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Method for preparing recombinant human coagulation factor VIII

A technology for human coagulation and hemophilia factors, applied in the biological field, can solve the problems of complex cell line construction methods, failure to meet the needs of industrialization, and low expression of eight factors, avoiding the risk of virus contamination, low construction difficulty, The effect of building methods is simple

Inactive Publication Date: 2017-10-10
SHANGHAI MEDICILON INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The current two methods for large-scale preparation of recombinant factor VIII are flawed. The serum medium method requires exogenous addition of animal serum, which brings the risk of potential virus contamination.
However, in the co-expression method of VWF and FVIII genes, the expression level of the eight factors is relatively low, and the method of cell line construction is complicated, which cannot meet the needs of industrialization

Method used

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  • Method for preparing recombinant human coagulation factor VIII

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Experimental program
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Effect test

preparation example Construction

[0032] Preparation of blood coagulation factor eight products

[0033] The recombinant human blood coagulation factor eight product is isolated and purified from the obtained cell stock solution. As the separation method, conventional methods such as centrifugation and filtration can be used. The purification method may be, for example, nickel ion affinity chromatography purification or the like. VWF in the obtained recombinant human blood coagulation factor VIII can be removed or still retained to form a complex with FVIII.

Embodiment 1

[0036] 1) Preparation of expression cells

[0037] Construct eight-factor expressing cells; and domesticate them into serum-free cultured eight-factor expressing cells.

[0038] 2) Expansion culture of expressing cells

[0039] The eight-factor expressing cells acclimatized to serum-free culture were divided into 8×l0 5 Inoculate the Erlenmeyer shaker flask with cells / ml, the culture medium is serum-free medium, the culture temperature is 35°C, and the rotation speed of the shaker flask is 80 rpm; after about 4 days of serum-free culture, the cell density reaches 2×l0 6 cells / ml, passaged at a ratio of 1:5, cultured in shake flasks, cultured at 38°C, rotated at 60 rpm, until the cell density reached 2×l0 6 cells / ml.

[0040] 3) Preparation of VWF

[0041] Prepare VWF.

[0042] 4) Preparation of samples for eight-factor activity analysis

[0043] After culturing for 24 hours with recombinant VWF containing different activities, the cell culture fluid was collected for eig...

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Abstract

The invention relates to a method for preparing recombinant human coagulation factor VIII. The method comprises the following steps: constructing a cell strain expressing human coagulation factor VIII; inoculating the constructed cell strain expressing human coagulation factor VIII to a medium, and performing shaking culturing for a period until the cell density reaches to (1.5-2.5)*10<6> cells / mL; subculturing the cells according to a subculturing ratio of 1:5, and performing shaking culturing for a period until the cell density reaches to (1.5-2.5)*10<6> cells / mL, so as to obtain a seed liquid; inoculating the obtained seed liquid to a medium of a cell reactor, and performing culturing for a period until the cell density of the cell liquid reaches to (2-6)*10<6> cells / mL; then adding an exogenetic von Willebrand Factor into the cell liquid, and performing feeding culturing on the cell liquid, so as to obtain a cell stock solution, wherein during feeding culturing, the ratio of the activity of the von Willebrand Factor in the cell liquid to the activity of the human coagulation factor VIII in the cell liquid is controlled to be 2-12:1; and performing separation purification on the obtained cell stock solution, so as to obtain the recombinant human coagulation factor VIII.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing recombinant human blood coagulation factor eight. Background technique [0002] Hemophilia A is a blood coagulation disorder caused by the deficiency or abnormality of the eight coagulation factors in the human body. In the coagulation cascade reaction, coagulation factor 8 acts as a cofactor of activated factor 9 to activate factor 10, thereby stimulating the occurrence of endogenous coagulation reaction. [0003] Natural human coagulation factor VIII (FVIII) is a macromolecular glycoprotein composed of heavy chain and light chain, with a total molecular weight of 330KD and a plasma content of about 0.2mg / L to bind von Willebrand factor (VWF) form exists. Recombinant human FVIII has the same physiological, pharmacological and immune characteristics as natural FVIII, and has the advantage of eliminating HIV, HDV, proteovirus and other pathogenic sources. Ther...

Claims

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Application Information

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IPC IPC(8): C07K14/755C12N15/85C12N5/10
CPCC07K14/755
Inventor 陈春麟顾性初林筱芹王涛
Owner SHANGHAI MEDICILON INC