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A method for preparing recombinant human blood coagulation factor ⅷ

A human blood coagulation factor and human-like technology, applied in the field of bioengineering, can solve the problems of high technical barriers in cell culture technology, difficulty in maintaining cell state, serious cell clumping, etc., to improve cell suspension, reduce cell clumping and adhesion phenomenon, the effect of high mixing efficiency

Active Publication Date: 2021-12-14
CHIA TAI TIANQING PHARMA GRP CO LTD +1
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  • Description
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  • Application Information

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Problems solved by technology

[0003] As a kind of mammalian expression cell, human embryonic kidney cell (HEK293) expression system has been used to prepare many therapeutic proteins containing recombinant coagulation factor VIII, and the protein post-translational modification of the recombinant protein expressed by it is complete and the immune response is low, but The commonly used HEK293 cell culture process has high technical barriers, severe cell clumping, and difficult to maintain the cell state

Method used

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  • A method for preparing recombinant human blood coagulation factor ⅷ
  • A method for preparing recombinant human blood coagulation factor ⅷ
  • A method for preparing recombinant human blood coagulation factor ⅷ

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 investigates the cell culture effect of WAVE wave bioreactor and stirred stainless steel reactor

[0045] First-level seed solution preparation: take a frozen recombinant human coagulation factor VIII working cell bank cell from the liquid nitrogen tank (human embryonic kidney cell 293 / N27-7, self-made, capacity 1ml), thaw in a 37°C water bath, and transfer Into a 125ml cell culture shaker flask containing 20ml seed medium CDOptiCHO AGT (containing 4mmol / L glutamine (Life technologies company), 50μg / ml zeocin (Invotrogen company), Life technologies company), placed in 37 ℃, 6- 10%CO 2 Cultivate at 110-130rpm in a carbon dioxide constant temperature incubator. Observe the cell state every day, take samples for cell counting and detection of cell viability (trypan blue method), the cell density is about 3.0-4.0×10 6 Subculture at cells / ml, the subculture density is about 0.6-1.0×10 6 cells / ml, after 3-4 passages, the primary seed solution was inoculated in a ...

Embodiment 2

[0054] Embodiment 2 Purification of recombinant human blood coagulation factor VIII

[0055] (1) Harvest the cell culture medium

[0056] In the cell suspension (WAVE wave reactor cultivation) that embodiment 1 obtains, add the damping fluid (10mmol / L Hepes, 5mmol / L calcium chloride dihydrate, 4mol / L chloride) containing sodium chloride and calcium chloride Sodium, pH7.2), so that the final concentration of sodium chloride is about 0.5mol / L, at 2-8°C, mix the solution and let it stand for about 45 minutes, remove the cells by deep filtration and then filter (0.22 μm) to remove any remaining cellular debris and particulate matter.

[0057] (2) Unidirectional flow concentration

[0058] Concentrate the cell clarified liquid by one-way flow for about 3-4 times, and filter the concentrated liquid through a 0.2 μm filter.

[0059] (3) S / D virus inactivation

[0060] Virus inactivation was performed on the clarified cell harvest liquid obtained in step (2) using 0.3% tributyl phos...

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Abstract

A method for preparing recombinant human blood coagulation factor VIII comprises cultivating human embryonic kidney cell 293 / N27‑7 expressing recombinant human blood coagulation factor VIII in a wave bioreactor and separating and purifying recombinant human blood coagulation factor VIII from cell culture fluid. The wave bioreactor has high mixing efficiency, sufficient gas-liquid exchange, less foam and low shear force, which avoids the damage to the cells caused by the paddle end and air bubbles of the stirring stainless steel reactor.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for preparing recombinant human blood coagulation factor VIII, in particular to using screened human embryonic kidney cells to cultivate cells expressing recombinant human blood coagulation factor VIII through a wave-type bioreactor and extracting the recombinant human blood coagulation factor VIII from the cell culture medium. Isolation and purification of recombinant human coagulation factor VIII. Background technique [0002] Hemophilia is a genetic bleeding disorder in which congenital defects or mutations in clotting factor genes lead to coagulation dysfunction. According to the corresponding coagulation factors, hemophilia is divided into type A, type B, and type C (ie, hemophilia A, B, and C), of which hemophilia A accounts for 80%-85%. At present, alternative therapy such as plasma transfusion, blood coagulation factor VIII or IX is an effective measure for...

Claims

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Application Information

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IPC IPC(8): C12N5/22C12P21/02C07K14/755
CPCC12P21/02C07K14/755
Inventor 胡伟伟尚战强杜敏陆建胜苏贤德张哲文谢一龙费如翔吴鼎
Owner CHIA TAI TIANQING PHARMA GRP CO LTD
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