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Production method of human antithrombin III

A technology of antithrombin and production methods, which is applied in the direction of peptide preparation methods, chemical instruments and methods, protease inhibitors, etc., can solve problems such as increasing affinity chromatography, hidden dangers in product quality, and shedding of affinity ligands, etc. Achieve the effect of convenient handling, simple operation and large load

Active Publication Date: 2015-06-03
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gel of affinity chromatography is generally expensive and the production cost is high, and adding a step of affinity chromatography also increases the risk of affinity ligand falling off, which brings hidden dangers to product quality

Method used

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  • Production method of human antithrombin III
  • Production method of human antithrombin III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1 Fresh frozen human plasma was centrifuged to remove cryoprecipitate, and DEAE Sephadex-A50 gel was adsorbed;

[0036] 1.1 Plasma removal of cryoprecipitate

[0037] Fresh frozen plasma is fed, thawed, and centrifuged to separate cryoprecipitate, and the centrifuged plasma is collected. During the process, the plasma temperature should not exceed 4°C, and the output speed of the centrifuge should not exceed 2L / min.

[0038] 1.2 Adsorption of plasma DEAE Sephadex-A50 gel

[0039] The plasma from which cryoprecipitate was removed was filtered with a 1 μm filter element. Adjust the plasma pH to 7.00; add well-balanced A-50 gel and stir for adsorption for 45 minutes, the adsorption temperature is 4°C; filter out the gel. Equilibrium solution: 0.01mol / L sodium citrate, 0.20mol / L sodium chloride, pH 7.00, 4°C.

[0040] Add 1 times the gel volume of the washing solution, stir and wash twice, filter the washing solution with a 1 μm microporous filter element, and combine i...

Embodiment 2

[0069] plasma depth filtration

[0070] The filter material chooses 3M's 30LA and 60LA combination. Clean the filter material with an aqueous solution containing 5% EDTA at a dosage of 200L / m 2 . Then use 5% sodium citrate solution to clean the filter material, the dosage is 200L / m 2 . Among them, 50L / m is first used 2 Rinse the filter plate with sodium citrate solution, and then use 100L / m 2 Sodium citrate solution to wash out the above solution, and circulate for 30 minutes, and finally use 50L / m 2 Sodium citrate solution to wash out the aforementioned solution. After flushing, drain the solution in the device and perform plasma filtration.

[0071] The filtered plasma was subjected to on-line membrane filtration using a 3M PDA type 0.2 μm double-layer membrane filter element while undergoing column chromatography.

Embodiment 3

[0073] 1 Fresh frozen human plasma was centrifuged to remove cryoprecipitate, and DEAE Sephadex-A50 gel was adsorbed;

[0074] 1.1 Plasma removal of cryoprecipitate

[0075] Fresh frozen plasma is fed, thawed, and centrifuged to separate cryoprecipitate, and the centrifuged plasma is collected. During the process, the plasma temperature should not exceed 4°C, and the output speed of the centrifuge should not exceed 1L / min.

[0076] 1.2 Adsorption of DEAE Sephadex A-50 in plasma

[0077] The plasma from which cryoprecipitate was removed was filtered with a 1 μm filter element. Adjust the plasma pH to 7.15; add well-balanced A-50 gel and stir for adsorption for 60 minutes, the adsorption temperature is 2°C; filter out the gel. Equilibrium solution: 0.01mol / L sodium citrate, 0.20mol / L sodium chloride, pH 7.15, 2°C.

[0078] Add 1 times the gel volume of the washing solution, stir and wash twice, filter the washing solution with a 1 μm microporous filter element, and combine i...

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Abstract

The invention relates to a production method of human antithrombin III. The production method comprises the following steps: removing cryoprecipitate from fresh frozen human plasma to obtain a plasma supernatant, and carrying out adsorption treatment on the plasma supernatant by using a DEAE Sephadex-A50 gel; precipitating impurity protein of the plasma supernatant, and deeply filtering plasma; carrying out Capto Heparin affinity chromatography; carrying out S / D virus inactivation and Capto Q ion exchange chromatography, and removing an S / D reagent; and filtering by virtue of a nanometer film to remove viruses, preparing, freeze-drying and dry-heating. The process is capable of preparing the antithrombin III by virtue of affinity chromatography with only one step. According to the production method disclosed by the invention, the recovery rate can reach above 40 percent, and the specific activity can reach 8-10 IU / mg.

Description

technical field [0001] The invention relates to the technical field of production of biological products and blood products, and mainly relates to a method for separating and purifying human antithrombin III in the production of blood products. Background technique [0002] human antithrombin III alpha 2 Globulin, with a molecular weight between 58,000 and 65,000 Da, is an important anticoagulant substance in the human body, and can exert anticoagulant effect without heparin. The concentration of antithrombin III in the plasma of healthy adults is 100-300 μg / ml. When the level of antithrombin III in the human body is lower than 70% of the normal level, the risk of thrombosis will increase. Human antithrombin III products are suitable for the treatment of patients with antithrombin deficiency, such as congenital and acquired antithrombin deficiency, disseminated intravascular hemorrhage (DIC), sepsis, trauma, tumor, thromboembolism, concurrent sexual pregnancy etc. [0003...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K1/36C07K1/34C07K1/30C07K1/22C07K1/18
CPCC07K14/8128
Inventor 周安朱孟沼席智赢张翠萍马杰陈振陈晨菅长永马山
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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