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Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma

A technology to remove cold gelatin plasma and human coagulation factors, which is applied in coagulation/fibrinolytic factors, chemical instruments and methods, animal/human proteins, etc. It can solve the problems of incomplete product safety, single virus inactivation method, and thrombin activation. and other problems, to achieve the effect of reducing the probability of activation, high plasma utilization rate, and simple process

Inactive Publication Date: 2016-02-17
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

Clinical practice shows that the injection of high-purity human coagulation FIX is very safe for hemophilia B patients, but the injection of human prothrombin complex may cause serious side effects-thrombosis, so high-purity human coagulation FIX is the best choice for the treatment of hemophilia B. The drug is the first choice, but the traditional high-purity human blood coagulation FIX production process is complicated. During the preparation of FIX, the precious FVII is removed as an impurity, and the problem of thrombin activation is prone to occur during the production process, resulting in product failure. In addition, the virus inactivation method of the traditional process is single, which cannot fully guarantee the safety of the product, and the dry heat virus inactivation method often leads to a large loss of vitality

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  • Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma
  • Method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma

Examples

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Embodiment 1

[0051] 1. Preparation of decolonized plasma: Take 35 bags of frozen plasma, sterilize with 70% ethanol and rinse with water for injection, cut the bag, melt the frozen plasma in a jacketed tank, weigh 20kg, and centrifuge continuously at 0-3°C , remove the cold glue, collect the supernatant, and filter it with a Supradur50P filter plate produced by Pall in series with a 0.45 μm filter element to obtain clarified cold glue-free plasma; before filtering, use buffer solution (PH6.90-7.10, 0.02M Sodium, 0.1M sodium chloride) pre-wash the filter element;

[0052] 2. The first strong anion exchange column chromatography: put the clarified cold gelatinized plasma obtained in step 1 on the QSepharose4FF column, and the column is pre-filled with equilibrium buffer (PH6.90-7.10, 0.02M sodium citrate, 0.1M sodium chloride ,) equilibration; after loading the column, wash the column with the above equilibration buffer, and then wash with elution buffer (PH6.90-7.10, 0.02M sodium citrate, 0...

Embodiment 2

[0065] 1, the preparation of cold gelatin-free plasma: the same as in Example 1;

[0066] 2. The first strong anion exchange column chromatography: Put the clarified cold gel plasma obtained in step 1 on the Capto-Q column, and the column is pre-filled with an equilibration buffer (PH7.30-7.40, 0.02M sodium citrate, 0.1M chloride NaCl,) equilibrate; after loading the column, wash the column with the above equilibration buffer, and then wash the column with elution buffer (PH7.30-7.40, 0.02M sodium citrate, 0.8M sodium chloride, 0.02M arginine hydrochloride ) to elute the column and collect the eluate (rich in coagulation FVII and FIX);

[0067] 3. PEG precipitation to remove impurities: add 50% PEG solution to the above eluent to make the final PEG concentration to 6%, stir for 0.5 hours, and then filter with a 1.0 μm filter element in series with 0.45 μm to obtain a clear filtrate;

[0068] 4, S / D virus inactivation: with embodiment one;

[0069] 5. The second strong anion ...

Embodiment 3

[0075] 1, the preparation of cold gelatin-free plasma: the same as in Example 1;

[0076] 2. The first strong anion exchange column chromatography: Put the clarified cold gel plasma obtained in step 1 on the Capto-Q column, and the column is pre-filled with equilibration buffer (PH6.50-6.60, 0.02M sodium citrate, 0.15M chloride NaCl,) equilibrate; after loading the column, wash the column with the above equilibration buffer, and then wash the column with elution buffer (PH7.30-7.40, 0.02M sodium citrate, 0.6M sodium chloride, 0.02M arginine hydrochloride ) to elute the column and collect the eluate (rich in coagulation FVII and FIX);

[0077] 3. PEG precipitation to remove impurities: add 50% PEG solution to the above eluent to make the final PEG concentration to 4%, stir for 1 hour, and then filter with a 1.0 μm filter element in series with 0.45 μm to obtain a clear filtrate;

[0078] 4, S / D virus inactivation: with embodiment one;

[0079] 5. The second strong anion colum...

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Abstract

The invention discloses a method for preparing human blood coagulation factors IX and VII subcutaneously from cold-glue-removed blood plasma. The method comprises the following steps: 1, removing cold glue from blood plasma; 2, conducting strong anion-exchange column chromatography the first time; 3, conducting PEG sedimentation for removing impure protein; 4, conducting S / D viral inactivation; 5, conducting strong anion-exchange column chromatography the second time, and obtaining FVII eluent and FIX eluent; 6, conducting weak anion-exchange column chromatography, and concentration for purifying blood coagulation FVII; 7, conducting heparin affinity column chromatography for purifying blood coagulation FIX; 8, conducting ultrafiltration; 9, adding a stabilizing agent, and conducting adjustment; 10, conducting virus-removal filtration through nanofilms; 11, conducting sterilization, filtration and subpackage; 12, conducting freeze-drying; 13, conducting dry-hot viral inactivation. According to the method, PEG sedimentation is adopted for removing the impure protein, the target of preparing high-purity FVII and FIX simultaneously is achieved through combination of an ion-exchange column chromatography technology and an affinity chromatography technology, the process flow is simple, the production cycle is short, a product is subjected to three steps of virus eradicating measures, and use safety is high.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to the preparation of blood products, in particular to a method for simultaneously preparing human blood coagulation factors IX and VII from plasma without cold gelatin. Background technique [0002] Both human coagulation factor VII (FVII) and human coagulation factor IX (FIX) are vitamin K-dependent coagulation factors synthesized by the liver. The former is composed of 406 amino acid residues, with a molecular weight of about 50KD, an isoelectric point of 4.9, and a plasma concentration of 0.5 -2mg / L, the latter is composed of 416 amino acid residues, the molecular weight is about 57KD, and the concentration in plasma is about 5mg / L. Both coagulation factors play very important roles in the coagulation process. Among them, FVII is the initiating factor of the body's exogenous blood coagulation pathway. Whether it is primary or secondary FVII deficiency, it may cause bleeding and e...

Claims

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Application Information

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IPC IPC(8): C07K14/745C07K1/36C07K1/30C07K1/34C07K1/18
CPCC07K14/745
Inventor 李春洲
Owner 上海洲跃生物科技有限公司
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