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Method for preparing human fibrinogen by bilayer chromatography

A human fibrinogen and chromatography technology, which is applied in the field of biopharmaceuticals to achieve the effects of short process flow, high yield and small clinical side effects

Pending Publication Date: 2018-01-05
NANYUE BIOPHARMING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Preparation of Human Fibrinogen by Double Chromatography

Method used

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  • Method for preparing human fibrinogen by bilayer chromatography
  • Method for preparing human fibrinogen by bilayer chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Precipitation and dissolution of component Ⅰ: Weigh 2.0kg of the precipitate of component Ⅰ, cut it into small pieces with a knife and crush it, add 20kg of dissolving solution, stir in a water bath at 20-26°C for 1.5 hours until completely dissolved, filter with a plate and frame (filter plate The pore size is 0.7μm~1.0μm), and 20.2kg of the filtrate is collected. The solution is: every 1000ml of water for injection contains 12g of sodium citrate, 8.5g of sodium chloride, 4.5g of lysine hydrochloride, 3000IU of heparin sodium, adjusted to pH 6.9 with 1M HCl, and the temperature is 23°C.

[0026] (2) S / D virus inactivation: add S / D solution 2.0L in step (1) filtrate, make polysorbate 80 content be 1.0% in the mixed solution, tributyl phosphate content be 0.3%, in special S Carry out S / D inactivation in the / D inactivation tank, set the temperature at 24.5°C, stir at 100rmp, start timing when the temperature reaches 24.0°C and keep constant, and keep the temperature ...

Embodiment 2

[0035] (1) Precipitation and dissolution of component Ⅰ: Weigh 2.0kg of the precipitate of component Ⅰ, cut it into small pieces with a knife and crush it, add 30kg of dissolving solution, stir in a water bath at 20-26°C for 1.5 hours until completely dissolved, filter with a plate and frame (filter plate The pore size is 0.7μm~1.0μm), and 30.5kg of the filtrate is collected. The solution is as follows: every 1000ml of water for injection contains 12g of sodium citrate, 10g of sodium chloride, 6g of lysine hydrochloride, and 5000IU of heparin sodium. The pH is adjusted to 6.9-7.1 with 1M HCl, and the temperature is 20-26°C.

[0036](2) S / D virus inactivation: add S / D solution 3.05L in step (1) filtrate, make polysorbate 80 content be 1.0% in the mixed solution, tributyl phosphate content be 0.3%, in special S Carry out S / D inactivation in the / D inactivation tank, set the temperature at 24.5°C, stir at 100rmp, start timing when the temperature reaches 24.0°C and keep constant,...

Embodiment 3

[0044] Embodiment 3: comparative example, traditional low temperature ethanol method, concrete preparation process is as follows:

[0045] (1) Primary suspension and filtration of component I: Weigh 2.0 kg of component I precipitate, cut into small pieces with a knife and crush, add 20 kg of primary suspension, stir in a water bath at 24°C to 26°C for 1.5 hours until completely dissolved, and plate Filtration (filter plate pore size 0.7 μm ~ 1.0 μm). The primary suspension is: 1000ml of water for injection contains 12g of sodium citrate, 8.5g of sodium chloride, 2.7g of Tris, 11g of sucrose, 4.4g of lysine hydrochloride, and the pH is adjusted to 6.7-7.0 with 1M HCl.

[0046] (2) S / D virus inactivation: add S / D solution by 1 / 10 of step (1) gained filtrate weight, make polysorbate 80 content be 1.0% in the mixed solution, tributyl phosphate content be 0.3%, Carry out S / D inactivation in a special S / D inactivation tank, set the temperature at 24.5°C, stir at 100rmp, start timin...

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Abstract

The invention discloses a method for preparing human fibrinogen by bilayer chromatography. The method comprises the following steps: precipitation, dissolution and filtration of a component I; inactivation of S / D virus; ion exchange chromatography; primary ultrafiltration concentration; heparin affinity chromatography; secondary ultrafiltration concentration; detection and preparation; packaging and freeze drying; capping and dry heating deactivation. According to the method disclosed by the invention, the ion exchange chromatography is combined with the heparin affinity chromatography, and the whole process is produced in a normal-temperature environment; the method has the advantages of short process flow, high purity of a product, high yield and short redissolving time.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a method for preparing human fibrinogen by double chromatography. Background technique [0002] Human fibrinogen (fibrinogen, Fg), that is, coagulation factor Ⅰ, is the coagulation factor with the highest content in plasma, and it is also the "central" protein in the coagulation system. Human fibrinogen is a precursor of fibrin that is synthesized by the liver. The molecular weight is 340,000, and the half-life is 4-6 days. The reference value in plasma is 2-4 g / L. Fibrinogen consists of three pairs of different polypeptide chains α, β, and γ, and the polypeptide chains are connected by disulfide bonds. It is associated with blood coagulation. Under the action of thrombin, the α-chain and β-chain release peptide A and peptide B, respectively, to generate fibrin monomers. Mainly used in congenital human fibrinogen reduction or deficiency; acquired fibrinogen reductio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/75C12N9/64C07K1/18C07K1/22
Inventor 资道凤岳跃飞曹希文陈治海
Owner NANYUE BIOPHARMING
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