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Preparation method of freeze-dried human thrombin

A technology of human thrombin and human prothrombin, applied in the field of preparation of freeze-dried human thrombin, which can solve the problems of turbidity of thrombin and low yield of target substances

Active Publication Date: 2017-03-08
SHANGHAI RAAS BLOOD PRODUCTS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It can be seen from the above technical scheme that two purification steps (i.e., two resin chromatography) have been implemented in the prior art, resulting in a low yield of the target substance (human thrombin) (about 600,000 IU / ton of plasma)
In addition, the freeze-dried human thrombin prepared by the prior art has turbidity after reconstitution

Method used

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  • Preparation method of freeze-dried human thrombin

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Step 1, according to the dilution ratio of 1:9, put 1.0kg of Fr.III precipitate into 9kg of HAc-NaAc buffer solution (pH value 5.00, 10°C), stir at a constant speed for 4 hours to fully dissolve, and then add silicon The alginate was stirred for 30 minutes, and then press-filtered at the same temperature to obtain about 0.81 kg of prothrombin precipitate.

[0039] Step 2, according to the dilution ratio of 1:4, put 0.81kg of prothrombin precipitate into 3.24kg Tris-HCL buffer solution (pH value is 6.50, 10°C), stir gently for 4 hours to fully dissolve and obtain a uniform suspension;

[0040] Step 3, add 0.5M CaCl to the above suspension 2 The solution was incubated at 15°C for 10 hours to activate prothrombin into thrombin;

[0041] Step 4, add PEG solution to the suspension after the above activation, stir for 1 hour, press filter, collect the supernatant, then use a 0.45 μm filter element for clarification and filtration, and collect 5.2 kg of filtrate;

[0042] S...

Embodiment 2

[0053] Step 1, according to the dilution ratio of 1:9, put 7.1kg of Fr.III precipitate into 63.9kg of HAc-NaAc buffer solution (pH value is 5.00, 10°C), stir at a constant speed for 4 hours to fully dissolve; add diatom Stir with soil for 30 minutes, then press filter at the same temperature to obtain about 6.2kg of prothrombin precipitate.

[0054] Step 2: According to the dilution ratio of 1:4, put 6.2kg of prothrombin precipitate into 24.8kg of Tris-HCL buffer solution (pH value is 6.50, 10°C), stir gently for 4 hours to fully dissolve;

[0055] Steps 3, 4 and 5 are the same as in Example 1 to obtain 39.2 kg of solution after S / D inactivation;

[0056] Step 6, add pre-balanced SP-Sephadex C-50 ion exchange resin to the solution after S / D inactivation, stir slowly for 2 hours, then filter with a sieve to collect the resin; then use 0.20M sodium chloride Tris -HCl buffer solution for washing; then use 0.6M sodium chloride Tris-HCl buffer solution for elution, and collect 10....

Embodiment 3

[0066] Step 1, according to the dilution ratio of 1:15, put 7.5kg of Fr.III precipitate into 112.5kg of HAc-NaAc buffer (pH value is 5.50, 15°C), stir at a constant speed for 2 hours to fully dissolve; add diatom Stir with soil for 30 minutes, then press filter to obtain about 5.6kg of prothrombin precipitate.

[0067] Step 2, according to the dilution ratio of 1:5, put 5.6kg of prothrombin precipitate into 28kg of Tris-HCL buffer solution (pH value is 7.50, 30°C), stir gently for 2 hours to fully dissolve;

[0068] Step 3, add 0.5M CaCl to the above solution 2 The solution was incubated at 30°C for 5 hours to activate prothrombin into thrombin;

[0069] Steps 4 and 5 are the same as in Example 2.

[0070] Step 6, add pre-balanced SP-Sephadex C-50 ion exchange resin to the solution after S / D inactivation, stir slowly for 1 hour, then filter with a sieve to collect the resin; then use 0.10M sodium chloride Tris -HCl buffer solution for washing; then use 1.0M sodium chloride ...

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Abstract

The invention relates to a preparation method of freeze-dried human thrombin. The preparation method comprises the following steps: (1) taking a Cohn component III as the raw material, and preparing human prothrombin by an isoelectric precipitation method; (2) using calcium chloride to activate prothrombin prepared in the step (1) to obtain a coarse product of thrombin; (3) subjecting the coarse product to S / D virus inactivation and SP-Sephadex C-50 resin chromatographic purification to obtain refined thrombin; and (4) subjecting the refined thrombin to ultrafiltration and concentration, adding an stabilizing agent into the refined thrombin, removing virus and filtering the refined thrombin by a nano membrane, sterilizing and filtering the refined thrombin, freeze-drying the refined thrombin, and carrying out a dry heating treatment to obtain the target product. Compared with the prior art, the preparation steps are concise, the product yield is high, and the production quality is good.

Description

technical field [0001] The invention relates to a method for preparing freeze-dried human thrombin, in particular to a method for preparing freeze-dried human thrombin comprising three steps of virus inactivation. Background technique [0002] Human thrombin (human thrombin) is a serine protease with a molecular weight of 37KD, which is composed of a 3kD light chain and another 31kD heavy chain connected by a disulfide bond. Its main function is to hydrolyze soluble fibrinogen into insoluble fibrin and activate coagulation factor F.XIII to cross-link fibrin to form a network to stop bleeding. [0003] Thrombin can directly convert fibrinogen into fibrin without going through the early stage of blood coagulation, so that blood coagulates rapidly and fills the bleeding point to achieve the purpose of hemostasis. It can be applied to bleeding symptoms caused by various reasons, such as digestive tract, trachea, and various surgical bleeding. Therefore, with the increasingly w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K9/19A61P7/04
Inventor 李春洲
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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