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Method for preparing pure ulinastatin from ulinastatin affinity chromatography medium

A chromatographic medium, ulinastatin technology, applied in peptide preparation methods, chemical instruments and methods, protease inhibitors, etc., can solve the problems of long purification time, low protein and activity recovery rate, etc., and achieve short consumption time. , suitable for large-scale promotion and application

Active Publication Date: 2014-06-25
NANCHANG WANHUA BIOCHEM PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Too many purification steps lead to longer purification time and lower recovery of protein and activity

Method used

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  • Method for preparing pure ulinastatin from ulinastatin affinity chromatography medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1 Ulinastatin affinity chromatography medium synthesis:

[0028] (1) Take 2000 g of sephadex G75, wash with 10 times the volume of purified water, filter, and then drain into a wet cake; add 2000 ml of activation buffer (0.5 M NaOH, 10% dimethyl sulfoxide, 6% epoxy Chloropropane), stirred at 40 °C for 2.5 h, then poured into a funnel, washed with 10 times the volume of purified water, filtered, and then sucked dry to form a wet cake.

[0029] (2) Add 2000 ml of an aqueous solution containing 5% 1,3-diamino-2-hydroxypropane to the activated Sephadex G75 medium, stir at 60 °C for 20 h, wash with 10 times the volume of purified water, and drain to form a wet cake.

[0030] (3) Add 2000 ml of 0.1M / L tripolychlorazine in water-acetone solution, adjust the pH of the system to 7.5 with 0.1M sodium hydroxide, stir in an ice bath at 4°C for 1 hour, wash with 10 times the volume of purified water, and drain to obtain Wet cake.

[0031] (4) Add 2000 ml of 0.15mol polylysine ...

Embodiment 2

[0038] 1 Ulinastatin affinity chromatography medium synthesis:

[0039] (1) Take 3000 g of sephadex G75, wash with 12 times the volume of purified water, filter, and then drain into a wet cake; add 3000 ml of activation buffer (0.5 M NaOH, 10% dimethyl sulfoxide, 6% epoxy Chloropropane), stirred at 45 °C for 3.5 h, then poured into a funnel, washed with 12 times the volume of purified water, filtered, and then sucked dry to form a wet cake.

[0040] (2) Add 2000 ml of an aqueous solution containing 10% 1,3-diamino-2-hydroxypropane to the activated sephadex G75 medium, stir at 70 °C for 20 h, wash with 12 times the volume of purified water, and drain to form a wet cake.

[0041] (3) Add 3000 ml of 0.1M / L tripolychlorazine in water-acetone solution, adjust the pH of the system to 8.5 with 0.1M sodium hydroxide, stir in an ice bath at 8°C for 1 hour, wash with 12 times the volume of purified water, and drain to obtain Wet cake.

[0042] (4) Add 3000 ml of 0.15mol polylysine...

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Abstract

The invention relates to a method for preparing pure ulinastatin from an ulinastatin affinity chromatography medium. The chromatography medium is formed by linking an ulinastatin affinity ligand and a common chromatography medium, wherein the affinity ligand is polylysine 3000; the common chromatography medium is sephadex; the affinity ligand and the chromatography medium are linked by a hydrophilic spacer arm, and then pure production is carried out on ulinastatin by utilizing the affinity chromatography medium. Ulinastatin with molecular weight of 37-43kDa is quickly separated from the raw materials and is purified via processes for adjusting the concentrations and pH values of various solutions and flow rates of washing and eluting and the like. One-step affinity chromatography is only needed in the whole process, and the yield and specific activity of pure ulinastatin obtained through purification are respectively more than 80% and more than 5000IU / mg.pr. The whole purifying step consumption time is short, and the activity recovery rate and the specific activity are relatively high, so that the method is suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to a method for preparing ulinastatin, in particular to a method for preparing pure ulinastatin through an ulinastatin affinity chromatography medium. Background technique [0002] Ulinastatin is a Kunitz-type broad-spectrum protease inhibitor that can effectively inhibit the activities of trypsin, chymotrypsin, lactase, lipase, hyaluronidase and other pancreatic enzymes. In addition, it has the functions of stabilizing lysosomal membrane, inhibiting the release of lysosomal enzymes, inhibiting the production of myocardial inhibitory factor (MDF), scavenging oxygen free radicals and inhibiting the release of inflammatory mediators. Ulinastatin can also improve immune function decline, abnormal protein metabolism and renal function reduction caused by surgical stimulation, prevent damage to internal organs and cells caused by surgical stimulation, and improve the circulatory state during shock. [0003] It is widely used in clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K1/22
CPCC07K14/8114
Inventor 陈备战辛瑜熊蔚刘兰花
Owner NANCHANG WANHUA BIOCHEM PHARMA
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