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Production method of high-purity human coagulation factor IX preparation

A technology of human coagulation factor and production method, which is applied in the field of high-purity human coagulation factor IX preparation production, can solve the problems of large gel filler consumption, high production cost, loss of target protein, etc. Time cost, the effect of reducing the amount of gel

Pending Publication Date: 2019-09-20
广东双林生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But because its production process is loaded down with trivial details, need to go through 4 times of chromatographic steps, as known to those skilled in the art, every time through 1 time of chromatographic purification steps, all will inevitably cause the loss of target protein, therefore, the product yield of this preparation process is relatively high. Low; in addition, 4 chromatographic steps need to consume a large amount of gel filler, the production cost is extremely high, and it is not convenient for large-scale promotion

Method used

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  • Production method of high-purity human coagulation factor IX preparation
  • Production method of high-purity human coagulation factor IX preparation
  • Production method of high-purity human coagulation factor IX preparation

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Removal of plasma cryoprecipitate: using fresh frozen human plasma as raw material, the cryoprecipitate in the plasma is removed through the processes of thawing, mixing, and continuous centrifugation to obtain 2 kg of plasma centrifugation supernatant without cryoprecipitation;

[0037] Gel batch adsorption: The plasma temperature is controlled at 15°C, according to 1.5g dry gel / L plasma, add the balanced DEAE A-50 gel to the cryoprecipitated plasma, stir and absorb for 45min, then turn off the stirring. The plasma after gel adsorption is filtered with a small closed filter, and the adsorbed A-50 gel is collected;

[0038] Column packing and elution: Pack the adsorbed A-50 gel into a small XK16 / 20 chromatographic column, and use the AKTA chromatographic system for online washing of the packed chromatographic column. The flow rate of the washing liquid is 2ml / min, and the washing time is 50min. The washed A-50 gel switches the eluent for online elution, the eluent flow...

Embodiment 2

[0044] Removal of plasma cryoprecipitate: Using fresh frozen human plasma as the raw material, the cryoprecipitate in the plasma is removed through the processes of thawing, mixing, and continuous centrifugation to obtain 4kg of plasma centrifugation supernatant without cryoprecipitation;

[0045] Gel batch adsorption: The plasma temperature is controlled at 10°C. According to 1.0g dry gel / L plasma, add the balanced DEAE A-50 gel to the cryoprecipitated plasma, stir and absorb for 45min, and then turn off the stirring. The plasma after gel adsorption is filtered with a small closed filter, and the adsorbed A-50 gel is collected;

[0046] Column packing and elution: Pack the adsorbed A-50 gel into a small XK16 / 20 chromatographic column, and use the AKTA chromatographic system for online washing of the packed chromatographic column. The flow rate of the washing liquid is 2ml / min, and the washing time is 60min. After washing, the A-50 gel switches the eluent for online elution, ...

Embodiment 3

[0053] A testing laboratory is commissioned to perform SDS-page electrophoresis testing on the finished product of the high-purity human coagulation factor IX preparation prepared in Example 2, so as to determine the distribution of main protein types in the finished product. Detection operation steps:

[0054] (1) Wash and dry the glass plate;

[0055] (2) Fix the glass plate on the glue-filling bracket. When fixing, force evenly on both sides to prevent the glass plate from being pinched;

[0056] (3) Configure 8% separating gel in proportion, quickly add it with a pipette, and add it to about 1cm from the upper edge, then add a little water to seal it overnight, and let it stand for about 40 minutes;

[0057] (4) Pour out the water and dry the remaining water with filter paper, prepare 5% concentrated gel, add the concentrated gel continuously and steadily to a distance of 5mm from the edge, quickly insert the sample comb, and let stand for 30-40min;

[0058] (5) After ad...

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Abstract

The invention discloses a production method of a high-purity human coagulation factor IX preparation, wherein the production method is les sin process step, low in production cost and high specific activity. The method includes the steps: (1) plasma cryoprecipitation removal; (2) DEAE sephadex A50 anion exchange chromatography; (3) S / D inactivated virus; (4) non-Ca2+dependent IX factor immune affinity chromatography; (5) adding of protective agents in matched liquid; (6) virus removal by a nano-filtration membrane; (7) sub-packaging and freeze drying. According to the method, a human coagulation factor IX production process is greatly simplified by the aid of novel high-adsorption specific immune affinity chromatography, the titer of a prepared high-purity human coagulation factor IX finished product can reach 114.2IU / ml, and the specific activity of an IX factor reaches up to 196.5IU / mg protein or more. Two-step virus inactivation is implemented by an S / D method with high maturity and safety and a nano-filtration method, and safety of the finished product is effectively ensured.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals and blood products, and in particular relates to a production method of a high-purity human blood coagulation factor IX preparation with few process steps, low production cost and high specific activity in the production of blood products. Background technique [0002] Human coagulation factor IX (h FIX) is a key coagulation factor, which belongs to vitamin K-dependent glycoprotein, and is the precursor of serine protease in the human endogenous coagulation cascade reaction. It plays a very important role in the blood coagulation pathway. [0003] Hemophilia B is a rare genetic disease with an incidence of about 1 in 25,000. The cause of hemophilia B is the lack of coagulation factor IX. Once the patient bleeds, it takes a very long time for the blood to clot. Some minor injuries can be life-threatening. In severe cases, joints and muscles often bleed spontaneously, causing pain. Th...

Claims

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Application Information

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IPC IPC(8): C12N9/64A61K38/48A61P7/04
CPCC12N9/6424C12Y304/21106A61K38/4846A61P7/04
Inventor 朱光祖罗观文胡川殷如杨波波庾昌文骆燕容
Owner 广东双林生物制药有限公司
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