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Pretreatment method of epothilone B fermentation liquor

A technology of epothilone and fermented liquid, applied in the direction of organic chemistry, etc., can solve the problems affecting the quality of epothilone B final product, cumbersome plate and frame filtration or centrifugation operation, easy to block resin column sieve plate, etc., and achieve improvement Extraction efficiency and yield, saving column time, saving organic solvent effect

Active Publication Date: 2020-04-07
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the process of collecting resin after fermentation, it is generally necessary to filter the fermentation broth through a plate frame or centrifuge for solid-liquid separation, but the plate frame filtration or centrifugation is cumbersome. The inventor tried to directly place the fermentation broth for a period of After a period of time, discard the fermentation supernatant, and pack the remaining bacteria and resin into the column for the next step of separation and purification; however, this method is easy to block the sieve plate of the resin column, making it difficult to carry out the next step of purification
The solid collected by plate-and-frame filtration or centrifugation contains bacteria and resin adsorbed with epothilone B. The bacteria does not contain epothilone B but contains impurities similar to its structure, and the retention time of the impurities is within angstroms Between bleomycin A and epothilone B, it is difficult to remove in the subsequent purification process, so as to affect the quality of the final product of epothilone B; in addition, what is collected by this method is resin and bacteria, bacteria The volume is large, and the amount of organic solvent used in the subsequent extraction is large

Method used

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  • Pretreatment method of epothilone B fermentation liquor
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  • Pretreatment method of epothilone B fermentation liquor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1.2m 3 Fermentation broth (epothilone B content 86.5mg / L) was left to stand for 30 minutes, and after the XAD-16N resin and bacteria settled, the fermentation broth was released from the upper layer for 1m 3 , the remaining 200L, add 40g of sodium chloride solid, stir slowly for 10min, after the sodium chloride solid is completely dissolved, take a sample to detect the remaining fermentation broth density 1.150g / cm 3 , stand still for 10-20 minutes, the bacteria are deposited in the lower layer, and the XAD-16N resin floats in the upper layer, and the fermentation broth with the bacteria is released from the lower layer, and 12Kg of the resin is collected and loaded into a stainless steel chromatography column.

[0044] Wash the stainless steel column with 50L purified water, then wash the stainless steel column with 75L volume fraction of 15% ethanol aqueous solution, and finally wash with absolute ethanol until the effluent does not contain epothilone B, and the washi...

Embodiment 2

[0046] 1.2m 3 Fermentation broth (epothilone B content 85.7mg / L) was left to stand for 30 minutes, and after the XAD-16N resin and bacteria settled, the fermentation broth was released from the upper layer for 1m 3 , the remaining 200L, add 50g of zinc sulfate heptahydrate solid, stir slowly for 10min, after the zinc sulfate heptahydrate solid is completely dissolved, take a sample to detect the remaining fermentation broth density 1.159g / cm 3 , stand still for 10-20 minutes, the bacteria are deposited in the lower layer, and the XAD-16N resin floats in the upper layer, and the fermentation broth with the bacteria is released from the lower layer, and 12Kg of the resin is collected and loaded into a polypropylene chromatography column.

[0047] Rinse the polypropylene chromatography column with 50L of purified water, then rinse the polypropylene chromatography column with 100L volume fraction of 10% ethanol aqueous solution, and finally rinse with 90% volume fraction of ethano...

Embodiment 3

[0049] 1.2m 3 Fermentation broth (epothilone B content 44.6mg / L) was left to stand for 30min, after the X-5 resin and bacteria settled, the fermentation broth was released from the upper layer for 1m 3 , the remaining 200L, add 60g of magnesium sulfate heptahydrate solid, stir slowly for 10min, after the magnesium sulfate heptahydrate solid is completely dissolved, take a sample to detect the remaining fermentation broth density 1.145g / cm 3 , stand still for 10-20 minutes, the bacteria are deposited in the lower layer, and the X-5 resin floats in the upper layer, and the fermentation broth with the bacteria is released from the lower layer, and 12Kg of the resin is collected and loaded into a polypropylene chromatography column.

[0050] Rinse the polypropylene chromatography column with 50L of purified water, then rinse the polypropylene chromatography column with 70L volume fraction of 20% methanol aqueous solution, and finally rinse with anhydrous methanol until the effluen...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses a pretreatment method of epothilone B fermentation liquor. An inorganic salt is added into the fermentation liquor,so that resin and thalli are thoroughly separated, only the resin phase remains after the thalli are removed, the subsequent extract is reduced to 1 / 3 of the original mixture, an organic solvent for further extraction is saved, and impurities in the thalli are prevented from being introduced into the subsequent purifying process; and impurities with the retention time similar to that of epothiloneB are removed from the source. In addition, the separated resin phase does not need to be centrifuged or filtered by a plate frame and is directly loaded into a column without blocking a sieve plate,so that the column passing time is saved. According to the pretreatment method of the epothilone B fermentation liquor, the thalli and the resin are well separated, subsequent extraction steps are simplified, the usage amount of subsequent extraction organic solvents is reduced, the extraction efficiency and yield are improved, the universality is high, and the industrialization cost is low.

Description

technical field [0001] The invention belongs to the technical field of bioseparation, and in particular relates to a pretreatment method of a macrolide antitumor drug epothilone B fermented liquid. Background technique [0002] Epothilones (epotuilones) were discovered in the secondary metabolites of the myxobacteria Sorangium cellulosum in 1993. Its structure is based on the sixteen-membered lactone macrocycle as the center body and the thiazole ring ligand as the side chain. A class of cytotoxic compounds, an antineoplastic drug with tubulin polymerization and microtubule stabilizing effects similar to paclitaxel. Paclitaxel has achieved great success at the end of the 20th century, but its source is extremely scarce, its own toxic side effects and multidrug-resistant cell lines and other factors also limit its use. Compared with paclitaxel, epothilone has no restriction of plant source, no disadvantages such as complex structure and water insolubility of paclitaxel, and ...

Claims

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Application Information

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IPC IPC(8): C07D493/04
CPCC07D493/04
Inventor 杨家森王钦超朱兵峰
Owner LUNAN PHARMA GROUP CORPORATION
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