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The purification process of thrombin

A thrombin and process technology, applied in the field of thrombin purification process, can solve the problems of nano-membrane pore blockage and the like

Active Publication Date: 2019-10-11
浙江杭康药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention provides a purification process of thrombin, which solves the problem that the enzyme liquid is directly subjected to nano-filtration in the traditional process, which easily causes the blockage of the nano-membrane pores

Method used

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  • The purification process of thrombin
  • The purification process of thrombin
  • The purification process of thrombin

Examples

Experimental program
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Effect test

Embodiment 1

[0029] (1) Take pig blood, add anticoagulant, and centrifuge to obtain plasma and serum; the anticoagulant is sodium citrate, and the weight ratio of plasma to anticoagulant is 200:1.

[0030] (2) Adsorb prothrombin in the plasma by ion exchange resin, and elute to obtain a prothrombin solution; for details, refer to the method disclosed in CN105039295A. _

[0031] (3) Add rabbit cephalin powder into the eluent at a ratio of 1%, let it stand at room temperature for 10 hours, activate prothrombin and convert it into thrombin, and collect the crude thrombin solution by filtration.

[0032] (4) Filtrating the crude thrombin is mainly to remove macromolecular substances and bacteria in the enzyme solution, but cannot remove lipid-enveloped viruses and non-lipid-enveloped viruses.

[0033] (5) Add tributyl phosphate in the final filtrate, the addition amount is 0.5% of the weight of the filtrate, then add Tween-80, the addition amount is 1% of the weight of the filtrate, stir at n...

Embodiment 2

[0043] Phosphate buffer solution with a pH value of 7.5 is used for the diafiltration of the membrane package, the diafiltration multiple is 9 times, and the tangential flow rate of the diafiltration is 5mL / min m 2 , the membrane pressure is 30psi; the tangential flow rate of membrane bag concentration is 5mL / min·m 2 , the membrane pressure is 45psi, concentrated 9 times. The nano-membrane has a pore size of 20nm and a membrane pressure of 5bar. All the other are with embodiment 1.

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Abstract

The invention discloses a purification process for thrombin. The purification process comprises a step of nano filtration; before nano filtration is performed, a membrane bag is washed and filtered to remove impurity protein of which the molecular weight is greater than 200KD. According to the purification process disclosed by the invention, macromolecular proteins in a thrombin solution can be removed by washing and filtering the membrane bag, so that the condition that a nano-membrane pore channel is blocked by macromolecular substances is avoided; in addition, filtering conditions of the membrane bag are mild, so the condition that the activity of the thrombin is reduced is avoided, and the aim of removing non-lipid-enveloped viruses is achieved.

Description

technical field [0001] The invention relates to the technical field of biological product production, in particular to a process for purifying thrombin. Background technique [0002] Thrombin is a serine protease and the main effector protease in the blood coagulation cascade, exhibiting procoagulant and anticoagulant properties. Clinically, thrombin is suitable for ligation of small blood vessels, capillaries and solid organs that are difficult to stop bleeding. Thrombin used in trauma, surgery, oral cavity, ENT, urology, burns, orthopedics and other bleeding can directly act on fibrinogen in the blood to promote the transformation into fibrin, accelerate blood coagulation and stop bleeding. [0003] The preparation of thrombin is usually to obtain the crude product of prothrombin through a series of procedures, and then activate it in a specific way to convert the prothrombin into thrombin, then purify the thrombin, obtain the thrombin liquid through ultrafiltration and c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74
Inventor 付存雷徐新胜方伟国李倩吴倩倩柯益娜卢文义范罗莹李强
Owner 浙江杭康药业有限公司
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