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Human fibrinogen preparation method

A technology of human fibrinogen and solution, applied in the field of preparation of human fibrinogen, can solve problems such as product stability decline, and achieve the effect of efficient separation and simplified process flow

Active Publication Date: 2018-03-23
INST OF PROCESS ENG CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, plasminogen also plays an important role in the process of fibrin degradation, and its residue in fibrinogen products will lead to a decrease in product stability

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] A preparation method for human fibrinogen, comprising the steps of:

[0071] (1) Dissolution of component I: crush component I of human plasma cryogenic ethanol precipitation, add to the extract at a mass volume ratio of 10 g / L, stir and dissolve at 20° C. for 1.5 h to obtain component I solution;

[0072] The extract is: an aqueous solution for injection containing 17.8g / L trisodium citrate, 11g / L sucrose, 1.3g / L Tris, 8.5g / L NaCl, and the pH is adjusted to 7.5 with HCl;

[0073] (2) S / D inactivation: Mix the component I solution obtained in step (1) with the S / D solution at a volume ratio of 1:10, and stir at 25°C for 6h;

[0074] Wherein the S / D solution comprises polysorbate 80 of 110g / L and tributyl phosphate of 33g / L;

[0075] (3) Lysine affinity chromatography: the lysine affinity chromatography medium Lysine Sepharose 4B gel is equilibrated with 20mM, pH7.5 phosphate buffer, and then the solution obtained in step (2) is loaded on the column Carry out chromatog...

Embodiment 2

[0083] A preparation method for human fibrinogen, comprising the steps of:

[0084] (1) Dissolution of component I: crush component I of human plasma low-temperature ethanol precipitation, add it to the extract at a mass volume ratio of 6 g / L, stir and dissolve at 24°C for 1 hour, and obtain component I solution;

[0085] The extract is: an aqueous solution for injection containing 15g / L trisodium citrate, 15g / L sucrose, 1.5g / L Tris, 8g / L NaCl, and the pH is adjusted to 7.5 with HCl;

[0086] (2) S / D inactivation: Mix the component I solution obtained in step (1) with the S / D solution at a volume ratio of 1:8, and stir at 24° C. for 5 h;

[0087]Wherein the S / D solution comprises polysorbate 80 of 110g / L and tributyl phosphate of 33g / L;

[0088] (3) Lysine affinity chromatography: the lysine affinity chromatography medium Lysine Sepharose 4B gel is equilibrated with 50mM, pH7.2 phosphate buffer, and then the solution obtained in step (2) is loaded on the column Carry out chr...

Embodiment 3

[0094] A preparation method for human fibrinogen, comprising the steps of:

[0095] (1) Dissolution of component I: crush component I of human plasma low-temperature ethanol precipitation, add it to the extract at a mass volume ratio of 3.5 g / L, stir and dissolve at 25°C for 1.5 hours, and obtain component I solution;

[0096] The extract is: an aqueous solution for injection containing 20g / L trisodium citrate, 10g / L sucrose, 2g / L Tris, 5g / L NaCl, and the pH is adjusted to 6.9 with HCl;

[0097] (2) S / D inactivation: Mix the component I solution obtained in step (1) with the S / D solution at a volume ratio of 1:9, and stir at 26° C. for 4 h;

[0098] Wherein the S / D solution comprises polysorbate 80 of 110g / L and tributyl phosphate of 33g / L;

[0099] (3) Lysine affinity chromatography: equilibrate the lysine affinity chromatography medium Lysine Sepharose 4B gel with 50mM, pH6.5 phosphate buffer, and then put the solution obtained in step (2) on the column Carry out chromatog...

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Abstract

The invention provides a human fibrinogen preparation method which comprises the following steps: (1) dissolving a human plasma low-temperature ethanol precipitation component I into an extracting solution to obtain a component I solution; (2) performing S / D inactivation on the component I solution obtained in the step (1); (3) balancing a lysine affinity chromatography medium by equilibrium liquid, then performing column chromatography on the solution obtained in the step (2) and collecting a penetration peak; (4) balancing a cation exchange chromatography medium by the equilibrium liquid, then performing column chromatography on the penetration peak obtained in the step (3), leaching a chromatography column by leachate after chromatography finishes, finally eluting by an eluant and collecting an elution peak to obtain human fibrinogen. The method disclosed by the invention has simpleness in operation and can effectively remove plasminogen; a prepared human fibrinogen product has theadvantages of higher purity and stability.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a preparation method of human fibrinogen. Background technique [0002] Human fibrinogen is a coagulation factor synthesized by the liver, and its content in plasma is about 2-4g / L. The most important physiological function is to participate in the coagulation process. Human fibrinogen is a fibrous protein with a symmetrical dimeric structure, two monomeric amino acids form a central E domain, and the carboxyl terminus forms two enlarged terminal D domains. Under the action of thrombin, the E and D domains of human fibrinogen are activated, exposing the binding site. Each E domain combines with two adjacent D structures of human fibrinogen, and finally forms a network structure to realize blood coagulation function. [0003] At present, the production of human fibrinogen mainly adopts the low-temperature ethanol method (CN 102286095A), using blood plasma ...

Claims

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Application Information

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IPC IPC(8): C07K14/75C07K1/22C07K1/18C12N9/68
CPCC07K14/75C12N9/6435C12Y304/21007
Inventor 罗坚马小伟苏志国张宝献陈颖李增兰
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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