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Method for preparing recombinant human blood coagulation factor VIII

A human blood coagulation factor and bioreactor technology, applied in the field of bioengineering, can solve the problems of high technical barriers in cell culture technology, difficulty in maintaining the state of cells, and serious cell clumping, so as to improve production, protein activity, cell viability and Increased protein expression and high mixing efficiency

Active Publication Date: 2017-10-24
NANJING SHUNXIN PHARM CO LTD OF CHIATAI TIANQING PHARM GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 HEK293 cell culture technology has high technical barriers, severe cell clumping, and difficult to maintain cell state

Method used

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  • Method for preparing recombinant human blood coagulation factor VIII
  • Method for preparing recombinant human blood coagulation factor VIII
  • Method for preparing recombinant human blood coagulation factor VIII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Investigating the cell culture effect of WAVE wave bioreactor and stirred stainless steel reactor

[0033] First-level seed solution preparation: take a frozen recombinant human coagulation factor VIII working cell bank cell (self-made, 1ml) from the liquid nitrogen tank, thaw it in a water bath at 37°C, and transfer it to CD OptiCHO AGT containing 20ml seed medium ( In a 125ml cell culture shake flask containing 4mmol / L glutamine (Life technologies company), 50μg / ml zeocin (Invotrogen company, Life technologies company), placed in 37 ℃, 6~10%CO 2 Cultivate in a carbon dioxide constant temperature incubator at 110-130rpm. Observe the cell state every day, take samples for cell count and detect 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 to 4 passages, inoculate the primary seed solution in the WAVE wave reactor (GE Healthcare, model 20 / 50EHT...

Embodiment 2

[0041] Example 2 WAVE wave bioreactor to investigate the effect of different culture periods on cell viability and protein expression

[0042] Refer to the preparation method of Example 1 to obtain the secondary seed liquid.

[0043] Inoculate the secondary seed solution at a ratio of 1 / 4 to 1 / 3, 0.6 to 1.0×10 6 The seeding density of cells / ml, the medium is CDOptiCHO AGT (containing 4mmol / L glutamine (Life technologies company), 0.02% antifoam C (SIGMA company), 1g / L Kolliphor P188 (sigma company), Life technologies company), For fed-batch culture, add 200mmol / L glutamine to a concentration of 2-4mmol / L at the 24th to 72nd hour of the culture cycle, and add 200g / L glucose (Sigma Company) solution until the glucose content is 2-4g / L. L, harvested at the 120th hour of the culture cycle. During the culture process, samples were taken every 24 hours for cell counting to detect cell viability (trypan blue method) and protein activity (one-phase method). WAVE wave reactor cell c...

Embodiment 3

[0045] Example 3 Purification of recombinant human blood coagulation factor VIII

[0046] (1) Harvest the cell culture medium

[0047] 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, so that the conductivity at 2~8°C is 40~50mS / cm, mix the solution and let it stand for about 30 minutes, and filter it through deep layer Cell removal was followed by sterile filtration (0.22 μm) to remove any remaining cell debris and particulate matter.

[0048] (2) S / D virus inactivation

[0049] Virus inactivation was performed on the clarified cell harvest liquid obtained in step (1) using 0.3% tributyl phosphate (TNBP) (v / v) and 1% Triton X-100. Stir at 20-25°C for 45 minutes to inactivate the virus.

[0050] (3) Affinity chromatogr...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to a method for preparing a recombinant human blood coagulation factor VIII, in particular to a method for culturing cells for expressing a recombinant human blood coagulation factor VIII by using a wave type bioreactor and separating and purifying the recombinant human blood coagulation factor VIII from a cell culture liquid. The WAVE bioreactor is high in mixing efficiency, sufficient in gas-liquid exchange, small in foam amount and low in shearing force, damage of paddles of a stirring type stainless steel reactor and foams to cells is avoided, and thus the cell state, the cell survival rate and the protein activity of the WAVE bioreactor are all superior to those of the stirring type 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 coagulation factor VIII, in particular to cultivating cells expressing recombinant human coagulation factor VIII through a wave bioreactor and separating and purifying recombinant human coagulation factor VIII from cell culture fluid factor. 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, replacement therapy such as plasma transfusion, blood coagulation factor VIII or IX is an effective measure for the treatment of hemophilia, but because blood coagulation products derived from plasma can easily cause blood-borne virus conta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02
CPCC07K14/755
Inventor 赵伟吕海丽蒋丹张哲文秦宇徐霆郭康平张喜全程艳菊
Owner NANJING SHUNXIN PHARM CO LTD OF CHIATAI TIANQING PHARM GRP
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