Method for separating and extracting alfapsin

A technology of chymotrypsin and chymotrypsinogen, which is applied in the field of biopharmaceuticals, can solve the problems of reducing investment and production costs, less reusable times, and expensive affinity chromatography media, so as to reduce investment and production costs and benefit the environment Effects of protection and reduction of process load

Active Publication Date: 2008-11-12
BEIJING GEYUANTIANRUN BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for separating and extracting chymotrypsin, which can solve the disadvantages of expensive affinity chromatography medium and few reusable times; it can reduce the burden of chromatography procedures, reduce The amount of medium used not only reduces investment and production costs, but also can obtain high specific activity of chymotrypsin, which is suitable for large-scale production

Method used

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  • Method for separating and extracting alfapsin

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Effect test

Embodiment 1

[0023] ① Prepare 4000L 0.125mol / L sulfuric acid solution in a 6000L reaction tank. Cut 1000kg of fresh bovine pancreas from -18°C to -20°C cold storage into slices with a slicer, then crush it into a slurry with a high-pressure homogenizer, add it to the above-mentioned reaction tank, and add a 5-15°C refrigerant to the jacket of the reaction tank Liquid protection, stirring and extracting for 24 hours, after the extraction is completed, add filter aid and stir well, pump the extract into 40m 2 Plate and frame filter press for liquid-solid separation. After separation, the solid residue is bagged and disposed of according to environmental protection requirements. The resulting supernatant should be clear without turbidity and pumped into the ultrafiltration tank. Use a 40KD ultrafilter to perform ultrafiltration separation on the liquid in the ultrafiltration tank. After the ultrafiltration is over, use a 10KD ultrafilter to perform ultrafiltration on the above ultrafiltrat...

Embodiment 2

[0031] ① Prepare 2500L 0.125mol / L sulfuric acid solution in a 5000L reaction tank. Cut 500 kg of fresh bovine pancreas stored in a cold storage at -18°C to -20°C into slices with a microtome, and then pulverize into a slurry with a high-pressure homogenizer. Put it into the above reaction tank, add 5 ~ 15 ℃ refrigerant liquid protection in the jacket of the reaction tank, stir and extract for 18 hours, after the extraction is completed, add filter aid and stir well, pump the extract into 30m 2 Plate and frame filter press for liquid-solid separation. After separation, the solid residue is bagged and disposed of according to environmental protection requirements. The resulting supernatant should be clear without turbidity and pumped into the ultrafiltration tank. Use a 40KD ultrafilter to perform ultrafiltration separation on the liquid in the ultrafiltration tank. After the ultrafiltration is over, use a 10KD ultrafilter to perform ultrafiltration on the above ultrafiltrate...

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Abstract

The invention discloses a method for separating and extracting chymotrypsin, comprising the following steps of: pulverizing and filtering beef pancreas gland and extracting a filtrate; removing hetero-protein from the filtrate, and refining and concentrating the filtrate; adding ammonium sulphate and regulating pH value with sodium hydroxide for culture to separate chymotrypsinogen by crystallization; carrying out extraction and filtration to the culture to extract chymotrypsinogen; recycling the extracting and the filtering liquid of the culture for later use; adding trypsinase to activate zymohydrolysis; and obtaining a first semi-finished product solution after filtering; filling chromatographic column with CM-sopharose FF gel medium; balancing the chromatographic column with acetic acid buffer solution; then loading the extracting and filtering liquid of the culture after balancing; washing the column with balancing liquid until effluent liquid OD(280) value is less than 0.1; eluting the column with monosodium orthophosphate -disodium hydrogen phosphate buffer liquid; and collecting the eluting peak to obtain a solution which is a second semi-finished product; mixing the first semi-finished product with the second semi-finished product; subjecting the mixture to desalting and concentration, aseptic filtration and freeze vacuum drying; and obtaining the finished product of chymotrypsin. The method solves the problems of expensive affinity chromatography medium and less times of repeated use, and is applicable for large scale production.

Description

technical field [0001] The invention relates to a separation and extraction method of chymotrypsin, belongs to the field of biopharmaceuticals, and relates to a biochemical separation technology of medicinal protease. Background technique [0002] Chymotrypsin is a protease isolated and purified from bovine pancreas or porcine pancreas. Its pharmacological and clinical functions mainly include two aspects: 1. Anti-inflammation: it is effective for various inflammations, inflammatory edema, hematoma, ulcers and thrombus. Better curative effect. 2. Anti-cancer: It can promote the penetration of anti-cancer drugs into the lesion, and promote the effect of chemotherapy drugs. It is reported that the anti-cancer synergistic effect is more than 50%, and the effect is stronger when it is used locally in large doses. Large-dose use can treat a variety of tumors, such as breast cancer, cervical cancer, gastric cancer, etc., without any toxic side effects or adverse reactions. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
Inventor 吕向广
Owner BEIJING GEYUANTIANRUN BIOTECH
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