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Method for purifying chymotrypsin through affinity chromatography stepwise elution and pharmaceutical composition containing chymotrypsin

A chymotrypsin and segmented elution technology is applied in the directions of drug combinations, biochemical equipment and methods, and medical preparations containing active ingredients, which can solve problems such as low specificity and achieve cost savings and economic benefits Effect

Inactive Publication Date: 2015-04-22
QINGDAO KANGYUAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Chymotrypsin, also known as chymotrypsin, has a molecular weight of 42,000 Daltons. It is used for the enzymatic hydrolysis of peptide chains. It specifically hydrolyzes peptide bonds. It belongs to endopeptidases and has a high hydrolysis yield, but its specificity is lower than that of trypsin.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1) Multiple crystallization

[0044] 1.1) Crystal I

[0045] Put 30kg of chymotrypsinogen into the reaction tank, add 210L of purified water and stir to dissolve, add 500mL of 2.5mol / L sulfuric acid and stir for 8 hours, then adjust the pH value to 2.5 with 2.5mol / L sulfuric acid; add 60L of saturated ammonium sulfate solution, stir After 15 minutes, adjust the pH value to 4.5 with 5mol / L NaOH, place it at 25°C, keep the temperature constant for 48 hours, and then filter to collect 23.6kg of filter cake;

[0046] 1.2) Crystal II

[0047] Put the obtained filter cake into the reaction tank, add 70.8L purified water and stir to dissolve the filter cake, adjust the pH value to 3.0 with 2.5mol / L sulfuric acid; add 23.6L saturated ammonium sulfate solution, stir for 15 minutes, adjust the pH with 5mol / L NaOH value to 5.0, put it under the condition of 25 ℃, filter after constant temperature for 24 hours, and collect 20.5kg of filter cake;

[0048] 1.3) Crystal III

[004...

Embodiment 2

[0075] 1) Multiple crystallization

[0076] 1.1) Crystal I

[0077] Put 28kg of chymotrypsinogen into the reaction tank, add 196L of purified water and stir to dissolve, add 500mL of 2.5mol / L sulfuric acid and stir for 8 hours, then adjust the pH value to 2.5 with 2.5mol / L sulfuric acid; add 56L of saturated ammonium sulfate solution, stir After 15 minutes, adjust the pH value to 4.5 with 5mol / L NaOH, place it at 25°C, keep the temperature constant for 48 hours, and then filter to collect 23.2kg of filter cake;

[0078] 1.2) Crystal II

[0079] Put the obtained filter cake into the reaction tank, add 69.6L purified water and stir to dissolve the filter cake, adjust the pH value to 3.0 with 2.5mol / L sulfuric acid; add 23.2L saturated ammonium sulfate solution, stir for 15 minutes, adjust the pH with 5mol / L NaOH value to 5.0, put it under the condition of 25 ℃, filter after constant temperature for 24 hours, and collect 20.2kg of filter cake;

[0080] 1.3) Crystal III

[008...

Embodiment 3

[0107] Take 4.4 million units of chymotrypsin prepared in Example 2, weigh 15g of mannitol, add an appropriate amount of water for injection at 8°C to dissolve, mix, adjust the pH value to 5.8 with 0.05mol / L phosphate buffer, and then add Water for injection to 1000ml, sterile filtered with polyethersulfone ultrafiltration membrane, divided into 1000 treated vials, freeze-dried and capped.

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Abstract

The invention discloses a method for purifying chymotrypsin through affinity chromatography stepwise elution and a pharmaceutical composition containing chymotrypsin. The method comprises the following process steps: (1) performing multiple-crystallization; (2) activating; (3) salting out; (4) performing ultrafiltration; (5) performing primitive heat treatment; (6) dialyzing; (7) performing affinity chromatography; and (8) draining, and preparing the chymotrypsin. The chymotrypsin production process is continuously improved, particularly various process parameters are subjected to repeated experiment research and are continuously optimized, and finally, a set of scientific large-scale production process is determined. The titer of the chymotrypsin obtained by affinity chromatography purification is 1500-1800iu / mg, and each index accords with the provision in the Chinese pharmacopoeia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying chymotrypsin by using multiple crystallization, activation, salting out, dialysis, affinity chromatography and the like. Background technique [0002] Chymotrypsin, also known as chymotrypsin, has a molecular weight of 42,000 Daltons. It is used for the enzymatic hydrolysis of peptide chains. It specifically hydrolyzes peptide bonds. It belongs to endopeptidases and has a high hydrolysis yield, but its specificity is lower than that of trypsin. . Its solid state is relatively stable, showing white rod-like crystals; while its aqueous solution is extremely unstable, when the pH is 5-8, its activity is the strongest, and it is also most likely to lose its activity. [0003] Chymotrypsin is a proteolytic enzyme secreted by the pancreas, which can quickly decompose denatured proteins. Its function and use are similar to trypsin, but it has stronger decomposition a...

Claims

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Application Information

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IPC IPC(8): C12N9/76A61K38/48A61P17/02A61P29/00
CPCC12N9/6427A61K38/00C12Y304/21001
Inventor 刘冠男林晓磊刘翠珍
Owner QINGDAO KANGYUAN PHARMA
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