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Preparing method for pig thrombin freeze-dried powder

A technology for 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 contamination risk of exogenous virus, complex prothrombin activation method, and many process steps, etc. Product safety, improving product safety, and the effect of simple process steps

Active Publication Date: 2016-01-20
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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  • Preparing method for pig thrombin freeze-dried powder
  • Preparing method for pig thrombin freeze-dried powder
  • Preparing method for pig 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-SephadexA50 gel dry powder, and use 3.75mmol / LNa 2 B 4 o 7 and 85mmol / LH 3 BO 3 , after pH 8.0 buffer was swelled and equilibrated, it was added to the plasma, stirred and adsorbed at room temperature for 2 hours; the above-mentioned gel with adsorbed prothrombin was collected by filtration with double-layer 400-mesh silk cloth, and packed into a chromatographic column; Wash with water until the baseline becomes flat, then wash with 0.1mol / LNaCl until the baseline becomes flat, and finally wash with 0.4mol / LNaCl and 2mmol / LBaCl 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 br...

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 / LNa 2 B 4 o 7 -85mmol / LH 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 is flat, then wash with 0.1mol / LNaCl until the baseline is flat, and finally wash with 0.4mol / LNaCl-2mmol / LBaCl 2 Elution, the elution peak was collected...

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 preparing method for pig thrombin freeze-dried powder. The preparing method comprises the following steps that anticoagulant pig plasma and gel are mixed and stirred for adsorption and pass through a column for elution, and a prothrombin solution is obtained; normal saline is added to rabbit brain powder, then the prothrombin solution and CaCl2 are added for zymogen activation, a crude thrombin enzyme solution is obtained, subjected to ultrafiltration for desalination and concentrated, viral inactivation is carried out, an obtained crude enzyme solution passes through DEAE-Sepharose Fast Flow chromatographic column, elution is carried out, target peaks are collected, and pig thrombin is obtained; the pig thrombin is added with mannitol or dextran 40, filtering, sterilizing, subpackaging, freeze drying and vacuum tamponing are carried out, an aluminum plastic combined cover is rolled, and a pig thrombin freeze-dried product is obtained; the freeze-dried product is subjected to dry heat treatment at 100 DEG C for secondary virus inactivation, and the pig thrombin freeze-dried powder is obtained after packaging. The specific activity of pig thrombin in the product is not lower than 130 U / mg, the whole process is simple in step and easy to implement, the product safety is improved, and the preparing method 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 Applications(China)
IPC IPC(8): A61K9/19A61K38/48A61P7/04C12N9/74
Inventor 汤华东吴红梅朱德芳陈亚陈煌
Owner WUHAN HITECK BIOLOGICAL PHARMA
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