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Method for purifying ulinastatin based on anion exchange resin

A technology of exchange resin and ulinastatin, which is applied in the field of medicine, can solve problems such as the influence of ulinastatin yield and complicated purification process, and achieve the effects of increasing molecular weight cut-off, simple purification process and improving accuracy

Inactive Publication Date: 2016-08-10
GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But for a certain volume of urine, the more complex the purification process, the purity of ulinastatin can be improved to a certain extent, but the yield of ulinastatin is affected

Method used

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  • Method for purifying ulinastatin based on anion exchange resin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1) Get 1t of clear urine, adjust the pH to 6, add 12kg of silica gel, stir, filter to obtain the filtrate, adjust the pH of the filtrate to 3, continue to add 8kg of chitosan-chitin mixture (chitosan: chitin) in the filtrate =3:1), stirred until the adsorption is complete, sequentially eluted with ammonia water, salted out with ammonium sulfate, allowed to stand, precipitated, and dried to obtain the crude product of ulinastatin;

[0041] 2) Add sodium acetate-acetic acid buffer solution of pH=5 to the crude product of ulinastatin until completely dissolved, continue to add sodium alginate (molecular weight 20,000), stirring, leaving standstill, centrifuging to obtain the lower sediment 1, adding ammonium sulfate buffer solution to the sediment 1 to make pH=6, ultrafiltration to obtain the retentate 1; wherein, the ultrafiltration pressure is 0.2MPa, The temperature is 30°C, the molecular weight cut-off is 5×10 4 ;

[0042] 3) Adjust the pH of the retentate 1 to 6, pa...

Embodiment 2

[0047] 1) Get 1t of clear urine, adjust the pH to 6.5, add 12kg of silica gel, stir, filter to obtain the filtrate, adjust the pH of the filtrate to 5, continue to add 12kg of chitosan-chitin mixture (chitosan: chitin) in the filtrate =8:1), stirred until the adsorption is complete, sequentially eluted with ammonia water, salted out with ammonium sulfate, allowed to stand, precipitated, and dried to obtain the crude product of ulinastatin;

[0048] 2) Add sodium acetate-acetic acid buffer solution of pH=5.5 to the crude product of ulinastatin until completely dissolved, continue to add sodium alginate (molecular weight 50,000), stirring, standing, and centrifugation to obtain the lower sediment 1, adding ammonium sulfate buffer solution to the sediment 1 to make pH=6.5, and ultrafiltration to obtain the retentate 1; wherein, the ultrafiltration pressure is 0.2MPa, The temperature is 30°C, the molecular weight cut-off is 5×10 4 ;

[0049] 3) Adjust the pH of retentate 1 to 6,...

Embodiment 3

[0054] 1) Get 1t of clear urine, adjust the pH to 6.5, add 12kg of silica gel, stir, filter to obtain the filtrate, adjust the pH of the filtrate to 5, continue to add 12kg of chitosan-chitin mixture (chitosan: chitin) in the filtrate =5:1), stirred until the adsorption is complete, sequentially eluted with ammonia water, salted out with ammonium sulfate, allowed to stand, precipitated, and dried to obtain the crude product of ulinastatin;

[0055] 2) Add sodium acetate-acetic acid buffer solution of pH=5.5 to the crude product of ulinastatin until completely dissolved, continue to add sodium alginate (molecular weight 20,000), stirring, standing, and centrifuging to obtain the lower sediment 1, adding ammonium sulfate buffer solution to the sediment 1 to make pH=6, and ultrafiltration to obtain the retentate 1; wherein, the ultrafiltration pressure is 0.5MPa, The temperature is 25°C, the molecular weight cut-off is 7×10 4 ;

[0056] 3) Adjust the pH=6 of the retentate 1, pa...

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Abstract

The invention belongs to the technical field of medicine and particularly relates to a method for purifying ulinastatin based on anion exchange resin. The method comprises the steps that firstly, a mixture of chitosan and chitin is used for adsorption separation, and a crude ulinastatin product is obtained; then, the crude product and alginate are mixed and then subjected to ultrafiltration, anion exchange resin purification and ultrafiltration in sequence; finally, interception liquid obtained after ultrafiltration is subjected to freezing drying, and a pure ulinastatin product is obtained. According to the method for purifying ulinastatin based on anion exchange resin, the purifying process is shortened, and the yield and purity of ulinastatin are high, wherein the yield of ulinastatin reaches 87%, and the specific activity reaches 4900 IU / mg protein.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for purifying ulinastatin based on anion exchange resin. Background technique [0002] Ulinastatin is an acidic glycoprotein isolated and purified from male urine. According to literature reports, it is composed of 143 amino acids, with sugar chains on the 10th serine and 45th aspartic acid, and the surface of the structure contains a large number of non-polar hydrophobic groups, such as glycine, alanine, valine , leucine, etc. The molecular weight of ulinastatin is 60-70KD determined by HPLC, 40-50KD determined by SDS-PAGE, and the isoelectric point is between 2-3. [0003] Since the two active functional regions in the structure have a broad spectrum of enzyme inhibition, it can simultaneously inhibit trypsin and phospholipase A 2 , hyaluronidase, elastase and other hydrolytic enzyme activities; moreover, the low molecular weight components formed by th...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/30C07K1/18
CPCC07K14/8114
Inventor 王旭郑少亮田友军肖益热
Owner GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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