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Preparation method of human thrombin

A technology of human thrombin and prothrombin, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve problems such as the inability to guarantee the long-term stability of thrombin activity and the impact of thrombin activity, and achieve obvious practical significance , good long-term stability and high vitality

Inactive Publication Date: 2016-10-12
SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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  • Abstract
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Problems solved by technology

Since the patent uses the dry heat virus inactivation method to treat the freeze-dried product of thrombin, it may affect the activity of thrombin, and the long-term stability of thrombin activity cannot be guaranteed

Method used

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  • Preparation method of human thrombin

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Experimental program
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Effect test

Embodiment 1

[0036] 1. 80 kilograms of component III obtained by the ethanol fractionation method of 2.3 tons of healthy human plasma obtained 11.5 kilograms of prothrombin complex solution through SD virus inactivation and ion exchange resin separation and purification (see the prothrombin complex virus for specific methods. Research on inactivation [J]. Chinese Journal of Biological Products, 1995, 8 (3): 101-104; Virus inactivation and verification of human prothrombin complex [J]. Chinese Journal of Blood Transfusion, 1998, 11 (4 ):195-197). After 10mM calcium ion activation, when the detected thrombin activity is greater than 1000IU / ml and tends to be stable, the calcium ion incubation is stopped, and the cation exchange resin is used to separate and purify thrombin. The chromatographic medium is SP-Sephadex C50, the equilibrium buffer is 0.1M NaCl, 0.02M Tris-HCL, pH6.5 solution, and the elution buffer is 0.5M NaCl, 0.02M Tris-HCL, pH6. 5 solution. The eluent was 15.2 kg of thrombi...

Embodiment 2

[0040] 1. 81 kilograms of component III obtained by ethanol fractionation of 2.3 tons of healthy human plasma obtained 12 kilograms of prothrombin complex solution through SD virus inactivation and ion exchange resin separation and purification (see the prothrombin complex virus for specific methods. Research on inactivation [J]. Chinese Journal of Biological Products, 1995, 8 (3): 101-104; Virus inactivation and verification of human prothrombin complex [J]. Chinese Journal of Blood Transfusion, 1998, 11 (4 ):195-197). After 100mM calcium ion activation, when the detected thrombin activity is greater than 1000IU / ml and tends to be stable, the calcium ion incubation is stopped, and the cation exchange resin is used to separate and purify thrombin. The chromatography medium is SP-Sepharose FF, the equilibration buffer is 0.15M NaCl, 0.01M Tris-HCL, pH7.0 solution, and the elution buffer is 1.0M NaCl, 0.01M Tris-HCL, pH7. 0 solution. Obtained 15.4 kg of thrombin eluate with a ...

Embodiment 3

[0044] Place the finished thrombin product prepared in Example 2 at 25°C, take out samples at 0, 1, 2, and 3 months respectively, redissolve with 2ml of water, detect thrombin activity, and observe the stability of thrombin. The results are shown in the following table :

[0045]

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Abstract

The invention relates to the field of blood products and particularly relates to a preparation method of human thrombin. The invention provides a preparation method of human thrombin, which comprises the following steps: 1) performing redissolution and virus inactivation of a component III obtained by Cohn fraction of human plasma; and purifying the obtained liquid through anion exchange resin to obtain prothrombin complex eluent; 2) adding calcium ions into the eluent obtained in the step 1), and incubating until the prothrombin is effectively activated; 3) separating and purifying the liquid obtained in the step 2) through cation exchange resin; and 4) performing nano membrane filtration of the liquid obtained in the step 3) to obtain a finished product of human thrombin. In the preparation method provided by the invention, by adopting the thrombin product of the technology market, the human thrombin has high vigor, high yield and good long-term stability, meets the requirements on safety and effectiveness of clinical application and is of relatively obvious economic value and practical significance.

Description

technical field [0001] The invention relates to the field of blood products, in particular to a preparation method of human thrombin, which can be used for large-scale production of human thrombin from human plasma. Background technique [0002] Thrombin belongs to the serine proteolytic enzyme, which is composed of 308 amino acid residues and has a molecular weight of 36KD. It is a double-chain molecule formed by connecting the light chain and the heavy chain through disulfide bonds. Among them, the light chain has 49 amino acid residues, which play an important role in the structural stability of thrombin; the heavy chain has 259 amino acid residues, which is the enzymatic functional region of thrombin. The precursor of thrombin is prothrombin (FII), a vitamin K-dependent protein synthesized in the liver, with a plasma content of about 10-15 mg / dL and a molecular weight of 68KD. [0003] The formation of thrombin plays a central role in the overall coagulation mechanism. ...

Claims

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Application Information

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IPC IPC(8): C12N9/74
CPCC12N9/6429C12Y304/21005
Inventor 徐俊施炜夏正祥王弘远占德国周雪峰程露
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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