Preparation method of high-purity human coagulation factor IX

A technology of human blood coagulation factor and blood coagulation factor, which is applied in the field of biomedicine, can solve problems such as high cost, low product safety, and complicated process, and achieve the effects of less loss, reduced probability of thrombin activation, and simple process operation

Inactive Publication Date: 2015-12-23
上海洲跃生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for preparing high-purity human blood coagulation factor IX, which solves the problem of preparing human blood coagulation factor IX in the prior art. The method process of IX is complicated, cost is high, and the technical problem of product safety is not high

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  • Preparation method of high-purity human coagulation factor IX
  • Preparation method of high-purity human coagulation factor IX

Examples

Experimental program
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Effect test

Embodiment 1

[0025] A method for preparing high-purity human coagulation factor IX includes the following steps:

[0026] 1) Take 35 bags of frozen plasma, rinse the surface with 70% ethanol and rinse with cold water for injection, cut the bags, pour them into a stainless steel melting slurry bucket, melt in a 0-3℃ water bath, then accurately weigh 20kg and remove by centrifugation Cryoprecipitate to obtain 19.8 kg of cold-gelatinized plasma, and then heat the cold-gelatinized plasma to 13-15°C in a 15°C water bath;

[0027] 2) Weigh 20 grams of DEAE-Sephadex A-50 gel and place it in a gel bucket, first fully swell it with about 2kg of hot water for injection (about 80°C), and then rinse and cool it with about 2kg of cold water for injection (about 15°C). Then equilibrate with about 1kg buffer (PH6.50, 0.01MNa-citrate, 0.075MNaCL, 15℃), then add it to the cold glue-free plasma, stir slowly for 1 hour, and then stand for 10 minutes;

[0028] 3) Filter the cold glue plasma with filter cloth in the...

Embodiment 2

[0040] A preparation method of high-purity human coagulation factor FIX includes the following steps:

[0041] 1) Same as Example 1;

[0042] 2) The dosage, swelling and cooling of DEAE-Sephadex A-50 gel are the same as those in Example 1, and then equilibrate with about 1kg buffer (PH7.35, 0.01MNa-citrate, 0.15MNaCL, 15℃), and then add to the decold gel In the plasma, stir slowly for 1 hour and then stand for 20 minutes;

[0043] 3) Filter the cold glue plasma with filter cloth in the gel bucket, collect DEAE-SephadexA-50 gel, and then add 2 liters of washing buffer (PH7.35, 0.015MNa-citrate, 0.2MNaCL) to the gel bucket Stir for about 5 minutes, after filtering through a filter cloth, drain the washing solution from the bottom of the bucket, and repeatedly wash the gel 6 times; then wash with elution buffer (PH7.35, 0.015MNa-citrate, 0.02MArginineHydrochloride, 1.0MNaCL) Degelling 4 times, 1 liter each time, collect about 3988 g of the eluate, filter the eluate with a 0.45 μm filt...

Embodiment 3

[0055] A method for preparing high-purity human coagulation factor IX includes the following steps:

[0056] 1) Same as Example 1;

[0057] 2) The dosage, swelling and cooling of DEAE-Sephadex A-50 gel are the same as in Example 1, and then equilibrate with about 1kg buffer (PH7.05, 0.01MNa-citrate, 0.10MNaCL, 15℃), and then add to the cold gel In the plasma, stir slowly for 1 hour and then stand for 20 minutes;

[0058] 3) Filter the cold glue plasma with filter cloth in the gel bucket, collect DEAE-SephadexA-50 gel, and then add 2 liters of washing buffer (PH7.05, 0.01MNa-citrate, 0.15MNaCL) to the gel bucket Stir for about 8 minutes, after filtering through a filter cloth, drain the washing solution from the bottom of the bucket, and wash the gel 5 times repeatedly, and then wash with elution buffer (PH7.05, 0.01MNa-citrate, 0.02MArginineHydrochloride, 0.65MNaCL) Degelling 3 times, 1 liter each time, collect about 2955g of the eluate, filter the eluate with a 0.45μm filter eleme...

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Abstract

The invention relates to a preparation method of a high-purity human coagulation factor IX, which comprises the following steps: melting refrigerated plasma, and carrying out low-temperature centrifugation; adsorbing with a DEAE (diethylaminoethanol) Sephadex A-50 gel to remove the coagulation factor IX in the cold-glue plasma; removing impure proteins in the solution by using polyethyleneglycol; carrying out S / D virus inactivation; carrying out anion exchange column chromatography to obtain a purified coagulation factor IX solution; passing through a heparin affinity column for further chromatography to obtain a high-purity coagulation factor IX solution; carrying out ultrafiltration, dialysis and concentration, and adding arginine hydrochloride and glycinate as protective agents; filtering through a 20nm filter element to remove viruses; carrying out freeze-drying; and carrying out dry heat virus inactivation. The protein protective agents are added during the gel adsorption, column chromatography and ultrafiltration dialysis, thereby lowering the activation probability of the FIX product thrombin and enhancing the qualification rate of the product. The technique has high product yield; the FIX specific activity can reach 150 IU / mg or so which is much higher than that of the traditional product; and by performing the three-step virus inactivation, the product is safe and reliable to use.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and relates to a human coagulation factor IX, specifically a method for preparing high-purity human coagulation factor IX. Background technique [0002] Human coagulation factor IX (FIX) is one of the thirteen types of coagulation factors in the human body and plays a very important role in the coagulation mechanism of the human body. Blood coagulation disorder caused by the lack of coagulation factor IX is hemophilia B. This disease is a genetic disease. The patient is mainly male, and the incidence is about 1 / 30000 of male live infants. It is spontaneous or traumatic. Bleeding is characterized by bleeding, which is mainly in joints, soft tissues and muscles. Delayed or insufficient treatment can cause various complications, including arthropathy and synovitis associated with repeated joint bleeding, which can lead to joint deformities and dysfunction in severe cases. In addition, more severe bleeding, e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/34C07K1/22C07K1/18
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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