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A kind of preparation method of high-purity human coagulation factor VIII

A human coagulation factor, high-purity technology, applied in the preparation method of peptide, coagulation/fibrinolysis factor, factor VII, etc., can solve the problems of decreased coagulation effect, low specific activity and low specific activity of FVIII, etc., and improve the safety of use. properties, avoid protein damage, and reduce the effect of denaturation and inactivation

Active Publication Date: 2020-09-11
上海洲跃生物科技有限公司
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AI Technical Summary

Problems solved by technology

Foreign recombinant human coagulation factor VIII has entered the Chinese market in 2007, but its high price discourages ordinary patients, and more and more clinical data show that patients who frequently inject recombinant human coagulation factor VIII will produce inhibitors in their bodies , so that the coagulation effect is significantly reduced
[0004] At present, domestic blood product companies supply a very limited number of clinical FVIII products, which is difficult to meet the use requirements of domestic patients with hemophilia A
The reason is mainly that the traditional FVIII production process is unstable, resulting in low product yield. In addition, due to the high content of impurity protein, the specific activity of FVIII is low, the product is unstable, the appearance is poor, and the thermal stability is not good, which often leads to product scrapping
[0005] The present invention develops a new method for separating and purifying FVIII from cryoprecipitate, optimizes the parameters of the preparation process, especially adopts a two-step chromatographic purification process, and overcomes the low specific activity (generally lower than 50IU / mg), low yield (generally 100,000 IU / ton of plasma) and poor product appearance (the reconstituted solution of freeze-dried powder has precipitates and obvious opalescence), with high specific activity, stable quality, and qualified rate High advantages, can be produced on a large scale

Method used

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  • A kind of preparation method of high-purity human coagulation factor VIII
  • A kind of preparation method of high-purity human coagulation factor VIII

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Cryoprecipitate dissolution: put 1kg cryoprecipitate into 5kg dissolution buffer (0.02M TRIS-HCL, 0.1MNacL, glycine 1.5% (wt%), PH6.50-6.60), add heparin to the dissolution buffer in advance to 2000IU / kg, the temperature is controlled at 15-20°C, and stirred for 3 hours to fully dissolve;

[0030] 2. Al(OH)3 adsorption and pressure filtration: Add 0.6kg of Al(OH)3 gel to the above solution, stir thoroughly for 1 hour, then filter with Supradur 50P filter plate produced by Pall Company in series with 1.0μm filter element, Collect the clarified filtrate, and pre-wash the filter plate and filter element with the dissolution buffer described in step 1 before filtering;

[0031] 3. S / D virus inactivation: Add Tween80 to 1.0% (wt%) and TNBP (tributyl phosphate) to 0.3% (wt%) to the above filtrate, stir well, heat up to 24-26°C, and keep warm for 6 hours, then clarify and filter with a 0.45μm filter element;

[0032] 4. Anion-exchange column chromatography: the filtrate ...

Embodiment 2

[0041] 1. Cryoprecipitate dissolution: Put 1 kg of cryoprecipitate into 10 μL dissolution buffer (0.02M TRIS-HcL, glycine 0.5%, (wt%), 0.1M NaCL, PH7.30-7.40), pre-added to the dissolution buffer Heparin to 10000IU / kg, temperature controlled at 25-30°C, stirred for 1.5 hours to fully dissolve;

[0042] 2, Al(OH)3 adsorption and pressure filtration: add 1.65kg of Al(OH)3 gel to the above solution, stir thoroughly for 1.5 hours, then filter with Supradur 50P filter plate produced by Pall Company in series with 1.0μm filter element, Collect the clarified filtrate, and pre-wash the filter plate and filter element with the dissolution buffer described in step 2 before filtering;

[0043] 3, S / D virus inactivation: with embodiment one;

[0044] 4. Anion-exchange column chromatography: the filtrate after virus inactivation is put on Capto DEAE column, and the column is fully equilibrated with equilibration buffer in advance. ),0.0l5M CaCl 2 , PH7.30-7.40; after loading the column...

Embodiment 3

[0050] 1, Cryoprecipitate dissolution: put 1kg cryoprecipitate into 7kg dissolution buffer (0.02M TRIS-HCL, 0.15MNaCL, glycine 1.0% (wt%), PH6.90-7.10), add heparin to the dissolution buffer in advance to 6000IU / kg, the temperature is controlled at 20-25 ℃, stirred for 2 hours to fully dissolve;

[0051] 2, Al(OH)3 adsorption and pressure filtration: add 0.7kg of Al(OH)3 gel to the above solution, stir thoroughly for 0.5 hours, then filter with Supradur 50P filter plate produced by Pall in series with 1.0μm filter element, Collect the clarified filtrate, and pre-wash the filter plate and filter element with the dissolution buffer described in step 2 before filtering;

[0052] 3, S / D virus inactivation: with embodiment one;

[0053] 4. Anion exchange column chromatography: the filtrate after virus inactivation was put on Q SePharose 4FF column, and the column was fully equilibrated with equilibration buffer in advance. wt%) , 0.01M CacL2 , PH6.90-7.10; after loading the colu...

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Abstract

The invention discloses a method for preparing a high-purity human coagulation factor VIII from a cryoprecipitate of a human plasma fraction. The method comprises the following steps: (1) cryoprecipitate dissolution; (2) aluminum hydroxide gel adsorption and filter pressing; (3) S / D virus inactivation; (4) anion exchange resin column chromatography; (5) hydrophobic column chromatography; (6) ultrafiltration dialysis and concentration; (7) addition of one or more stabilizers and titer adjustment; (8) nano-membrane virus-removing filtration; (9) sterile filtration and sub-packaging; (10) freeze-drying; (11) dry-heat virus inactivation. The method has the advantages that a human coagulation factor VIII is purified through the two column chromatography steps, so that the prepared high-purity product can reach a specific activity of about 300 IU / mg, considerably higher than about 50 IU / mg in the prior art, and the product appearance and the heat stability are obviously improved; in the preparation process, three virus removing modes are adopted, so that the clinical use safety of the product can be greatly improved.

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 preparation method of high-purity human blood coagulation factor VIII. Background technique [0002] Human coagulation factor VIII ( F VIII ) is one of the most important coagulation factors in the human body. It is 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, the two peptide chains are separated by 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 / L. [0003] FVIII deficiency can cause coagulation disorders and lead to hemophilia A. It is a genetic disease of coagulation dysfunction caused by mutations in the gene encoding FVIII leading to functional defects of the coagulation factor. It is an X-linked recessive in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/755C07K1/36C07K1/34C07K1/30C07K1/22C07K1/20C07K1/18
CPCC07K14/755
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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