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Method for preparing fidaxomicin by flash chromatography

A fidaxomicin and chromatographic technology, which is applied in the field of rapid chromatography to prepare fidaxomicin, can solve the problems of high requirements for equipment and filler specifications, high cost investment, etc., and achieve the effects of fast and convenient acquisition, low cost and simple operation

Inactive Publication Date: 2017-05-10
FUJIAN INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is simple to operate, and the purity and yield of fidaxomicin are high. However, due to the adoption of dynamic axial compression column (DAC) technology, the requirements for equipment and packing specifications are relatively high, and the cost investment is relatively large.

Method used

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  • Method for preparing fidaxomicin by flash chromatography
  • Method for preparing fidaxomicin by flash chromatography
  • Method for preparing fidaxomicin by flash chromatography

Examples

Experimental program
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Effect test

preparation example Construction

[0047] a. Preparation and cultivation of slant colony: connect the actinomycetes thalli to the slant medium, and cultivate it at 30°C for 10 days; the slant medium is ISP medium, and its formula is: yeast extract 4g / L, malt extract 10g / L, glucose 4g / L, agar 20g / L, pH7.2;

[0048] B, preparation and cultivation of seed culture medium: adopt platinum ring to move the single bacterium colony gained on the slant culture medium into the seed culture medium, and under 30 ℃ of culture temperature, 240rpm shaker vibration culture 48h, obtain fidaxomicin fermentation primary seed liquid;

[0049] Seed medium formula: soluble starch 20g / L, glucose 5g / L, peptone 5g / L, yeast powder 5g / L, soybean powder 5g / L, ammonium sulfate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, seven Magnesium sulfate hydrate 0.5g / L, calcium carbonate 2g / L, pH 7.2, the volume of the shake bottle is 50mL / 500mL.

[0050] C, the preparation and cultivation of fermentation medium: move the primary seed liquid obt...

Embodiment 1

[0054] Get 10L of Fidaxomycin fermented liquid, centrifuge the fermented liquid with 4500rpm for 10 minutes, obtain 2kg mycelia (wet weight); Centrifuge after extracting for 4 hours, collect the extract containing Fidaxomycin (4.5L, containing Fidaxomycin 1179mg), and the HPLC chromatogram of the extract is as follows: figure 1 As shown; the extract was diluted with water to an ethanol concentration of 20%, and then introduced into the nano-micro NM-200 polystyrene resin column for adsorption. The resin loading capacity was 300mL, and the flow rate was 2BV / h. %, 50%, 60% ethanol solution for gradient desorption, the flow rate is 2BV / h, and the desorption solution with a fidaxomicin content of more than 45% is collected in sections. The HPLC chromatogram of the desorption solution is as follows figure 2 shown.

[0055] Combine the collected desorption solution with a fidaxomicin content of more than 45%, add water to dilute to an ethanol concentration of 50%, and then introdu...

Embodiment 2

[0059] Get 20L of fidaxomicin fermentation broth, centrifuge the fermentation broth at 4500rpm for 10 minutes to obtain 3.9kg mycelium (wet weight); add 8L of 60% ethanol to the mycelium, and stir with an electric stirrer (350rpm) at room temperature Centrifuge after leaching for 4 hours, collect the extract containing Fidaxomycin (9L, containing Fidaxomycin 2358mg), and the HPLC chromatogram of the extract is as follows: figure 1 As shown; the extract was diluted with water to an ethanol concentration of 40%, and then introduced into the nano-micro NM-200 polystyrene resin column for adsorption. The resin loading capacity was 500mL, and the flow rate was 2BV / h. %, 50%, 60% ethanol solution for gradient desorption, the flow rate is 2BV / h, and the desorption solution with a fidaxomicin content of more than 45% is collected in sections. The HPLC chromatogram of the desorption solution is as follows Figure 5 shown.

[0060] Combine the collected desorption solution with a fidax...

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Abstract

The invention discloses a method for preparing fidaxomicin by flash chromatography. The method comprises the following steps: firstly, extracting fidaxomicin fermentation mycelia with 60 to 80 percent ethanol, roughly separating extract liquid through polystyrene resin, and collecting a fraction which contains fidaxomicin; then, performing reverse-phase bonding silica gel rapid column chromatography separation twice on the fraction, wherein elution solvents of different selectivity are adopted; finally obtaining a non-fidaxomicin refined product of which the purity is over 98.5 percent. By adopting the method, the purity and yield of the obtained fidaxomicin are high; moreover, the method is relatively easy to operate, is relatively low in cost, and is suitable for industrial production.

Description

[0001] 【Technical field】 [0002] The invention belongs to the field of microbial pharmacy, and in particular relates to a method for preparing fidaxomicin by flash chromatography. [0003] 【Background technique】 [0004] Clostridium difficile is a Gram-positive anaerobic bacillus widely distributed in the natural environment and in animal and human feces. When it overgrows and releases toxins, it can cause Clostridium difficile infection (CDI), leading to colon inflammation, severe diarrhea and even death. [0005] Fidaxomicin (fidaxomicin) is a new narrow-spectrum macrolide antibiotic developed by Optimer Pharmaceuticals. It was approved by the FDA in May 2011 and launched in the United States, becoming the first drug mainly used in the treatment of Clostridium difficile in 30 years. Antibiotics for associated diarrhea (CDAD). Fidaxomycin kills bacteria by inhibiting the RNA polymerase of Clostridium difficile and blocking the synthesis of ribonucleic acid. Compared with t...

Claims

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Application Information

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IPC IPC(8): C07H17/08C07H1/06
CPCC07H17/08C07H1/06
Inventor 江宏磊连云阳陈名洪严凌斌王传喜彭飞
Owner FUJIAN INST OF MICROBIOLOGY
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