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A kind of preparation method of porcine thrombin freeze-dried powder

A technology of porcine thrombin and freeze-dried powder, which is applied in the field of preparation of porcine thrombin freeze-dried powder, can solve the problems of unavoidable secondary pollution risk of exogenous virus, complex activation method of prothrombin, and many process steps, and achieve improvement Product safety, improved product safety, simple process steps

Active Publication Date: 2018-05-08
WUHAN HITECK BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has the disadvantages of (1) many and complicated process steps, (2) the prothrombin activation method is complex and cumbersome, and (3) the risk of secondary contamination by exogenous viruses cannot be avoided during the thrombin preparation process.

Method used

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  • A kind of preparation method of porcine thrombin freeze-dried powder
  • A kind of preparation method of porcine thrombin freeze-dried powder
  • A kind of preparation method of porcine thrombin freeze-dried powder

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Preparation of porcine thrombin freeze-dried powder preparation of the present invention

[0025] 1. Preparation of porcine thrombin

[0026] (1) Preparation of prothrombin

[0027] Take 40L of anticoagulated pig plasma; take another 40g of QAE-Sephadex A50 gel dry powder, and use 3.75mmol / L Na 2 B 4 o 7 and 85mmol / L H 3 BO 3 , pH 8.0 buffer solution swelled and balanced, added to the plasma, stirred at room temperature for 2 hours; filtered with double-layer 400-mesh silk cloth to collect the gel adsorbed with prothrombin, and filled the chromatography column; Wash until the baseline becomes flat, then wash with 0.1mol / L NaCl until the baseline becomes flat, and finally wash with 0.4mol / L NaCl and 2mmol / L BaCl 2 The eluent was eluted, and the elution peak was collected to obtain 8570 mL of prothrombin solution.

[0028] (2) Preparation of Thromboplastin Solution

[0029] Take 5g of rabbit brain powder, add 100mL of physiological saline preheated to...

Embodiment 2

[0040] Embodiment 2 Preparation of porcine thrombin freeze-dried powder preparation of the present invention

[0041] Such as figure 1 As shown in the schematic diagram, the porcine thrombin freeze-dried powder preparation of the present invention is prepared.

[0042] 1. Preparation of porcine thrombin

[0043] (1) Preparation of prothrombin

[0044] Take 35L of anticoagulated pig plasma; take another 52.5g of QAE-SephadexA50 gel dry powder, and use 3.75mmol / L Na 2 B 4 o 7 -85mmol / L H 3 BO 3 , pH 8.0 buffer solution was swollen and balanced, then added to the plasma, stirred and adsorbed at room temperature for 2 hours; the above-mentioned gel adsorbed with prothrombin was collected by filtration with double-layer 400-mesh silk cloth, and packed into a chromatographic column; Wash with purified water until the baseline becomes flat, then wash with 0.1mol / L NaCl until the baseline becomes flat, and finally wash with 0.4mol / L NaCl-2mmol / L BaCl 2 Elution, the elution pea...

Embodiment 3

[0057] Example 3 Porcine thrombin purification effect evaluation

[0058] Take 35L of anticoagulant pig plasma, adopt the process method of Example 2 to extract, separate and purify porcine thrombin, repeat three times in a row, and comprehensively evaluate process indicators and quality indicators such as thrombin purification effect, yield and S / D residual amount. The experimental results are shown in Table 1.

[0059] Table 1 The experimental results of the evaluation of the purification effect of porcine thrombin

[0060]

[0061] According to the results in Table 1, after two-step chromatography, the specific activity of thrombin can reach more than 130 U / mg, which is an order of magnitude higher than the national pharmacopoeia standard (10 U / mg); more than 80,000 U of thrombin can be produced per liter of pig plasma Pure product, and the residues of Tween-80 (Tween-80) and tributyl phosphate (TNBP) are in line with national standards (Tween-80<100μg / mL, TNBP<10μg / mL)...

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Abstract

The invention discloses a preparation method of porcine thrombin freeze-dried powder, comprising the following steps: mixing, stirring and adsorbing anticoagulant porcine plasma and gel, eluting through a column to obtain a prothrombin solution; adding physiological saline to rabbit brain powder, and then adding The prothrombin solution and CaCl2 were added to activate the zymogen to obtain a crude thrombin enzyme solution, which was desalted by ultrafiltration and concentrated to inactivate the virus. That is to obtain pig thrombin; add mannitol or dextran 40 to the pig thrombin, filter and sterilize, sub-pack, freeze-dry, vacuum stopper, and roll the aluminum-plastic combination cover to obtain a freeze-dried product of pig thrombin; the freeze-dried product is at 100 Dry heat treatment is carried out at ℃ to inactivate the virus again, and the porcine thrombin freeze-dried powder is obtained by packaging. The specific activity of porcine thrombin in the product is not less than 130U / mg, the whole process steps are simple and easy to implement, the safety of the product is improved, and it is suitable for industrial production.

Description

technical field [0001] The invention relates to a preparation process of porcine thrombin freeze-dried powder, in particular to a preparation method of porcine thrombin freeze-dried powder. Background technique [0002] Thrombin is a serine proteolytic enzyme that plays an important role in the blood coagulation system. It can hydrolyze the 4 Arg-Gly peptide bonds of fibrinogen, transform the fibrinogen in the sol state into fibrin in the gel state, and promote Blood coagulation can also promote platelet aggregation, further accelerate blood coagulation, and achieve the purpose of rapid hemostasis. [0003] At present, most of the thrombin preparations used clinically are prothrombin extracted from bovine blood or pig blood, which is activated by prothrombin activator to obtain a sterile freeze-dried product of thrombin, which has high specificity. It is a fast-acting local hemostatic drug, which is widely used in gastrointestinal bleeding and surgical hemostasis. (Song Ho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74A61K9/19A61K38/48A61P7/04
Inventor 汤华东吴红梅朱德芳陈亚陈煌
Owner WUHAN HITECK BIOLOGICAL PHARMA