Method for effectively expressing recombinant human coagulation factor VIII

A high-efficiency expression and human blood coagulation technology, applied in the field of bioengineering, can solve the problems of low expression of the eight factors, complex cell line construction, and expression levels that cannot reach industrialization, and achieve low construction difficulty, improved accumulation and stability, and easy Magnified effect

Active Publication Date: 2012-11-14
上海泰龙生物医药科技有限公司
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Problems solved by technology

As for the co-expression of VWF and FVIII genes, people found in the study of the expression of recombinant human blood coagulation factor 8 that the co-expression of VWF and FVIII, the expression level of 8 factors is relatively low, and the construction of cell lines is also relatively complicated. The expression level of cell lines expressing eight factors in serum-free medium is generally 0.1-0.5 international units / day / 10 6 cells, the expression level is far from the requirements of industrialization

Method used

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  • Method for effectively expressing recombinant human coagulation factor VIII
  • Method for effectively expressing recombinant human coagulation factor VIII
  • Method for effectively expressing recombinant human coagulation factor VIII

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Experimental program
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Effect test

Embodiment 1

[0033] 1. Experimental method

[0034] 1) Acclimate into serum-free cultured eight-factor expressing cells (construction of eight-factor expressing cells reference "Expression of blood coagulation factor FVIII in mammalian cells", Wang Qi et al., Pharmaceutical Biotechnology, 2002, 9 (5) : 251~255; the serum-free acclimatization method of cell lines can refer to the cell culture manual, such as the cell culture operation part in the appendix of the product manual of Invitrogen Company), with 9×10 5 cells / ml to inoculate the Erlenmeyer shaker flask, the culture temperature is 32°C, and the shaker flask speed is 60 rpm; after about 3 days of serum-free culture, the cell density reaches 2×10 6 cells / ml, passaged at a ratio of 1:2, cultured in shake flasks, cultured at 38°C, rotated at 60 rpm, until the cell density reached 1-2×10 6 cells / ml. The serum-free cell culture medium is commercialized SFMII302 medium produced by SIGMA Company.

[0035]2) Change the culture medium cont...

Embodiment 2

[0058] 1) Construction of cell lines expressing human coagulation factor VIII

[0059] The preparation method of the cell line expressing human coagulation factor VIII is the same as that in Example 1.

[0060] 2) Expansion culture of cells expressing eight factors

[0061] Take 1×10 5 Inoculate the Erlenmeyer shake flask with cells / ml, culture at 37℃ without serum for about 4 days, and the cell density reaches 1~2×10 6 cells / ml; subculture at a passage ratio of 1:3, and continue to shake the flask at 37°C until the cell density reaches 1-2×10 6 cells / ml to obtain the seed solution; the rotation speed of the shaker flask was 150 rpm.

[0062] 3) Eight factor expression cell reactor culture

[0063] After inoculating the seed solution in the serum-free medium of the cell reactor with a 10% inoculation amount, culture it in a tank until the cell density is about 2×10 6 cells / ml, then feed-culture the cell liquid; in the feed-culture stage, the activity of the eight-factor i...

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Abstract

The invention relates to the technical field of biological engineering, in particular to a process method for effectively expressing a recombinant human coagulation factor VIII by utilizing mammalian cell culture. The method specifically comprises the following steps: adding an exogenous angiohemophilia factor to cell culture liquid for expressing the recombinant human coagulation factor VIII, and controlling an active ratio of the angiohemophilia factor to the human coagulation factor VIII in the cell culture liquid to be 1-10: 1 in the culture process. According to the invention, by utilizing a VWF (Von Willebrand Factor) molecule partner, which is necessary in an expression process of the recombinant human coagulation factor VIII, the newly generated human coagulation factor VIII is stabilized, the accumulating effect of the human coagulation factor VIII is improved, the technical difficulty is reduced, and the expression effect of a target protein is improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a process method for expressing recombinant factor eight efficiently by using mammalian cell culture. Background technique [0002] Natural human blood 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. In the blood coagulation reaction, activated FVIII greatly increases the enzyme activity of FIX, catalyzes the activation of FX, and combines to form a complex, which catalyzes the agglutination chain reaction to proceed. Deletion or lack of FVIII molecules leads to hemophilia A, and supplementation with active FVIII is an effective measure for the treatment of hemophilia A. [0003] Recombinant human FVIII has the same physiological, pharmacological and immune characteristics as na...

Claims

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Application Information

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IPC IPC(8): C12P21/02
Inventor 李军辉杨松峰许必雄
Owner 上海泰龙生物医药科技有限公司
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