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Method for extracting human fibrinogen from component I through column chromatography

A technology of human fibrinogen and column chromatography, which is applied to the preparation methods of fibrinogen and peptides, animal/human proteins, etc., can solve the problems of poor quality stability, low purity, and low yield, and achieve enhanced stability , to ensure stability and improve quality

Active Publication Date: 2011-10-12
BANGHE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Human fibrinogen is a blood product with national standards. It is included in the three volumes of "Chinese Pharmacopoeia" in the 2010 edition. The drug has been used clinically for many years in China, and its production technology is relatively mature. The patent application number is the production method of human fibrinogen of 200910237204.6, but most of the blood product manufacturers at home and abroad basically use the low-temperature ethanol method to extract human fibrinogen from component I of the Cohn6 method with plasma as raw material. The main defect of the method is that the purity of the product is low, generally 70-80%, the appearance is poor, and the quality stability is poor.
It is also reported that human fibrinogen is extracted from the cryoprecipitate. Because the content of fibrinogen in the cryoprecipitate is low, the final yield of the product is not high from FI, and the cryoprecipitate is as the starting material of human blood coagulation factor VIII. For the production of human fibrinogen, it is bound to reduce the production of human coagulation factor VIII, and the yield is low

Method used

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  • Method for extracting human fibrinogen from component I through column chromatography
  • Method for extracting human fibrinogen from component I through column chromatography
  • Method for extracting human fibrinogen from component I through column chromatography

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Experimental program
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Embodiment 1

[0039] The present invention is realized by the following steps in concrete implementation:

[0040] (1) Dissolving and filtering of component I precipitation: use 5 times the volume of the first solution for the precipitation, stir and dissolve at 20°C for 1 hour, clarify and filter the dissolved product, and measure the volume of the product; the first solution is: Each 1000ml aqueous solution for injection contains: 10g trisodium citrate, 9g NaCl, 8g sucrose, 2g Tris, 3g lysine hydrochloride, adjust the pH value to 6.90 with HCl;

[0041](2) S / D inactivation: control the product temperature at 24°C, add 100ml per liter of filtrate, slowly add 11% S / D inactivator with a mass concentration under stirring, and keep the product temperature at 24°C6 Hour;

[0042] (3) The first glycine precipitation: measure the volume of the inactivation solution in step 2, add glycine powder at a concentration of 1.5mol / L, fully dissolve and cool to 2°C, perform continuous centrifugation at 4...

Embodiment 2

[0052] (1) Dissolving and filtering of component I precipitation: use 8 times the volume of the first solution for the precipitation, stir and dissolve at 23°C for 1 hour, clarify and filter the dissolved product, and measure the volume of the product; the first solution is: Each 1000ml aqueous solution for injection contains: 12g trisodium citrate, 9g NaCl, 10g sucrose, 3g Tris, 4g lysine hydrochloride, adjust the pH value to 7 with HCl;

[0053] (2) S / D inactivation: control the product temperature at 25°C, add 100ml per liter of filtrate, slowly add 11% S / D inactivator with a mass concentration under stirring, and keep the product temperature at 25°C6 Hour;

[0054] (3) The first glycine precipitation: measure the volume of inactivation solution in step 2, add glycine powder at a concentration of 1.8mol / L, fully dissolve and cool to 5°C, perform continuous centrifugation at 4200rpm for 40 minutes, and collect at 0°C precipitation;

[0055] (4) Dissolution and filtration o...

Embodiment 3

[0064] (1) Dissolving and filtering of component I precipitation: use 15 times the volume of the first solution for the precipitation, stir and dissolve at 26°C for 1 hour, clarify and filter the dissolved product, and measure the volume of the product; the first solution is: Each 1000ml aqueous solution for injection contains: 14g trisodium citrate, 9g NaCl, 12g sucrose, 4g Tris, 5g lysine hydrochloride, adjust the pH value to 7.10 with HCl;

[0065] (2) S / D inactivation: control the product temperature at 26°C, add 100ml per liter of filtrate, slowly add 11% S / D inactivator with a mass concentration under stirring, and keep the product temperature at 26°C6 Hour;

[0066] (3) The first glycine precipitation: measure the volume of inactivation solution in step 2, add glycine powder at a concentration of 2.2mol / L, fully dissolve and cool to 8°C, perform continuous centrifugation at 4200rpm for 40 minutes, and collect at 0°C precipitation;

[0067] (4) Dissolution and filtrati...

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Abstract

The invention relates to a method for extracting human fibrinogen from a component I through column chromatography. The problem of low purity, high stability and high yield of the human fibrinogen can be effectively solved. The method comprises the following steps of: (1) dissolving and filtering FI precipitate; (2) inactivating S / D ; (3) precipitating glycine for the first time; (4) dissolving and filtering the precipitate obtained in the precipitation for the first time; (5) performing column chromatography; (6) precipitating glycine for the second time; (7) dissolving and filtering the precipitate obtained in the precipitation for the second time; (8) precipitating; (9) packing the product in bags and freeze-drying the product; and (10) inactivating the product through hot air. In the method, glycine serves as a precipitator, so the finally obtained product contains about 0.5 percent of glycine; and the glycine and arginine hydrochloride are combined to serve as a stabilizer of the product, so the stability of freeze-dried product can be improved and the quality, the purity and the yield of the final product are improved. Therefore, the method is an innovation for preparing the human fibrinogen.

Description

1. Technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for extracting human fibrinogen from component I by column chromatography. 2. Background technology [0002] Human fibrinogen, human coagulation factor I, is one of the main components of plasma protein, with a molecular weight of about 340,000 and an isoelectric point of 5.5. It denatures quickly when heated to 57°C. It has a high content in plasma, about 2-4g / L, and is the main determinant of plasma viscosity, and the liver is the main place for the synthesis of fibrin. [0003] Human fibrinogen (Fg) can rapidly increase the content of fibrinogen in the blood. Through the action of thrombin, the fibrinogen is converted into insoluble fibrin, and the formed components such as blood cells in the network are coagulated into blood clots to achieve hemostasis. Purpose. It is used for the treatment of postpartum hemorrhage and coagulation disorders caused by fibrinogen...

Claims

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Application Information

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IPC IPC(8): C07K14/75C07K1/36C07K1/34C07K1/30C07K1/16
Inventor 雷文成时凯刘国荣江景玉皮川真陈惠珍
Owner BANGHE PHARMA CO LTD
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