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Method for preparing human prothrombin complex

A technology for human prothrombin and complexes, which is applied in the field of preparing human prothrombin complexes, can solve the problems of low FIX recovery rate, great influence of active ingredients, loss of activity recovery rate and the like, and achieves good application prospects, low cost, Less effect of non-specific adsorption

Pending Publication Date: 2016-08-03
CHENGDU RONGSHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Human prothrombin complex is a plasma product isolated from healthy human plasma. Appropriate reaction conditions and the use of protective agents have a significant impact on the yield of FIX in the process, but the current method for preparing human prothrombin complex has FIX Low recovery rate and great impact on other active ingredients in plasma
[0004] For example, Wei Shu et al., "Research on the Production Process of Freeze-dried Human Prothrombin Complex", Chinese Journal of Blood Transfusion, 2008, 21(4): 282-284 discloses a production process of human prothrombin complex. When the S / D virus is inactivated without taking a suitable protective agent, the activity recovery rates of blood coagulation factors II, VII, IX and X are respectively lost by about 20%.
[0005] The patent application with publication number 102151289B discloses a method for preparing human prothrombin complex, the FIX recovery rate is only 450,000 to 490,000 IU / ton of plasma, and no non-specific reduction of human serum albumin and immunoglobulin is taken. Adsorption step, resulting in waste of precious plasma resources
[0006] The patent application whose publication number is 104109202A also discloses a method for preparing human prothrombin complex, which has a high recovery rate of FIX, but the multi-step clarification and filtration in the process increases the difficulty of operation and production cost, and according to the column bed volume and The ratio of the amount of plasma to be loaded is calculated as 1:10-1:50. To realize the processing of 1 ton of plasma / batch, the gel volume used is 20-100L. Calculated according to the market price of CaptoDEAE, the gel cost is 300-1500 RMB 10,000, far higher than the value of human prothrombin complex per ton of plasma, therefore, the cost is too high and the practical application value is not great

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 The preparation method of human prothrombin complex of the present invention

[0036] 1. Preparation method

[0037] (1) Pre-purification

[0038] Thaw the raw plasma, centrifuge to remove the cryoprecipitate, heat the cold supernatant plasma to 10°C, and adjust the pH to 7.0 with acetic acid buffer. Add the swollen gel according to the ratio of 1.4g A50 gel dry powder / L cold supernatant plasma, and the adsorption time is 45min. Collect the gel and treat the gel sequentially with equilibration, wash, and elution buffers. After the collected eluate is clarified and filtered, it is then subjected to ultrafiltration and dialysis, and the volume of the product is measured after completion.

[0039] (2) S / D virus inactivation

[0040] After adding heparin sodium at a ratio of 1 IU / ml to the product, add S / D stock solution according to the volume of the product, so that the final concentrations of Tween-80 and TNBP are 1% and 0.3%, respectively. Start timing...

Embodiment 2

[0062] Embodiment 2 The preparation method of human prothrombin complex of the present invention

[0063] 1. Preparation method

[0064] ⑴ Pre-purification

[0065] Thaw the raw plasma, centrifuge to remove the cryoprecipitate, heat the cold supernatant plasma to 13°C, and adjust the pH to 7.2 with acetic acid buffer. Add swelling gel according to the ratio of 1.5g A50 gel dry powder / L cold supernatant plasma, and the adsorption time is 50min. Collect the gel and treat the gel sequentially with equilibration, wash, and elution buffers. After the collected eluate is clarified and filtered, it is then subjected to ultrafiltration and dialysis, and the volume of the product is measured after completion.

[0066] ⑵S / D virus inactivation

[0067] After adding heparin sodium at a ratio of 2 IU / ml to the product, add S / D stock solution so that the final concentrations of Tween~80 and TNBP are 1% and 0.3%, respectively. Start timing when the temperature of the product reaches 24°...

Embodiment 3

[0090] Embodiment 3 The preparation method of human prothrombin complex of the present invention

[0091] 1. Preparation method

[0092] ⑴ Pre-purification

[0093] The raw plasma was melted, and the cryoprecipitate was removed by centrifugation. The temperature of the cold supernatant plasma was raised to 15°C, and the pH was adjusted to 7.4 with acetic acid buffer. Add swelling gel according to the ratio of 1.6g A50 gel dry powder / L cold supernatant plasma, and the adsorption time is 60min. Collect the gel and treat the gel sequentially with equilibration, wash, and elution buffers. After the collected eluate is clarified and filtered, it is then subjected to ultrafiltration and dialysis, and the volume of the product is measured after completion.

[0094] ⑵S / D virus inactivation

[0095] After adding heparin sodium at a ratio of 3 IU / ml to the product, add S / D stock solution so that the final concentrations of Tween~80 and TNBP are 1% and 0.3%, respectively. Start timi...

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Abstract

The invention discloses a method for preparing a human prothrombin complex .The preparation method comprises the following steps of 1 prepurifying; 2 S / D virus inactivating; 3 refining and purifying; 4 preparing, bacterium removing, subpackaging, freeze-drying and dry-heat treating .According to the method, the FIX recycling rate reaches up to about 580,000-670,000 IU per ton of plasma, and nonspecific adsorption on human albumin and immune globulin is little, so that precious plasma resources cannot be wasted; in addition, high-cost methods such as column chromatography do not need to be adopted, therefore, the cost is low, and the application prospect is good.

Description

technical field [0001] The invention belongs to the field of blood products, in particular to a method for preparing human prothrombin complex. Background technique [0002] Human prothrombin complex is mainly used for the treatment of congenital and acquired coagulation factor II, VII, IX, and X deficiency (individual or combined deficiency), including: 1. Coagulation factor IX deficiency (hemophilia B), And Ⅱ, Ⅶ, Ⅹ coagulation factor deficiency; 2. Anticoagulant overdose, vitamin K deficiency; 3. Hemorrhage patients caused by liver disease need to correct coagulation dysfunction; 4. Prolongation of prothrombin time caused by various reasons And it is intended for surgical patients, but it may not be effective for those with coagulation factor V deficiency; 5. Treatment of bleeding symptoms in patients with hemophilia A who have produced factor Ⅷ inhibitors; 6. Reversal of bleeding induced by coumarin anticoagulants. [0003] Human prothrombin complex is a plasma product i...

Claims

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Application Information

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IPC IPC(8): C12N9/64C07K14/75
CPCC12N9/6437C07K14/75C12N9/6432C12N9/644C12Y304/21006C12Y304/21021C12Y304/21022
Inventor 余伟鲁涛牟蕾李伟王黔川
Owner CHENGDU RONGSHENG PHARMA