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A method for adsorbing human prothrombin complex from plasma

A technology of human prothrombin and complexes, which is applied in the production of biological products and blood products, can solve the problems of gel leakage operation, cross-contamination, pollution, etc., achieve high flow rate, avoid pollution, and improve the yield

Active Publication Date: 2017-02-01
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, under the premise of ensuring that the plasma coagulation factors are not activated, large-scale deep filtration and fine filtration of plasma are realized, and gels suitable for adsorption and separation and purification of PCC are screened out and directly used in fixed-bed chromatography columns. Chromatography technology directly adsorbs and purifies PCC from plasma, which completely solves the problems of pollution, cross-contamination, gel leakage and complicated operation caused by traditional batch adsorption process

Method used

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  • A method for adsorbing human prothrombin complex from plasma
  • A method for adsorbing human prothrombin complex from plasma
  • A method for adsorbing human prothrombin complex from plasma

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Deep plasma filtration

[0027] Choose different filter plates and corresponding fixtures for assembly. Clean according to the cleaning method in the table below, and place the filtered plasma at 2~8°C for 24 hours to observe the coagulation of the plasma.

[0028]

Embodiment 2

[0029] Example 2: Verification of the recovery rate of human coagulation factor activity in plasma after filtration

[0030] Use 3M’s 90SP (14cm 2 )According to 10%EDTA 300L / m 2 , 10% sodium citrate 300L / m 2 After the method is cleaned, 200ml of plasma is filtered, and the coagulation factors II, VII, IX, X in the liquid before and after filtration are detected.

[0031]

[0032] The above results show that the use of filter plates treated with a filter plate balance solution containing sodium citrate components after plasma filtration did not cause the activity of the main components of the human prothrombin complex to coagulation factors II, VII, IX, and X. Activation or deactivation. Therefore, the filtered and clarified plasma can be used for the separation and purification of human prothrombin complex by fixed bed column chromatography.

Embodiment 3

[0033] Example 3: Separation and purification of PCC by fixed bed column chromatography

[0034] Load 1L Capto DEAE gel into a fixed-bed chromatography column, and use buffer A (25mmol / L sodium citrate, 20mmol / L arginine hydrochloride buffer, pH7.0) to equilibrate 2~5 column volumes. After deep filtration, 30L of plasma is filtered through a 0.2μm in-line membrane and pumped into the chromatography column. The sample flow rate is 60~120cm / h. Then wash the column with buffer A containing 160 mmol / L NaCl, and collect the flow-through and washing solution for the separation and purification of other proteins. Finally, the column was eluted with buffer A containing 500 mmol / L of NaCl to obtain PCC products.

[0035]

[0036] Among them, the specific activity of factor IX can reach more than 5.5IU / mg, which is more than 10 times that of the PCC produced by the traditional DEAESephadex A50 gel batch adsorption process.

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Abstract

The invention relates to a production method for adsorbing a human complex from plasma by a fixed bed column chromatography technique, which comprises the following steps: (1) cryoprecipitation plasma removal: filtering by using a cellulose deep filter plate which is cleaned by an EDTA (ethylene diamine tetraacetic acid) solution and a sodium citrate solution; (2) filtering the plasma subjected to deep filtration through a 0.2 mu m filter element membrane while fixed bed loading; (3) balancing 2-5 column volumes in a fixed bed chromatographic column filled with anion exchange gel Capto DEAE by using a buffer solution A at the plasma loading flow rate of 60-120 cm / hour, washing the chromatographic column with a buffer solution B, and eluting the chromatographic column with a buffer solution C to obtain a PCC (prothrombin complex concentrate) product. When the calculation is based on coagulation factor IX, the yield of the PCC can reach 75-90%, and the specific activity can reach 5.5 IU / mg above.

Description

Technical field [0001] The invention relates to the technical field of biological products and blood product production, and mainly relates to a fixed bed column chromatography separation and purification method of human prothrombin complex in the production of blood products. Background technique [0002] Human Prothrombin Complex (PCC) is extracted from healthy human plasma and mainly contains plasma protein products of human coagulation factors II, VII, IX, and X. The Chinese Pharmacopoeia requires that the titer of human coagulation factor IX in the product is not less than 10IU / ml, and its specific activity is not less than 0.5IU / ml. This product is mainly used to treat and prevent bleeding caused by the deficiency of factor II, VII, IX, X, such as hemophilia B, liver disease, etc. [0003] PCC is generally produced by DEAESephadex A50 gel. Because the swelling coefficient of the gel varies greatly under different salt concentrations, blood product companies at home and abroa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/745C07K1/36C07K1/34C07K1/18
CPCC12N9/6429C12N9/6432C12N9/6437C12N9/644C12Y304/21005C12Y304/21006C12Y304/21021C12Y304/21022
Inventor 马山张翠萍朱孟沼周安马杰董雪吴菲菲陈振陈晨席智赢菅长永
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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