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Method for preparing FEIBA (factor eight inhibitor bypassing activity) from human plasma Cohn component III serving as raw material

A technology of human plasma and raw materials, applied to the preparation method of peptides, chemical instruments and methods, thrombomodulin, etc.

Active Publication Date: 2016-03-30
SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although some researchers have claimed that Cohn components I+II+III, I, IIII, III-0, IV-1 and IV-4, which are rich in vitamin K-dependent proteins, may be used as raw materials for the preparation of FEIBA, but currently only There are reports on the successful preparation of FEIBA from the supernatant of fraction I and IV-1

Method used

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  • Method for preparing FEIBA (factor eight inhibitor bypassing activity) from human plasma Cohn component III serving as raw material
  • Method for preparing FEIBA (factor eight inhibitor bypassing activity) from human plasma Cohn component III serving as raw material
  • Method for preparing FEIBA (factor eight inhibitor bypassing activity) from human plasma Cohn component III serving as raw material

Examples

Experimental program
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Effect test

Embodiment 1

[0076] Preparation of FEIBA Forming Raw Materials from Component III

[0077] 1. Weigh 3 kg of the plasma Cohn fraction III precipitate, and put it into 12 kg of reconstitution buffer at a ratio of 1:4 (the buffer is 0.01M sodium citrate + 0.1M sodium chloride aqueous solution, pH7.5 ). Stir at a temperature of 25±1°C, adjust the pH to 7.0-7.5 with 1.0M aqueous sodium bicarbonate solution, and continue stirring until the precipitate is completely dissolved.

[0078] 2. Add PEG4000 aqueous solution (40wt%) to the above component III complex solution to a final concentration of 5%, and lower the temperature to 1-4°C, and continue stirring for 30 minutes. At this time, the pH of the solution is about 7.0-7.4.

[0079] 3. The complex solution of component III is centrifuged at low temperature to separate the precipitate and supernatant. Centrifuge at a speed of about 10000g, and centrifuge at a temperature of 1-4°C for 10 minutes. The supernatant after centrifugation was collec...

Embodiment 2

[0085] 1. Immerse the A50 gel that has been treated with acid and alkali and sterilized at 121°C in an aqueous solution of pH 7.5, 0.075M sodium chloride + 0.1M sodium citrate to balance. Measure the well-balanced A50 gel in a ratio of 1g / L, and put it into the 2Kg feed solution prepared in Example 1.

[0086] 2. After stirring and adsorbing for 1 hour, collect the gel by filtration, wash with pH 7.5, 0.1M sodium chloride + 0.01M sodium citrate aqueous solution for three consecutive times, and discard the filtrate. Finally, it was eluted with pH 7.5, 1M sodium chloride+0.01M sodium citrate aqueous solution, and the eluate was collected. This step operation is carried out at room temperature.

[0087] 3. Take 40ml of the eluate, use a 10KD ultrafiltration centrifuge tube, carry out ultrafiltration and concentration at 5000g, 4°C, and replace the liquid to 9.3ml. The replacement buffer is 0.05MTris-Hcl, 0.15MNaCl, pH7.0, A280 is 54.8.

[0088] 4. Put the concentrated solution...

Embodiment 3

[0093] 1. Take the 2kg S / D feed solution prepared in Example 1, put the pretreated A50 gel into it at a ratio of 1g / L, stir at room temperature for 1 hour, and collect the gel by filtration.

[0094] 2. Use 0.1mol / LNH at pH 7.6 for the gel in step 1 4 HCO 3 Wash about 200ml with aqueous solution, 0.2mol / LNH at pH7.6 4 HCO 3 Wash about 150 ml with aqueous solution, and about 200 ml with 0.15 mol / L NaCl aqueous solution, and discard the filtrate. Finally, the protein adsorbed by the gel was eluted with 1 mol / L NaCl aqueous solution, and about 150 ml of the eluate was collected.

[0095] 3. Under the condition of 4°C, use the replacement buffer solution (pH7.0, 0.05M Tris-HCl+0.15MNaCl aqueous solution) to carry out ultrafiltration concentration and exchange, and the ultrafiltration membrane pore size is 10KD. The final 150ml eluate was concentrated to about 30ml.

[0096] 4. Take a part of the concentrated solution and place it in an environment of 12°C. After incubating fo...

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Abstract

The invention relates to the pharmacy field of blood products, in particular to a method for preparing FEIBA (factor eight inhibitor bypassing activity) from a human plasma Cohn component III serving as a raw material. The method for preparing FEIBA from the human plasma Cohn component III serving as the raw material comprises steps as follows: (1), dissolution of the component III; (2), addition of a precipitator and centrifugalization; (3), inactivation of viruses; (4), gel adsorption; (5), washing and elution of gel; (6), condensation and solution replacement; (7) generation of FEIBA.

Description

technical field [0001] The invention relates to the pharmaceutical field of blood products, in particular to a preparation method of FEIBA (FactorEightinhibitorbypassingactivity) using human plasma Cohn component III as raw material. The preparation method uses human plasma Cohn method ethanol fraction III as a raw material to prepare the drug FEIBA obtained with anticoagulant factor inhibitors. Background technique [0002] The lack of coagulation factors is the root cause of hemostatic disorders in hemophiliacs. After the body's endogenous coagulation pathway is activated, the activated coagulation factor nine (FIXa) combines with its cofactor coagulation factor eight (FVIII), which makes the activity of FIXa catalyzing coagulation factor ten (FX) enhanced by about 100,000 to 1,000,000 times. It is one of the key steps that the blood coagulation system produces a large amount of thrombin (FIIa) to quickly achieve the purpose of hemostasis. Due to genetic defects in hemop...

Claims

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Application Information

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IPC IPC(8): C07K14/745C07K1/36C07K1/34C07K1/30C07K1/18
CPCC07K14/7455
Inventor 周雪峰徐俊黄敏易佳伟程露陆晖
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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