Preparation method of thrombin

A technology for thrombin and prothrombin, which is applied in the field of thrombin preparation, can solve the problems of increasing production steps and increasing production costs, and achieves the effects of improving utilization rate, increasing titer, and reducing production costs.

Active Publication Date: 2020-12-01
长春雷允上药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method also requires an additional virus removal step, which also increases production steps and increases production costs

Method used

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  • Preparation method of thrombin
  • Preparation method of thrombin
  • Preparation method of thrombin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of embodiment 1 prothrombin

[0044] 1. Take fresh pig blood, add 38g / L trisodium citrate aqueous solution to anticoagulate, use a centrifuge to centrifuge at a speed of 12000r / min, and filter to obtain plasma;

[0045] 2. Add adsorbent DEAE-sephadex-A50 ion exchange resin to plasma, wash with 0.2M aqueous sodium chloride solution and 3g / L trisodium citrate aqueous solution, wash with 2M aqueous sodium chloride solution and 3g / L trisodium citrate aqueous solution Elution, and finally dialyze the eluate with a dialysis bag with a molecular weight of 7000 to obtain a prothrombin dialysate.

Embodiment 2

[0046] Example 2 Recombinant lipidated tissue factor activates prothrombin

[0047] Add 1,000 mL of the prothrombin dialysate prepared in Example 1 into a stainless steel container, heat it to 37°C in a water bath, and add recombinant lipidated human tissue factor at a concentration of 0.1 mg / mL purchased from Beijing Boersi Technology Co., Ltd. The solution was 1 mL, the temperature of the solution was controlled at 37° C., stirred for 10 min, filtered, and placed in a 4° C. freezer for 1.0 h to obtain a solution containing thrombin.

[0048] Adjust the solution containing thrombin to pH 6 with acetic acid, place it in a freezer at 1°C for 20 minutes, centrifuge and discard the precipitate, and collect the supernatant; adjust the supernatant to pH 6.5 with saturated aqueous sodium bicarbonate , placed in a freezer at 6°C for 30 minutes, centrifuged and discarded, and the supernatant was collected.

[0049] The detection was repeated three times, and the titers of the obtaine...

Embodiment 3

[0050] Example 3 Recombinant lipidated tissue factor activates prothrombin

[0051] Add 1000 mL of the prothrombin dialysate prepared in Example 1 into a stainless steel container, heat it to 37°C in a water bath, and add recombinant lipidated rabbit tissue factor at a concentration of 0.1 mg / mL purchased from Shanghai Guandong Biotechnology Co., Ltd. The solution was 1 mL, the temperature of the solution was controlled at 37° C., stirred for 10 min, filtered, and placed in a 4° C. freezer for 1.0 h to obtain a solution containing thrombin.

[0052] Adjust the solution containing thrombin to pH 6 with acetic acid, place it in a freezer at 1°C for 20 minutes, centrifuge and discard the precipitate, and collect the supernatant; adjust the supernatant to pH 6.5 with saturated aqueous sodium bicarbonate , placed in a freezer at 6°C for 30 minutes, centrifuged and discarded, and the supernatant was collected.

[0053] The detection was repeated three times, and the titers of the o...

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Abstract

The present invention provides a preparation method of thrombin. The preparation method comprises that a prothrombin activator is added to a prothrombin-containing solution to activate prothrombin soas to obtain a thrombin-containing solution, and the prothrombin activator is selected from a recombinant lipidated tissue factor or a recombinant lipidated tissue factor and calcium ions. The methodfurther includes preparing the prothrombin-containing solution before activating prothrombin. The method further comprises the step of purifying and further treating the thrombin-containing solution to obtain a thrombin finished product. According to the preparation method disclosed by the invention, the recombinant lipidated tissue factor is used as an activator, so that compared with rabbit brain powder, the safety of a thrombin product can be remarkably improved, and the production cost is reduced; compared with soybean phospholipid and egg yolk phospholipid, the thrombin specific activityis obviously improved, and the method is very suitable for modern large-scale industrial production.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a preparation method of thrombin. Background technique [0002] Thrombin is an important extracellular serine protease, which consists of two peptide chains connected by disulfide bonds, and is an important part of the body's blood coagulation system. Thrombin is blood coagulation factor IIa, which can directly act on fibrinogen to convert fibrinogen into fibrin, so that blood coagulates rapidly, fills the bleeding point, and achieves the purpose of hemostasis. [0003] Thrombin is produced by the activation of prothrombin, that is, the presence of a prothrombin activator (activator) is required to activate prothrombin in the blood to form thrombin. In the extrinsic coagulation process, coagulation factor VII in the blood combines with coagulation factor III, and under the action of calcium ions, activates coagulation factor X to convert coagulation factor Xa; coagulation facto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74A61K38/48A61P7/04A61L15/38A61L15/32A61L15/44A61L15/42
CPCC12N9/6429C12Y304/21005A61K38/4833A61P7/04A61L15/38A61L15/32A61L15/44A61L15/425A61L2300/254A61L2300/418A61L2400/04C08L89/00
Inventor 朱振涛孙丽曹思瑶张娜辛欣咸思焕胡锡旺
Owner 长春雷允上药业有限公司
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