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Method for preparing human thrombin from cold-removing glue plasma

A technology for removing cold gelatin plasma and human thrombin, which is applied in the field of biomedicine, can solve the problems of incomplete virus inactivation product appearance, harsh activation conditions, and complicated process flow, and achieve stable activation process, short processing time, and high yield high effect

Inactive Publication Date: 2015-11-11
上海洲跃生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for preparing human thrombin from decolonized plasma. The method for preparing human thrombin from decolonized plasma solves the problem of existing The technical problems of the method for preparing human thrombin are complicated process, harsh activation conditions, long production cycle, incomplete virus inactivation and poor appearance of the product

Method used

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  • Method for preparing human thrombin from cold-removing glue plasma
  • Method for preparing human thrombin from cold-removing glue plasma
  • Method for preparing human thrombin from cold-removing glue plasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Step 1, weigh 10 grams of DEAE-SephadexA-50 dry gel, swell with 1 kg of hot water for injection, cool with about 1 kg of cold WFI, and then equilibrate with 1 kg of buffer A (0.01M sodium citrate-0.15M NacL buffer, pH7 .15, 10°C) balance, add the balanced resin to 10 liters of cold glue plasma, keep the plasma temperature at about 10-15°C, stir slowly for about 1 hour, then filter with the filter of the resin bucket, collect the resin, The filtered plasma enters other plasma separation processes;

[0046] Step 2, wash the resin continuously with about 1.2kg of equilibration buffer A (0.01M sodium citrate-0.15MNacL buffer, pH7.15, 10°C), and then use elution buffer A (0.01M sodium citrate-0.5MNacL buffer, pH7.15, 10°C, about 0.6kg slowly and continuously elute the resin, keep stirring the resin to make it evenly suspended, and collect 460g of the eluate;

[0047] Step 3, add 2.5 grams of diatomaceous earth, stir for 0.5 hours, at 20°C, add 0.5MCaCl 2 Solution 25g, stir...

Embodiment 2

[0061] Step 1, with embodiment 1;

[0062] Step 2, wash the resin continuously with about 1.8kg of equilibration buffer A (0.01M sodium citrate-0.15M NacL buffer, pH7.15, 12°C), and then use elution buffer A (0.01M sodium citrate-1M NacL buffer Liquid, pH7.15, 12°C, about 1.2kg slowly and continuously eluted the resin, kept stirring the resin to make it evenly suspended, and collected 850g of the eluent;

[0063] Step 3, add 8.5 grams of diatomaceous earth, stir for 1 hour, raise the temperature to 30°C, add 1MCaCl 2 Solution 180g, stirred gently for 4 hours;

[0064] Step 4, add 125g of 50% PEG solution and stir for 1 hour;

[0065] Steps 5,6,7 are the same as in Example 1;

[0066] Step 8, the temperature of the above solution was lowered to about 5°C, and the CM column (100ml) was used for chromatography. First wash with 8 times column volume of equilibration buffer B (0.02M sodium citrate-0.1M NacL buffer, pH7.15, 5°C,) to remove foreign proteins; then use 6 times of e...

Embodiment 3

[0073] Step 1, with embodiment 1;

[0074] Step 2, wash the resin continuously with about 1.5kg of equilibration buffer A (0.01M sodium citrate-0.15MNacL buffer, pH7.15, 15°C), and then use elution buffer A (0.01M sodium citrate-0.75MNacL Buffer, pH7.15, 15°C, about 0.9kg slowly and continuously elute the resin, keep stirring the resin to make it evenly suspended, and collect 675g of the eluent;

[0075] Step 3, add 3.35 grams of diatomaceous earth, stir for 0.5 hours, at 25°C, add 0.5MCaCl 2 Solution 80g, stirred gently for 6 hours;

[0076] Step 4, add 62g of 40% PEG solution and stir for 1 hour;

[0077] Steps 5,6,7 are the same as in Example 1;

[0078] Step 8, the temperature of the above solution was lowered to about 10° C., and the chromatography was carried out on SP-sepharose FF column (100 ml).

[0079] Wash with 5 times column volume equilibration buffer B (0.02M sodium citrate-0.15M NacL buffer, pH6.80, 10℃,) to remove impurities; then use 4 times elution buffe...

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Abstract

The invention discloses a method for preparing human thrombin from cold-removing glue plasma. Firstly, anion resins are used to absorb prothrombin in the cold-removing glue plasma, then elution and filtering are conducted, and a prothrombin solution is obtained; secondly, the prothrombin solution is activated through a CaCL2 solution, and a thrombin solution is obtained; thirdly, S / D viral inactivation is conducted on the thrombin solution; fourthly, chromatography is conducted through cation columns, and a purified thrombin solution is obtained; fifthly, ultrafiltration, dialysis and concentrate are conducted on the thrombin solution, and the thrombin solution with the titer meeting the product specification requirement is obtained; sixthly, a filter element of 20 nm in size is used to conduct virus removal filtering; seventhly, sterilization filtering is conducted through a filter element of 0.22 microns in size, and then subpackage is conducted according to the required specifications; eighthly, freeze-drying is conducted, and dry heat viral inactivation is conducted on freeze-drying powder, and a human thrombin product is obtained. According to the method, the thrombin is purified through the one-step cation columns, operation is simple, thrombin specific activity is high, and safety of clinic use of the product is guaranteed through a three-step virus elimination mode.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a human thrombin, in particular to a method for preparing human thrombin from cold-depleted plasma. Background technique [0002] Human thrombin (humanthrombin) is a serine protease with a molecular weight of 37KD. It 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 by activating coagulation factor XIII to cross-link fibrin to form a network, thereby playing a role in hemostasis. Under normal circumstances, the thrombin in the human blood circulation exists in an inactive form, that is, prothrombin. When bleeding tends to occur in the body or on the body surface, the prothrombin is converted into an activated form of thrombin through a series of complicated processes. . Using modern biopharmaceutical technology, prothrombin contained in plasma can be made into free...

Claims

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Application Information

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IPC IPC(8): C12N9/74
CPCC12N9/6429C12Y304/21005
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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