mixed solvent changes and cannot be reused, which increases the production cost; Reaction occurs or remains in the crystallization solution, resulting in a decrease in the purity of fidaxomicin powder
The disadvantage of this method is that multiple chromatographic columns are repeatedly used, the preparation method is too complicated and cumbersome, and the yield will be low after repeated chromatographic column separations. In addition, thin-layer preparative chromatography is used for enrichment, and the batch capacity is too small. Not suitable for large-scale production
WO2011146621A2 discloses a method for preparing high-purity Fidaxomycin. In this method, the preparation liquid is used to prepare Fidaxomicin crude product, and Fidaxomicin fine powder with a purity greater than 93% can be obtained. Although the steps of this method are simple, but The required equipment is relatively expensive, and the amount of fine powder prepared is small, so it is not suitable for industrial production

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Experimental program
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Embodiment 1

[0029] Preparation of Fidaxomycin fermentation broth:

[0030] The seed medium was inoculated with Actinomycetes mobilis strain N12W0304, and cultured at 32 °C and 220 rpm for 48 h to obtain seed liquid. The seed solution was inoculated with an inoculum volume of 10% by volume in a 50 L fermenter equipped with fermentation medium, the tank temperature was 28 °C, the tank pressure was 0.05±0.01 Mpa, the ventilation rate was 20 L / min, stirred at 400 rpm, and fermented for 120 h to obtain Fidaxomycin fermentation broth.

[0031] Wherein the preparation method of the seed medium is: 40.0 grams of cornstarch, 4.0 grams of glucose, 8.0 grams of peptone, 5.0 grams of beef extract, 5.0 grams of yeast extract powder, add tap water to dissolve, dilute to 1000 ml, pH value 7.0-7.2 , sterilized at 121°C for 30 min.

[0032] The preparation method of the fermentation medium is: 30.0 grams of glucose, 15.0 grams of defatted soybean powder, 3.0 grams of beef extract, K 2 HPO 4 0.6 g, Fe...

Embodiment 2

[0036] Fidaxomycin fermentation broth 10L, fermentation unit 620μg / mL. The fidaxomicin fermentation broth was filtered to obtain 2.6kg mycelium. Add 10 L of ethanol with a volume ratio concentration of 95% to the above-mentioned mycelia, stir and extract for 1 hour, then filter, and collect the fidaxomicin extract. The extract was diluted with water to an ethanol concentration of 35%, and then introduced into a macroporous decolorization resin LX98 column for decolorization. The resin loading capacity was 500mL, and the flow rate was 1000mL / h. The decolorization solution is introduced into the macroporous adsorption resin D312 column again for adsorption, the resin loading capacity is 500mL, and the flow rate is 1000mL / h. The saturated resin was eluted successively with 40% and 80% ethanol solutions at a flow rate of 500mL / h, and the eluent was collected in sections. The fidaxomicin analysis solution was concentrated, then extracted with butyl acetate, and the extract was co...

Embodiment 3

[0039] 30L of fidaxomicin fermentation broth, the fermentation unit is 570μg / mL. The fidaxomicin fermentation liquid was filtered to obtain 6.1 kg of mycelia. Add 18L of 90% ethanol to the above-mentioned mycelia, stir and extract for 1 hour, then filter, and collect the fidaxomicin extract. The extract was diluted with water to an ethanol concentration of 35%, and then introduced into a macroporous decolorizing resin D301 column for decolorization. The resin loading capacity was 1500mL, and the flow rate was 3000mL / h. The decolorization solution is introduced into the macroporous adsorption resin HZ816 column again for adsorption, the resin loading capacity is 1500mL, and the flow rate is 3000mL / h. The saturated resin was eluted successively with 40% and 80% ethanol solutions at a flow rate of 1500mL / h, and the eluent was collected in sections. The above fidaxomicin eluate was concentrated, then extracted with ethyl acetate, the extract was concentrated and dried to obtain ...

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Abstract

The invention discloses a method for separating and purifying fidaxomicin from fidaxomicin fermented mycelia. The method comprises the following steps of first filtering and extracting the fidaxomicin fermentation liquid, diluting the extracted liquor with water, then decoloring, adsorbing and analyzing the extracted liquor through macroporous resin, and concentrating, extracting and drying the analyzed liquor to obtain a fidaxomicin crude product; and dissolving the fidaxomicin crude product in a polarity organic solvent, injecting polymer microsphere columns to carry out the chromatographic separation to obtain the high-purity fidaxomicin fine powder. By adopting the method, the interference of fermented secondary metabolite can be effectively eliminated, and the quality and the yield of the fidaxomicin can be improved. The entire process is simple and easy to operate; the prepared fidaxomicin can be used for drugs; and the purity of the prepared fidaxomicin is more than 95 percent, and the whole-coarse total yield is more than 45 percent. The used solvent is a conventional solvent, small in toxicity and applicable to the industrial production.

Description

technical field [0001] The invention relates to a method for separating and purifying antibiotics, in particular to a method for separating and purifying fidaxomicin from fermented mycelium of fidaxomicin. Background technique [0002] Fidaxomicin is a new type of macrolide antibiotic with an 18-membered ring structure produced by the fermentation of cystic bacteria, with the molecular formula C 52 h 74 Cl 2 o 18 , the structural formula is as follows: [0003] [0004] Fidaxomicin structural formula [0005] Fidaxomycin is a narrow-spectrum antibacterial drug, mainly against Gram-positive aerobic and anaerobic bacteria, Clostridium difficile, and has good effect on methicillin-resistant Staphylococcus aureus, Clostridium perfringens and Enterococcus. Antibacterial effect, but very weak antibacterial activity against Gram-negative bacteria. MIC of Fidaxomycin against Clostridium difficile 50 and MIC 90 Both are significantly lower than vancomycin or metronidazole,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H17/08C07H1/08
Inventor 张雪霞任风芝段宝玲李晓露李宁张静岩
Owner NCPC NEW DRUG RES & DEV
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