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Preparation method for porcine thrombin

A technology of thrombin and prothrombin, which is applied in the field of preparation of porcine blood thrombin, can solve the problems of increasing operation difficulty, unfavorable industrial production, and easy restriction of clinical application of thrombin products, so as to reduce operation difficulty, improve purity, The effect of reducing production costs

Inactive Publication Date: 2018-02-16
ZHEJIANG FENGAN BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this preparation method only performs virus-removing treatment at the beginning. This treatment method needs to keep the product in a sterile state all the time in the later stage, which increases the difficulty of operation, is not conducive to industrial production, and easily limits the clinical application of thrombin products.

Method used

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  • Preparation method for porcine thrombin
  • Preparation method for porcine thrombin

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Experimental program
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Embodiment 1

[0047] Pig blood pretreatment

[0048] Step1 preparation concentration is the trisodium citrate solution of 38g / L;

[0049] Step2 Use a clean glass container to collect fresh pig blood that has been isolated for less than 10 minutes. While collecting the pig blood, add the trisodium citrate solution prepared in Step1 to the fresh pig blood while stirring to control the amount of pig blood and lemon in the glass container. The ratio of trisodium citrate is maintained at pig blood volume ratio: trisodium citrate volume ratio=400:2.5~3.0;

[0050] After Step3 to 40L of pig blood collection is completed, continue to add trisodium citrate solution until the concentration of trisodium citrate solution in the pig blood-trisodium citrate mixture is 0.29g / L, and the measured pH is 6.8;

[0051] Step4 Put the pig blood-trisodium citrate mixture in refrigerated at 4°C for 4 hours, take it out and put it into a horizontal centrifuge, set the centrifugal speed at 4020rpm, the centrifugati...

Embodiment 1 Embodiment 6

[0065] Carry out thrombin titer determination to embodiment one~embodiment six:

[0066] 1. Accurately weigh 30mg of fibrinogen, dissolve it with 1.5ml of 0.9% sodium chloride solution, add 0.1ml (3 units) of thrombin, shake quickly, leave at room temperature for about 1 hour until it is completely solidified, take out the coagulation, and wash with water Until the silver nitrate test solution added to the eluate does not produce turbidity, dry it at 105°C for 3 hours, weigh it, and calculate the content (%) of coagulum in fibrinogen. Then use 0.9% sodium chloride solution to make fibrinogen solution containing 0.2% coagulum, adjust the pH value to 7.0-7.4 with 0.05mol / L disodium hydrogen phosphate solution, and then dilute it with 0.9% sodium chloride solution to contain 0.1% coagulum fibrinogen solution, set aside;

[0067] 2. Take the thrombin standard product, and use 0.9% sodium chloride solution to make standard solution containing 5.0 units, 6.4 units, 8.0 units, and 1...

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Abstract

The invention discloses a preparation method for porcine thrombin. The invention aims to solve the problems of high operation difficulty and being adverse to industrial production of the thrombin extraction. The key point of the technical scheme comprises the following steps: adding trisodium citrate liquid into pig blood, thereby acquiring a pig blood-trisodium citrate mixed liquid; centrifugallylayering the pig blood-trisodium citrate mixed liquid, thereby acquiring the plasma of pig blood; adding anion exchanger into the plasma of the pig blood, stirring and absorbing, cleaning the anion exchanger and then filling into an eluting column; preparing a NaCl-trisodium citrate mixed liquid as an eluant, eluting and collecting the eluant containing prothrombin; desalting the eluant, removingsolvent and monitoring the conductivity of the eluant eluted from a desalting column, thereby acquiring a desalted liquid containing prothrombin; adding CaCl2 liquid into an activated liquid containing thrombin; removing viruses, thereby acquiring a thrombin liquid. The impurities, such as, impure proteins and viruses in the thrombin can be effectively reduced and the thrombin purity can be increased.

Description

technical field [0001] The invention relates to the extraction of thrombin, in particular to a preparation method of pig blood thrombin. Background technique [0002] Thrombin is an important coagulation factor involved in the coagulation process of the body, and exists in the form of prothrombin in the body. In the middle of the 20th century, people began to apply thrombin clinically. Due to its remarkable curative effect, its application is becoming more and more extensive. After that, the United States, Britain, Japan and other countries isolated thrombin from bovine plasma and used it clinically, and proved that animal thrombin did not appear thrombin antigenicity when it was applied to human body, and there was no allergic reaction or other adverse reactions when it was taken orally or topically. Thrombin extracted from animal plasma can be used clinically as the first choice drug for local hemostasis, and has the advantages of fast hemostasis and no side effects. At ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74
CPCC12N9/6429C12Y304/21005
Inventor 王福云泮闻吉张力超李佳辉吴秀燕
Owner ZHEJIANG FENGAN BIOPHARM
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