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Ulinastatin purification method based on affinity chromatography column

A technology of ulinastatin and a purification method, which is applied in the field of ulinastatin purification based on an affinity chromatography column, can solve problems such as high technical content, achieve simple operation, reduce production cost, and improve production efficiency

Inactive Publication Date: 2016-07-13
GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Chinese patent CN100528898 discloses a high-purity ulinastatin and its preparation method and a pharmaceutical composition containing ulinastatin, wherein the urine is treated and then purified by hydrophobic column adsorption and affinity chromatography column, and the use of metal The chelation column is combined with the metalloprotein and eluted by phosphate buffer to purify ulinastatin. Although a certain purity of ulinastatin can be obtained, the technical content is relatively high
[0009] The current ulinastat purification method cannot meet the existing requirements, so it is necessary to remove these impurities to further improve the safety of ulinastatin clinical application

Method used

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  • Ulinastatin purification method based on affinity chromatography column

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Experimental program
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Effect test

Embodiment 1

[0050] (1) Weigh 1TpH less than 6.5 clarified urine, stir continuously, slowly add 10kg chitosan, and measure with precision pH test paper, use acetic acid to adjust the urine pH to 6.0; 3.5kg of ammonium sulfate was salted out, left standing for 24 hours, placed in a vacuum desiccator with anhydrous magnesium sulfate water-absorbing agent after precipitation and centrifugation, and vacuum-dried to obtain the crude product of ulinastatin after drying;

[0051] (2) Hydrophobic column chromatography (XK50 / 20 chromatography column, PhenylSepharose6FastFlow (highsub) filler)

[0052] Pretreatment: Weigh 1kg of crude ulinastatin, dissolve it with 10mmol / LHAc-NaAc, stir for 30 minutes, centrifuge for 20 minutes, keep the centrifugate, adjust pH to 6.0 with acetic acid, add solid ammonium sulfate to adjust cd to 60ms / cm ;

[0053] Balance: use balance buffer (10mmol / LHAc-NaAc buffer, pH6.0, add solid sodium chloride to adjust cd60ms / cm), adjust the pump flow rate to 20.0ml / min, and ...

Embodiment 2

[0068] (1) Weigh 1TpH less than 6.5 clarified urine, stir continuously, slowly add 10kg chitosan, and measure with precision pH test paper, use acetic acid to adjust the urine pH to 6.0; 3.5kg of ammonium sulfate was salted out, left standing for 24 hours, placed in a vacuum desiccator with anhydrous magnesium sulfate water-absorbing agent after precipitation and centrifugation, and vacuum-dried to obtain the crude product of ulinastatin after drying;

[0069] (2) Hydrophobic column chromatography (XK50 / 20 chromatography column, PhenylSepharose6FastFlow (highsub) filler)

[0070] Pretreatment: Weigh 1kg of crude ulinastatin, dissolve it with 10mmol / LHAc-NaAc, stir for 30 minutes, centrifuge for 20 minutes, keep the centrifugate, adjust pH to 6.0 with acetic acid, add solid ammonium sulfate to adjust cd to 60ms / cm ;

[0071] Balance: use balance buffer (10mmol / LHAc-NaAc buffer, pH6.0, add solid sodium chloride to adjust cd60ms / cm), adjust the pump flow rate to 20.0ml / min, and ...

Embodiment 3

[0082] (1) Weigh 1TpH less than 6.5 clarified urine, stir continuously, slowly add 10kg chitosan, and measure with precision pH test paper, use acetic acid to adjust the urine pH to 6.0; 3.5kg of ammonium sulfate was salted out, left standing for 24 hours, placed in a vacuum desiccator with anhydrous magnesium sulfate water-absorbing agent after precipitation and centrifugation, and vacuum-dried to obtain the crude product of ulinastatin after drying;

[0083] (2) Hydrophobic column chromatography (XK50 / 20 chromatography column, PhenylSepharose6FastFlow (highsub) filler)

[0084] Pretreatment: Weigh 1kg of crude ulinastatin, dissolve it with 10mmol / LHAc-NaAc, stir for 30 minutes, centrifuge for 20 minutes, keep the centrifugate, adjust pH to 6.0 with acetic acid, add solid ammonium sulfate to adjust cd to 60ms / cm ;

[0085] Balance: use balance buffer (10mmol / LHAc-NaAc buffer, pH6.0, add solid ammonium sulfate to adjust cd60ms / cm), adjust the pump flow rate to 20.0ml / min, and...

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Abstract

The invention relates to a ulinastatin purification method based on an affinity chromatography column.The method comprises the following steps of urine processing, hydrophobic interaction column chromatography, affinity chromatography, gel filtration chromatography, washing and freeze-drying, and through a purification test on the finally obtained ulinastatin, it is found that the total yield of the ulinastatin is increased to 85% or above, and the total titer of the ulinastatin is 6500 iu / mg or above; the chromatography column is adopted for purification treatment, operation is easy, the production efficiency is improved through optimized packing and parameters such as the flow velocity, the production cost is greatly reduced, and further development and utilization values are achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying ulinastatin based on an affinity chromatography column. Background technique [0002] Ulinastatin, also known as Human Urinary Trypsin Inhibitor (HumanUrinaryTrypsinInhibitor), is a kind of trypsin inhibitor isolated and purified from human urine. In 1909, Beurer and Reich first reported the presence of this protein in human urine. A trypsin inhibitor. It is a glycoprotein composed of 143 amino acids, with alanine at the N-terminus, leucine at the C-terminus, and sugar chains at the 10th serine and 45th aspartic acid. The O-glycosidic chain on serine contains multiple chondroitin sulfate units (5 Ch4S and 10 Ch0S). Ulinastatin has a molecular weight of 60-70KD determined by HPLC, 40-50KD determined by SDS-PAGE, and an isoelectric point of 2.6. It is a heat-stable acidic protein. [0003] Ulinastatin has effective inhibitory effect on various hydrolytic enzym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/22C07K1/20C07K1/16
CPCC07K14/8114
Inventor 王旭郑少亮许文勤肖益热
Owner GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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