Epothilone B purification method

A technology of epothilone and purification method, which is applied in the field of biological separation, and can solve the problems of filtration and cleaning, time-consuming and labor-intensive problems

Active Publication Date: 2017-06-30
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Time-consuming and labor-intensive, and a large amount of organic solvents are used, which brings certain safety risks to large-scale production
In the extraction and purification process of other products, the method of low-temperature freezing filtration has been used to remove impurities. The filtration process needs to be carried out at a low temperature below -20°C, and the suspended impurities in the filtration process block the filter material, which is difficult for filtration and later cleaning. bring big problems

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0028] The preparation of fermented liquid used in the embodiment:

[0029] The epothilone B producing bacterium is Somatocystis cellulosus ATCC25532. The cultivation method is:

[0030] 1) Solid plate culture: the solid medium consists of 0.1% glucose, 0.2% peptone, 0.1% magnesium sulfate, 0.2% calcium chloride, 0.2% dipotassium hydrogen phosphate, and 0.3% yeast extract. Adjust the pH to 7.2, sterilize at 121°C for 30 minutes, spread the bacteria on the solid plate medium, and incubate it upside down at 30°C for 6-7 days.

[0031] 2) Shake flask expansion: plate inoculation in the shake flask, the components of the shake flask medium are glucose 0.1%, soluble starch 2%, magnesium sulfate 0.1%, calcium chloride 0.2%, dipotassium hydrogen phosphate 0.2%, yeast Extract 0.3%. Adjust the pH to 7.2, sterilize at 121°C for 30 minutes, and culture at 30°C at 200rpm for 4-5 days.

[0032] 3) Seed culture: inoculate the shake flask into the seed tank, the culture medium is the sam...

Embodiment 1

[0035] 1. Preparation of concentrate

[0036]Collect 32.30 kg of macroporous adsorption resin in 3 tons of fermentation broth, wash twice with water and once with 15% ethanol, add ethanol for 2 hours and filter to collect the first extraction, add ethanol again for 2 hours and filter to collect the second extraction , the combined extracts were concentrated under reduced pressure at 40°C to an alcohol-free liquid, added ethyl acetate and then added an appropriate amount of purified water for extraction, left to separate layers for 2h, collected the ethyl acetate layer and concentrated under reduced pressure at 40°C to 2.90 L of concentrate containing 65.30 g of epothilone B.

[0037] 2. Freezing to remove impurities

[0038] Take 2.90 L of the above concentrate, fully dissolve it in 21.80 L of acetonitrile, let it stand at -15°C for 15 hours, slowly add 5.50 L of acetonitrile, and let it stand for 30 minutes to collect the supernatant. The yield of epothilone B is 96.68%.

...

Embodiment 2

[0047] 1. Preparation of concentrate

[0048] Collect 32.12 kg of macroporous adsorption resin in 3 tons of fermentation broth, wash twice with water and once with 15% ethanol, add ethanol for 2 hours and filter to collect the first extraction, add ethanol again for 2 hours and filter to collect the second extraction , the combined extracts were concentrated under reduced pressure at 40°C to an alcohol-free liquid, added ethyl acetate and then added an appropriate amount of purified water for extraction, left to separate layers for 2h, collected the ethyl acetate layer and concentrated under reduced pressure at 40°C to 2.79 L of concentrate, containing 63.52 g of epothilone B.

[0049] 2. Freezing to remove impurities

[0050] Take 2.79 L of the above concentrate, fully dissolve it in 31.76 L of acetonitrile, let it stand at -20°C for 24 hours, slowly add 10.59 L of acetonitrile, let it stand for 30 minutes to collect the supernatant, and the yield of epothilone B is 98.62%. ...

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PUM

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Abstract

The invention relates to an improved method for epothilone B extraction and purification, and belongs to the technical field of biological separation. According to the method, most of impurities are removed by a low-temperature refrigeration method, solid-liquid separation is achieved by changing density, the purity of finished products is further improved by a two-time crystal method, rate-limiting steps such as silica-gel column chromatography are decreased, an epothilone B extraction process is simplified, operation cost is reduced, and the method has a wider application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological separation, and in particular relates to a method for extracting and purifying epothilone B. Background technique [0002] The discovery of paclitaxel has pushed the research of anticancer drugs into a new field, but with the in-depth research and clinical application, paclitaxel has exposed many shortcomings and drawbacks, including the difficulty of synthesis, the lack of extraction resources and the lack of yew trees in clinical applications. side effect. Later, in 1985, the GBF Institute in Germany isolated a myxobacteria, Somatocystis cellulosus Soce90 strain, from the soil of the Zambezi River beach in South Africa in 1985, and measured its culture to have antifungal activity and cytotoxicity. Around 1995, Daniel M.Bollag of the West Point Merck Research Laboratory in Pennsylvania, USA, etc., in order to seek compounds with tubulin (Tubulin) polymerization activity, obtained from the J.E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/04
CPCC07D493/04
Inventor 张贵民沈书庆杨家森
Owner LUNAN PHARMA GROUP CORPORATION
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