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Method for adsorbing human prothrombin complex from plasma

A technology of human prothrombin and complex, which is used in the production of biological products and blood products, can solve the problems of gel leakage operation, cross-contamination, contamination, etc., and achieves the effects of high flow rate, avoiding contamination and improving yield.

Active Publication Date: 2014-10-22
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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  • Method for adsorbing human prothrombin complex from plasma
  • Method for adsorbing human prothrombin complex from plasma
  • Method for adsorbing human prothrombin complex from plasma

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: Plasma deep layer filtration

[0027] Select 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 cleaning according to the method, 200ml of plasma was filtered, and coagulation factors II, VII, IX, and X in the feed liquid before and after filtration were detected.

[0031]

[0032] The above results show that after plasma filtration using the filter plate treated with the filter plate balance solution containing sodium citrate, there is no activity of coagulation factors II, VII, IX, and X, which are the main components of the human prothrombin complex. Activate or deactivate. Therefore, the plasma after filtration and clarification can be used for the separation and purification of human prothrombin complex by fixed bed column chromatography.

Embodiment 3

[0033] Embodiment 3: fixed bed column chromatography separates and purifies PCC

[0034] Pack 1 L of 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 for 2 to 5 column volumes. The 30L plasma after deep layer filtration is then pumped into the chromatographic column after 0.2μm online membrane filtration, and the sample loading flow rate is 60~120cm / h. The chromatographic column was then washed with buffer A containing 160 mmol / L NaCl, and the flow-through and washing liquid were collected for separation and purification of other proteins. Finally, the chromatographic column was eluted with buffer A containing 500 mmol / L 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 the specific activity of factor IX in PCC produced by using the traditional DEAESephadex A50 ge...

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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 production of biological products and blood products, and mainly relates to a method for separating and purifying human prothrombin complex by fixed bed column chromatography in the production of blood products. Background technique [0002] Human prothrombin complex (Prothrombin Complex, PCC) is extracted from healthy human plasma, mainly containing plasma protein products of human coagulation factors II, VII, IX, and X. The Chinese Pharmacopoeia requires that the titer of human blood coagulation factor IX in this product is not less than 10IU / ml, and its specific activity is not less than 0.5IU / ml. This product is mainly used for the treatment and prevention of bleeding caused by deficiencies of II, VII, IX, and X factors, such as hemophilia B, liver disease, etc. [0003] PCC is generally produced by using DEAESephadex A50 gel. Because the gel swelling coefficient varies greatly under different salt c...

Claims

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Application Information

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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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