Method for removing endotoxin in trypsinase

A technology of trypsin and filter cake, which is used in the field of removing endotoxin in trypsin, can solve the problems of long time consumption and inactivation of trypsin, and achieve the effect of improving production efficiency and reducing operation volume.

Inactive Publication Date: 2014-09-03
SPH NO 1 BIOCHEM & PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] The technical problem to be solved by the present invention is to provide a method for removing endotoxin in trypsin in order to overcome the defects in the prior art that the endotoxin in trypsin cannot be completely removed, easily causes trypsin inactivation, and takes a long time. Methods

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  • Method for removing endotoxin in trypsinase
  • Method for removing endotoxin in trypsinase
  • Method for removing endotoxin in trypsinase

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Embodiment 1

[0039]The batch number of trypsin raw material is 120901, which is produced in the processing department of beef and sheep organs in Jining, Inner Mongolia. The trypsin raw material is fresh bovine pancreas soaked in sulfuric acid, minced, salted out and crystallized, and its mass percentage purity should not be lower than 10%. Feed 4000g of protease raw material, stir and dissolve with 5 times the volume (V / W) of purified water first, add 5000mL of 2.5mol / L sulfuric acid solution to adjust the pH to 3.0 to help it dissolve. Measure the volume of the solution, add solid ammonium sulfate into it according to the ratio of 415g / L to make it reach 0.7 saturation, after it dissolves, let it stand for precipitation for 8 hours. Filter the solution with a Buchner funnel and three layers of medium-speed filter paper, weigh the filter cake weight 9115g, add 27345ml of purified water according to three times the weight of the filter cake (V / W), stir and dissolve, add 2.5mol / L sulfuric ac...

Embodiment 2

[0046] Trypsin raw material batch number 120902, produced in Inner Mongolia Jining cattle and sheep organ processing department, trypsin raw material is fresh bovine pancreas soaked in sulfuric acid, minced, salted out and crystallized, its mass percentage purity should not be lower than 10%; Feed 4500g of protease raw material, first stir and dissolve with 5 times the volume (V / W) of purified water, add 2.5mol / L sulfuric acid solution to adjust the pH to 3.0 to help it dissolve. Measure the volume of the solution, add solid ammonium sulfate into it according to the ratio of 415g / L to make it reach 0.7 saturation, after it dissolves, let it stand for precipitation for 8 hours. Filter the solution with a Buchner funnel and three layers of medium-speed filter paper, weigh the filter cake weight 9500g, add 28500ml of purified water according to 3 times the filter cake weight (V / W), stir and dissolve, add 2.5mol / L sulfuric acid solution Adjust the pH to 3.0, add 19000ml of liquid ...

Embodiment 3

[0053] Trypsin raw material batch number 120903, produced in Inner Mongolia Jining cattle and sheep organ processing department, trypsin raw material is fresh bovine pancreas soaked in sulfuric acid, minced, salted out and crystallized, its mass percentage purity should not be lower than 10%; Feed 4500g of protease raw material, first stir and dissolve with 5 times the volume (V / W) of purified water, add 2.5mol / L sulfuric acid solution to adjust the pH to 3.0 to help it dissolve. Measure the volume of the solution, add solid ammonium sulfate into it according to the ratio of 415g / L to make it reach 0.7 saturation, after it dissolves, let it stand for precipitation for 8 hours. Filter the solution with a Buchner funnel and three layers of medium-speed filter paper, weigh the filter cake to 10,000 g, add 30,000 ml of purified water according to three times the weight of the filter cake (V / W), stir and dissolve, then add 2.5 mol / L sulfuric acid solution Adjust the pH to 3.0, add ...

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Abstract

The invention discloses a method for removing endotoxin in trypsinase. The method comprises the following steps: dissolving a trypsinase filter cake in water, carrying out ultrafiltration concentration to obtain a concentrated solution, sequentially adding a phosphate water solution and a calcium salt water solution, regulating the pH value to 8-9, and centrifugating. On the premise of not influencing the activity and yield of the trypsinase, the method for removing endotoxin in the trypsinase production process effectively removes the endotoxin pollutant contained in the trypsinase production process, and has been applied to trypsinase raw drug production at present; and the endotoxin content conforms to the related requirement.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for removing endotoxin in trypsin. Background technique [0002] Trypsin is an important proteolytic enzyme secreted by the pancreas of animals. It can hydrolyze proteins and polypeptides, and can selectively hydrolyze the peptide chains of arginine and lysine. It is an endopeptidase. Diseases such as inflammation, ulcer, pus, hematoma, empyema, skin and digestive tissue cells have indirect and direct therapeutic effects. As a kind of intramuscular injection drug, in order to ensure the safety of its clinical application, its endotoxin content should be controlled. Endotoxin is produced in the outer cell wall of Gram-negative bacteria, and its main components are lipopolysaccharide (LPS) and lipid A, which are the active parts of pyrogens. Their relative molecular mass is generally 10,000 to 25,000, and they form associations in aqueous solution, and their relative mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/76
CPCC12N9/6427C12Y304/21004
Inventor 黄臻辉周建华周伟洁琚姝王克平汪雅雯孙源远
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
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