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Method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen

A technology of human fibrinogen and human coagulation factor, applied in the direction of fibrinogen, coagulation/fibrinolytic factor, VII factor, etc., can solve the problems of FVIII white loss, human fibrinogen throwing away, etc., to reduce loss and reduce The effect of increasing labor intensity and yield

Inactive Publication Date: 2016-02-10
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

[0008] The traditional production process mostly uses cryoprecipitate to produce human coagulation factor VIII and component I to produce human fibrinogen. The disadvantage of this process is that the human fibrinogen contained in cryoprecipitate (accounting for about 20% of plasma Fg ) was thrown away as production waste; likewise, a large amount of FVIII contained in fraction I was lost without recycling

Method used

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  • Method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen
  • Method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen

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Experimental program
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Effect test

Embodiment 1

[0058] 1. Prepare cryoprecipitate and component I precipitation according to the method described in step 1 of the process flow, weigh 0.5kg each of cryoprecipitate and component I precipitation, and put them into 10kg of pre-prepared dissolution buffer 1 together, and dissolution buffer 1 is composed of 0.02MTRIS-HCL, 0.06M sodium citrate, 0.02M lysine hydrochloride, 0.15MNaCL, PH6.90-7.10; heparin sodium was pre-added to 6000IU / kg in the dissolution buffer 1; the temperature was controlled at 25-30 ℃, stir for 2 hours to fully dissolve; then filter with a Supradur50P filter plate produced by Pall in series with a 0.45 μm filter element to collect the filtrate; pre-wash the filter plate and filter element with dissolution buffer 1 before filtering;

[0059] 2. Add DEAESephadexA-50 gel which was pre-swelled, cooled and balanced with the dissolution buffer 1 described in the previous step to the above filtrate. The amount of the gel added is 5.5g based on dry gel; stir slowly fo...

Embodiment 2

[0064] 1, with embodiment one;

[0065] 2. Add DEAESephadexA-50 gel that was pre-swelled, cooled and balanced with the dissolution buffer 1 described in the previous step to the above filtrate. The amount of the gel added is 11g based on dry gel, pH6.90-7.10; slowly stir for at least 45 minutes , after standing for 30 minutes; then filter with filter cloth and collect the filtrate;

[0066] 3, with embodiment one;

[0067] 4. Put the filtrate after virus inactivation above on the CaptoDEAE column. The column is fully equilibrated with equilibration buffer 1 in advance. The composition of equilibration buffer 1 is 0.02MTRIS-HCL, 0.06M sodium citrate, 0.02M lysine hydrochloride, 0.1 MNaCl, 0.01M CaCl 2 PH7.40-7.50); ​​use a stainless steel tank with a refrigerant jacket to collect the flow-through liquid, after loading the column, rinse the column with equilibration buffer 1, and merge the washing liquid into the flow-through liquid; then use washing buffer 1 (0.02MTRIS- HCl,...

Embodiment 3

[0070] 1~3 are with embodiment one;

[0071] 4. The above virus inactivated filtrate is put on the QSepharose4FF column. The column is fully equilibrated with equilibrium buffer 1 in advance. The composition of equilibrium buffer 1 is 0.02MTRIS-HCL, 0.05M sodium citrate, 0.04M lysine hydrochloride, 0.15 MNaCl, 0.015MCaCl 2 PH6.60-6.80); use a stainless steel tank with a refrigerant jacket to collect the flow-through liquid; after loading the column, wash the column with equilibration buffer 1, and merge the washing liquid into the flow-through liquid; then use washing buffer 1 (0.02MTRIS- HCl, 0.2M NaCl, 0.01M CaCl 2 , PH6.60-6.80) to wash the column, then use elution buffer 1 (0.02MTRIS-HCL, 1.0MNaCL, 0.003MCaCL 2 , pH6.60-6.80) to elute, filter with a 0.45μm filter element in time, and collect the filtrate; add NaCL to 1.8M, put PhenylSepharose6FF (LS), and the column is fully equilibrated with equilibrium buffer 2 in advance, and the composition of equilibrium buffer 2 is...

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Abstract

The invention discloses a method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen by cryoprecipitate and component I precipitation, mixing and feeding. The method comprises the following steps: (1) simultaneous feeding and dissolution of a cryoprecipitate and a component I; (2) DEAE Sephadex A-50 gel adsorption; (3) S / D virus inactivation; (4) anion exchange column chromatography; (5) two-step low-temperature ethanol precipitation and purification, sterile filtration, subpackage, freeze-drying and dry heat virus inactivation of a chromatographic penetration liquid to obtain a human fibrinogen; (6) further hydrophobic column chromatography of a chromatographic eluant; (7) ultrafiltration, nanofilm filtration, sterile filtration, subpackage, freeze-drying and dry heat virus inactivation of a hydrophobic eluant to obtain a high-purity human coagulation factor VIII. By the adoption of the process, FVIII and Fg in the two raw materials are extracted simultaneously, so that the yields of the two products are greatly improved, the yield of the human coagulation factor VIII can reach 200,000 IU / ton plasmas, the yield of the human fibrinogen exceeds 2,000 bottles / ton plasmas, and the yields are both far higher than those of a traditional process.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to the preparation of a blood product, in particular to a method for simultaneously preparing high-purity human coagulation factor VIII and human fibrinogen. Background technique [0002] Human blood coagulation factor VIII (FVIII) is one of the human blood coagulation factors synthesized in the liver. It has 2332 amino acid residues and is composed of two peptide chains. The molecular weight of the heavy chain is 90-200kDa; the molecular weight of the light chain is 80kDa. Ca 2+ connect. The half-life in the human body is 8-12 hours, and the content in plasma is only 0.05-0.1 mg / liter. FVIII deficiency can lead to the occurrence of hemophilia A, which is a genetic disease. Most patients are male, with an incidence of about 1 / 10000~1 / 5000. The bleeding sites of patients are concentrated in joints, muscles and internal organs. Among them, repeated joint bleeding can lead to bone def...

Claims

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Application Information

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IPC IPC(8): C07K14/755C07K14/75C07K1/36C07K1/34C07K1/30C07K1/18
CPCC07K14/755C07K14/75
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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