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Method for large-scale production of high-purity ulinastatin (namely human urinary trypsin inhibitor)

An ulinastatin, high-purity technology, applied in the field of medicine, can solve the problems of unfavorable large-scale industrial production, high cost of purification media, complicated process steps, etc. The effect of efficiency

Active 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

[0010] However, the above methods all need to be combined with multi-step chromatography, and the process steps are complicated and difficult to scale up; in addition, the purification medium used in multi-step chromatography is expensive, which is not conducive to large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1) Take 1 ton of clarified urine, adjust the pH to 6, add 8kg of silica gel, stir, filter to obtain the filtrate, adjust the pH of the filtrate to 4.5, add 8kg of chitosan (molecular weight: 30,000) and 0.4kg sodium alginate (molecular weight: 20,000), stirred, stood still, discarded the supernatant, and obtained the lower sediment 1;

[0041] 2) Add water to the precipitate 1 to rinse, discard the supernatant, add ammonium sulfate buffer solution, adjust the pH to 5 to obtain the mixed solution 1, and ultrafilter the mixed solution 1 to obtain the retentate 1, wherein the volume of the retentate 1 It is 1 / 5 of the volume of the mixed solution 1, and the molecular weight cut-off is 5×10 4 ;

[0042] 3) Add solution A to the retentate 1 and adjust the pH to 7.5, stir to obtain the mixed solution 2, and carry out secondary ultrafiltration on the mixed solution 2 to obtain the retentate 2, wherein the solution A is triethylamine and isopropanol The volume ratio of the mi...

Embodiment 2

[0047] 1) Take 1 ton of clarified 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 6, add 12kg of chitosan (molecular weight: 30,000) and 0.36kg sodium alginate (molecular weight: 50,000), stirred, stood still, discarded the supernatant, and obtained the lower sediment 1;

[0048] 2) Add water to the precipitate 1 to rinse, discard the supernatant, add ammonium sulfate buffer solution, adjust the pH to 6 to obtain the mixed solution 1, and ultrafilter the mixed solution 1 to obtain the retentate 1, wherein the volume of the retentate 1 It is 1 / 5 of the volume of the mixed solution 1, and the molecular weight cut-off is 6×10 4 ;

[0049] 3) Add solution A to the retentate 1 and adjust the pH to 8, stir to obtain the mixed solution 2, and carry out secondary ultrafiltration on the mixed solution 2 to obtain the retentate 2, wherein the solution A is triethylamine and isopropanol The volume ratio of the m...

Embodiment 3

[0054] 1) Take 1t of clarified urine, adjust the pH to 6.5, add 10kg of silica gel, stir, and filter to obtain the filtrate, adjust the pH of the filtrate to 5, add 10kg of chitosan (molecular weight: 50,000) and 0.1kg sodium alginate (molecular weight: 50,000), stirred, stood still, discarded the supernatant, and obtained the lower sediment 1;

[0055] 2) Add water to the precipitate 1 to rinse, discard the supernatant, add ammonium sulfate buffer solution, adjust the pH to 7, and obtain the mixed solution 1, and ultrafilter the mixed solution 1 to obtain the retentate 1, wherein the volume of the retentate 1 It is 1 / 5 of the volume of the mixed solution 1, and the molecular weight cut-off is 7×10 4 ;

[0056] 3) Add solution A to the retentate 1 and adjust the pH to 7, stir to obtain a mixed solution 2, and perform secondary ultrafiltration on the mixed solution 2 to obtain a retentate 2, wherein the solution A is triethylamine and isopropanol The volume ratio of the mixed...

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Abstract

The invention belongs to the technical field of medicines, and particularly relates to a method for large-scale production of high-purity ulinastatin (namely human urinary trypsin inhibitor). The method comprises the following steps: mixing chitosan and alginate with urine after preliminary filtration so as to obtain a mixture, and then purifying the mixture in a manner combining ultrafiltration and ethanol fractional precipitation. The method provided by the invention is convenient to operate, high in controllability, simple in purification technology, and suitable for large-scale production; besides, the yield of the ulinastatin is as high as 72%, the molecular weight of protein obtained through purification is 66800D-67200D, and the activity of the ulinastatin for restraining trypsin is higher than 4780IU / mg protein.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for large-scale production of high-purity ulinastatin. Background technique [0002] Ulinastatin, also known as human urinary trypsin inhibitor (humanurinary trypsin inhibitor, H-UTI) is a serine protease inhibitor with an apparent relative molecular weight of 66-68kD and an isoelectric point of 2-3. It is a glycoprotein with a sugar content as high as 20%-30%. [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 the decomposition of ulinastatin also have a strong inhibitory effect on hydrolytic enzymes. Ulinastatin is not only a broad-spectrum protease inhibitor, but also has the functions of inhibiting the production of...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/30
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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