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Method for preparing freeze-dried human blood coagulation factor VIII

A human coagulation factor and freeze-drying technology, which is applied in the preparation method of peptides, coagulation/fibrinolytic factors, factor VII, etc., can solve the problems of no medicine available, poor product appearance, severe opalescence, etc., and improve the safety of use Sexuality, avoiding denaturation and inactivation, and the effect of clarifying the reconstituted solution

Inactive Publication Date: 2016-02-03
上海洲跃生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hemophilia A is a hereditary disease, and there is no cure at present. The only alternative treatment is the infusion of FVIII preparations. However, as long as the FVIII preparations are infused regularly, patients with hemophilia A can live like normal people. , will not affect work and study
[0004] At present, the number of FVIII products supplied by domestic blood product companies to the market is very limited, which cannot meet the use requirements of domestic patients with hemophilia A, and there are often cases where there is no drug available, which seriously endangers the health and life safety of patients
At present, the yield of FVIII preparation process of domestic blood product manufacturers is generally low, generally about 100,000 IU / ton of plasma (FVIII content is generally 700,000-800,000 IU / ton of plasma), and the product purity is not high, and the specific activity is generally not higher than 50IU / mg, in addition, the appearance of the product is not good, the freeze-dried powder does not form a cake, the opalescence is serious after reconstitution, and some have protein precipitates
[0005] In view of this, the present invention has developed a new process for preparing freeze-dried human blood coagulation factor VIII from cryoprecipitate, which overcomes the shortcomings of low yield, low specific activity and poor product appearance in the current production process. The advantages of simple process, high purity and high yield can be produced on a large scale

Method used

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  • Method for preparing freeze-dried human blood coagulation factor VIII
  • Method for preparing freeze-dried human blood coagulation factor VIII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Dissolving and filtering cryoprecipitate: put 1kg cryoprecipitate into 9kg dissolution buffer (0.02MTRIS-HCL, 0.15MNaCL, pH6.50-6.60), add heparin to the dissolution buffer to 2000IU / kg in advance, and control the temperature at 15 Stir at -20°C for 4 hours to make it fully dissolve, then filter with a Supradur50P filter plate produced by PALL in series with a 0.45 μm filter element, wash the filter element afterward, and finally collect 10.8 kg of clarified filtrate, pre-wash the filter plate and filter element;

[0028] 2. DEAEsephadexA-50 gel adsorption: Add 10.8g of DEAEsephadexA-50 gel to the above filtrate, and use hot water for injection above 70°C to swell and equilibrate the buffer before adding the gel (0.02MTRIS-HCL, 0.15MNaCL, PH6.50 -6.60) Pre-balanced, fully stirred for 1.5 hours after adding, then filtered with a filter cloth, collected the filtrate, and reused after the gel was regenerated;

[0029] 3. S / D virus inactivation: add Tween80 to 1.0% (wt%...

Embodiment 2

[0037] 1. Dissolving and filtering cryoprecipitate: put 1kg cryoprecipitate into 19kg dissolving buffer (0.02MTRIS-HcL, 0.075MNaCL, pH7.40-7.50), add heparin to the dissolving buffer in advance to 10000IU / kg, and control the temperature at 25 Stir at -30°C for 2 hours to make it fully dissolve, then filter with a Supradur50P filter plate produced by PALL in series with a 0.45 μm filter element, wash the filter element afterward, and finally collect 21.2 kg of clarified filtrate, pre-wash the filter plate and filter element;

[0038] 2. DEAESephadexA-50 gel adsorption: Add 10.6g of DEAESephadexA-50 gel to the above filtrate, and use hot water for injection above 70°C to swell and equilibrate the buffer before adding the gel (0.02MTRIS-HCL, 0.075MNaCL, PH7.40 -7.50) pre-balanced, fully stirred for 1 hour after adding, then filtered with a filter cloth, collected the filtrate, and reused after the gel was regenerated;

[0039] 3, S / D virus inactivation: with embodiment one;

[...

Embodiment 3

[0045] 1. Cryoprecipitate dissolution: put 1kg cryoprecipitate into 14kg dissolution buffer (0.02MTRIS-HcL, 0.1MNacL, pH6.90-7.10), add heparin to the dissolution buffer in advance to 6000IU / kg, and control the temperature at 20-25 ℃, stirring for 3 hours to make it fully dissolved; then filter with a Supradur50P filter plate produced by PALL in series with a 0.45 μm filter element, wash the filter element afterward, and finally collect 15.9 kg of clarified filtrate, and pre-wash the filter plate and filter element with dissolution buffer before filtration;

[0046] 2. DEAESephadexA-50 gel adsorption: Add 12g of DEAESephadexA-50 gel to the above filtrate, and use hot water for injection above 70°C to swell and equilibrate the buffer before adding the gel (0.02MTRIS-HCL, 0.1MNaCL, PH6.90- 7.10) Balance in advance, stir thoroughly for 45 minutes after adding, then filter with filter cloth, collect the filtrate, regenerate the gel and reuse it;

[0047] 3, S / D virus inactivation:...

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Abstract

The invention discloses a method for preparing a freeze-dried human blood coagulation factor VIII through cryoprecipitation. The method comprises the following steps of (1) dissolving and carrying out filter pressing in cryoprecipitation; (2) deactivating S / D viruses; (3) removing a blood coagulation factor depended by vitamin K by using DEAE SephadexA-50 gel; (4) purifying FVIII through carrying out column chromatography on anion exchange resin; (5) carrying out ultrafiltration and dialysis, and concentrating an eluent; (6) adding a stabilizing agent in a concentrated solution, and regulating the titer and PH value of FVIII; (7) removing viruses on a nanofilm, and filtering; (8) degerming, filtering and subpackaging; (9) carrying out freeze drying; and (10) carrying out dry heat deactivation on the viruses. According to the method disclosed by the invention, the blood coagulation factor depended by vitamin K is removed through adsorption of the DEAE SephadexA-50 gel, the traditional aluminum hydroxide and PEG sedimentation way is replaced, and therefore, the method has the advantages of production stability, high yield and good quality; and the safety of the product is greatly improved due to the adoption of three-step virus removing measures.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a preparation method of blood products, in particular to a preparation method of freeze-dried human blood coagulation factor VIII. Background technique [0002] Human coagulation factor VIII (FVIII) 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 lead to the occurrence of hemophilia A, which is a hereditary disease, most of the patients are male, and the incidence rate is about 1 / 10000-1 / 5000. About 400,000 people, and the number of hemophiliacs in China is about 100,000. At p...

Claims

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Application Information

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