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Method for inactivating virus contained in trypsin inhibitor extracted from human urine

A trypsin inhibition and inactivation technology, applied in the directions of protease inhibitors, peptide preparation, inactivation/attenuation, etc., can solve the problems affecting drug safety, residues, drug safety effects, etc., and improve drug safety , Safety improvement, no organic solvent residue effect

Active Publication Date: 2013-05-15
SHANGHAI FENGHUA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zhou Xipeng and others verified the virus inactivation method adopted in the production process of human urinary trypsin inhibitors by using a 60°C water bath for 10 hours and ethanol treatment for 3 hours (Chinese Journal of Biochemical Medicine [J], 2009, 6, 407-410), although It has a good inactivation effect, but the organic solvent (ethanol) will remain in the final product, which will affect the safety of the drug
[0006] Although the prior art has conducted preliminary research on the inactivation of viruses contained in human urinary trypsin inhibitors, these technologies may be applicable to the inactivation of hepatitis viruses, or may still have an impact on drug safety

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1) Pour the freshly collected urine into the stirring tank, adjust the pH to 8.5 while stirring, and take the supernatant urine after standing still;

[0036] 2) After adjusting the pH of the supernatant urine to 4.5, add 25 kg of chitosan per ton of supernatant urine, stir while adding, take the precipitate after standing still, and filter to dry to obtain the adsorbate.

[0037] 3) The obtained adsorbate is rinsed with 3 times the weight of water and filtered to dry, repeating 2 times;

[0038] 4) Add 1.0 liter of 3N ammonia solution with a pH of 10.5 per kilogram of adsorbate to elute the adsorbate, and collect the ammonia water eluate;

[0039] 5) According to adding 400 solid ammonium sulfate per liter of ammonia water eluent, until all dissolved, let stand overnight;

[0040] 6) Take diatomaceous earth as the bottom, and filter with suction to obtain the suction filtrate;

[0041] 7) Add 10 liters of water to dissolve every kilogram of suction filtrate, adjust t...

Embodiment 2

[0048] 1) Pour the freshly collected urine into the stirring tank, adjust the pH to 9.0 while stirring, and take the supernatant urine after standing;

[0049] 2) After adjusting the pH of the supernatant urine to 6.5, add 25 kg of chitosan per ton of supernatant urine, stir while adding, take the precipitate after standing still, and filter to dry to obtain the adsorbate.

[0050] 3) Add 3 times the weight of water to the obtained adsorbate to rinse and filter to dry, repeat 3 times;

[0051] 4) Add 2.0 liters of 3N ammonia solution with a pH of 12.0 per kilogram of adsorbate to elute the adsorbate, and collect the ammonia water eluate;

[0052] 5) According to adding 450g of solid ammonium sulfate per liter of ammonia eluent, until all dissolved, let stand overnight;

[0053] 6) Take diatomaceous earth as the bottom, and filter with suction to obtain the suction filtrate;

[0054] 7) Add 10 liters of water to dissolve per kilogram of suction filtrate, adjust the pH to 6.5,...

Embodiment 3

[0060] 1) Pour the freshly collected urine into the stirring tank, adjust the pH to 9.0 while stirring, and take the supernatant urine after standing;

[0061] 2) After adjusting the pH of the supernatant urine to 6.5, add 25 kg of chitosan per ton of supernatant urine, stir while adding, take the precipitate after standing still, and filter to dry to obtain the adsorbate.

[0062] 3) Add 3 times the weight of water to the obtained adsorbate to rinse and filter to dry, repeat more than 2 times;

[0063] 4) Add 1.5 liters of 3N ammonia solution with a pH of 11.0 per kilogram of adsorbate to elute the adsorbate, and collect the ammonia water eluate;

[0064] 5) According to adding 450g of solid ammonium sulfate per liter of ammonia eluent, until all dissolved, let stand overnight;

[0065] 6) Take diatomaceous earth as the bottom, and filter with suction to obtain the suction filtrate;

[0066] 7) Add 10 liters of water to dissolve per kilogram of suction filtrate, adjust the ...

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Abstract

The invention relates to a method for inactivating virus contained in a trypsin inhibitor extracted from human urine. The method comprises the following steps of dissolving a raw urinary trypsin inhibitor extracted from human urine in a buffer solution and heating at 60 DEG C; then eluting after being adsorbed on a DEAE (diethyl-aminoethanol) chromatographic column so as to obtain DEAE eluate; next sampling the DEAE eluate in ammonium sulfate solution and adsorbing on a hydrophobic chromatography column, then eluting with ammonium sulfate solution of 1M so as to obtain hydrophobic eluate; and after ultrafiltration of the hydrophobic eluate, incubating at low pH, and carrying out gel filtration chromatography to obtain the urinary trypsin inhibitor with virus inactivated. According to the method, a plurality of virus activation methods are utilized comprehensively, especially the two virus inactivation methods of thermally treating firstly and then incubating at low pH, and other separating medium are utilized for realizing inactivation of various virus, so that the inactivation effect of virus contained in the trypsin inhibitor extracted from human urine is further improved, and drug use safety is improved.

Description

technical field [0001] The present invention relates to a method for preparing pharmaceutical biological products, in particular to a method for inactivating viruses contained in trypsin inhibitor extracted from human urine. Background technique [0002] Human Urinary Trypsin Inhibitor (hUTI) is a glycoprotein isolated and purified from the fresh urine of healthy adult males. 45 kDa (Eur. J. Biochem., 158, 417-422). It belongs to protease inhibitors and has inhibitory effects on serine proteases such as trypsin and α-chymotrypsin, granulocyte elastase, hyaluronidase, sulfhydrylase and plasmin and other enzymes. In addition, it also has the functions of stabilizing lysosomal membranes, 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. Human urinary trypsin inhibitors can also improve immune function decline, abnormal protein metab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K1/20C07K1/18C07K1/16C12N7/04
Inventor 纪向阳
Owner SHANGHAI FENGHUA PHARMA CO LTD
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