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A perfusion culture method for expressing recombinant factor eight

A technology of perfusion culture and factors, which is applied in the field of perfusion culture for high-efficiency expression of eight recombinant factors, to achieve the effects of high retention efficiency, large perfusion efficiency, easy cleaning and sterilization

Active Publication Date: 2018-10-30
SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many types of methods that can be applied, these methods all have very obvious defects.

Method used

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  • A perfusion culture method for expressing recombinant factor eight
  • A perfusion culture method for expressing recombinant factor eight

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Inoculation of cells

[0040] A cell line expressing recombinant FVIII (preservation number: CCTCC C2015214) was revived in the cell bank, cultured and expanded in a shake flask with serum-free medium, and inoculated into a reactor of a 5L culture system after the number of cells was sufficient. The cell density after inoculation is 0.8*10 6 a / ml;

[0041] (2) Initial growth stage

[0042] After the cells were inserted into the reactor, various culture parameters were set, such as temperature 37°C, pH 6.9, dissolved oxygen (DO) 30-50%, and initial rotation speed 90 rpm. Sampling was aseptically taken every day to detect the content of glucose and FVIII.

[0043] (3) Enter the perfusion culture mode

[0044] When the glucose content is lower than 1g / L, turn on the Spin filter (CelliGen Plus, New Brunswick Scientific) to enter the perfusion culture mode, and the perfusion volume is to keep the glucose content at about 1g / L. Remove part of the cells from the react...

Embodiment 2

[0046] (1) Inoculation of cells

[0047] A cell line expressing recombinant FVIII (same as in Example 1) was revived in the cell bank, cultured and amplified in a shake flask with a serum-free medium, and inoculated into a reactor of a 5L culture system after the number of cells was sufficient. The cell density after inoculation is 0.8*10 6 a / ml;

[0048] (2) Initial growth stage

[0049] After the cells were inserted into the reactor, various culture parameters were set, such as temperature 37°C, pH 6.9, dissolved oxygen (DO) 30-50%, and initial rotation speed 90 rpm. Sampling was aseptically taken every day to detect the content of glucose and FVIII.

[0050] (3) Enter the perfusion culture mode

[0051] When the glucose content is lower than 1g / L, turn on the audio frequency perfusion device (10L Biosep, Applikon) for perfusion culture. The perfusion volume is to keep the glucose content at about 1g / L. Remove part of the cells from the container and keep the cells at 1...

Embodiment 3

[0053] (1) Inoculation of cells

[0054] A cell line expressing recombinant FVIII (same as in Example 1) was revived in the cell bank, cultured and amplified in a shake flask with a serum-free medium, and inoculated into a reactor of a 5L culture system after the number of cells was sufficient. The cell density after inoculation is 0.8*10 6 a / ml;

[0055] (2) Initial growth stage

[0056] After the cells were inserted into the reactor, various culture parameters were set, such as temperature 37°C, pH 6.9, dissolved oxygen (DO) 30-50%, and initial rotation speed 90 rpm. Sampling was aseptically taken every day to detect the content of glucose and FVIII.

[0057] (3) Change the training mode

[0058] When the glucose content is lower than 1g / L, turn on the spin filter first, and carry out perfusion culture. The perfusion volume is to keep the glucose content at about 1g / L, and maintain the cells at 1-2*10 7 pieces / ml.

[0059] (4) Continuous perfusion culture stage

[006...

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Abstract

The invention relates to the technical field of biology, in particular to a perfusion culture method for efficiently expressing a recombinant factor VIII. The perfusion culture method for efficiently expressing the recombinant factor VIII comprises the following steps that 1, seeds of a recombinant FVIII cell strain are inoculated into a reactor for batched culturing; 2, when the glucose content in a culturing system ranges from 0.5 g / L to 1.5 g / L, a rotary filtering perfusion method is utilized to enter a perfusion culture mode; 3, when cells in the reactor are in a steady-state phase, an audio perfusion method and the rotary filtering perfusion method are simultaneously used for perfusion culture, and the cell density is maintained to be 2-3*10<7> / ml. According to the perfusion culture method for efficiently expressing the recombinant factor VIII, the audio perfusion method and the rotary filtering perfusion method are combined for the perfusion culture, the survival rate of the cells in the whole stable expression period is always maintained to 90 percent or above, the cell density can reach about 2.5*10<7> / ml, and the average expression quantity of the factor VIII is 18 IU / ml.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a perfusion culture method for highly expressing recombinant factor eight. Background technique [0002] Coagulation factor VIII is currently the only treatment for hemophilia A (Hemophilia A, HA). HA patients have reduced FVIII expression or functional defects due to gene defects, which manifest as coagulation dysfunction and spontaneous bleeding. Human blood FVIII and recombinant FVIII are currently commercially available. However, human blood FVIII products still have the possibility of spreading unknown blood-borne pathogens. Therefore, with the increasing development and progress of recombinant DNA technology, recombinant FVIII products have received more and more attention. [0003] Recombinant FVIII uses gene recombination technology to insert the gene fragment encoding FVIII protein into a plasmid vector, and then transfer the plasmid into mammalian cells capable of expressi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/91
CPCC07K14/755
Inventor 程露郑继岱陆晖周雪峰
Owner SHANGHAI RAAS BLOOD PRODUCTS CO LTD
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