Method for conducting affinity chromatography, stepwise elution and purification on chymotrypsin and medicine composition for improving stability of chymotrypsin

A technology of chymotrypsin and segmented elution, which is applied in the directions of drug combination, biochemical equipment and method, peptide/protein composition, etc., can solve problems such as low specificity, and achieve the effect of saving cost and improving economic benefit.

Inactive Publication Date: 2016-03-16
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 For 15 minutes, use 5mol / L NaOH to adjust the pH value to 4.5, place it at 25°C, and filter after 48 hours at a constant temperature 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 value with 5mol / L NaOH to 5.0, placed at 25°C, kept at constant temperature for 24 hours and then filtered to collect 20.5kg of filter cake;

[0048] 1.3) Crystal III

[0049] Put the obtain...

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 For 15 minutes, use 5mol / L NaOH to adjust the pH value to 4.5, 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 value with 5mol / L NaOH to 5.0, placed at 25°C, kept at constant temperature for 24 hours and then filtered to collect 20.2kg of filter cake;

[0080] 1.3) Crystal III

[0081] Put th...

Embodiment 3

[0107] Take 4.4 million units of chymotrypsin prepared in Example 2, weigh 15g of mannitol and 3.6g of disodium edetate, add an appropriate amount of water for injection at 8°C to dissolve, mix, and adjust with 0.05mol / L phosphate buffer Adjust the pH value to 5.8, then add water for injection to 1000ml, filter aseptically with a polyethersulfone ultrafiltration membrane, divide into 1000 treated vials, lyophilize and cap.

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Abstract

The invention discloses a method for conducting affinity chromatography, stepwise elution and purification on chymotrypsin and a medicine composition for improving the stability of chymotrypsin. The method includes the following process steps: 1, multiple crystallization; 2, activation; 3, salting out; 4, ultrafiltration; 5, pyrogen treatment; 6, dialysis; 7, affinity chromatography; 8, freeze-drying and preparation of the chymotrypsin. According to the method for conducting affinity chromatography, stepwise elution and purification on chymotrypsin and the medicine composition for improving the stability of chymotrypsin, the production process of the chymotrypsin is continuously improved, particularly repeated experimental study is conducted on all process parameters, continuous optimization is conducted, finally a scaled production process which is more scientific is established, the titer of the chymotrypsin obtained through affinity chromatography and purification reaches as high as 1500-1800 iu/mg, and all indexes meet the requirements of 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/76A61K38/48A61P17/02A61P29/00
CPCA61K38/4826C12N9/6427C12Y304/21001
Inventor 刘乃山林晓磊刘翠珍
Owner QINGDAO KANGYUAN PHARMA
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