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Method for continuously massively producing recombinant adenoviruses

A recombinant adenovirus, a large-scale technology, applied in the direction of supporting/immobilizing microorganisms, biochemical equipment and methods, viruses, etc., can solve the problems of poor cycle and poor relative stability of cells, achieve low cost and improve cell growth environment , the effect of high density

Active Publication Date: 2018-12-14
WUHAN CEKG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And from acclimatization of adherent cells to suspension culture cells, the cycle is poor, and the relative stability of cells is poor

Method used

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  • Method for continuously massively producing recombinant adenoviruses
  • Method for continuously massively producing recombinant adenoviruses
  • Method for continuously massively producing recombinant adenoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] A method for continuous large-scale production of recombinant adenovirus provided in this embodiment comprises the following steps:

[0060] Step 1. Cell culture

[0061] 1) Cell recovery: Take a cryopreservation tube of HEK293 cells and put it in warm water at 37°C for instant dissolving, take it out immediately when it melts until only a small ice nucleus remains, and wipe the surface of the cryopreservation tube with 70%-75% alcohol cotton ball After disinfection, open the tube cover on the ultra-clean workbench, use a 5ml pipette to suck out the cell suspension and gently inject it into a sterile centrifuge tube containing DMEM medium, and mix gently; freeze the cells with a 5ml pipette Aspirate the stored solution into a 15ml centrifuge tube, centrifuge (800 rpm, 4 minutes), discard the supernatant, add DMEM medium to resuspend the cells; add the resuspended cells to the square bottle (sampling, counting) , placed in an incubator for cultivation, the conditions of...

Embodiment 2

[0074] This example is similar to the experimental steps adopted in the method for continuous large-scale production of recombinant adeno-associated virus described in Example 1, the difference is that:

[0075] The carrier filling number in the cell culture bag in step 2 and step 3 is 10g / L;

[0076] The cell inoculation density in step 2 and step 3 is 1E+5 cells / ml;

[0077] In step 2, the daily sugar consumption of the cells in the 3L cell culture bag is 4.0±0.5g. When the cells are expanded for culture, some of them are kept in the 3L cell culture bag for culture, and some are inoculated into the 30L cell culture bag for scale-up culture;

[0078] In step 3, when the cells in the 30L cell culture bag are inoculated with adenovirus, the daily sugar consumption is 40±5g, the total number of cells is 5.3E+10; the MOI of the virus-infected host cells is 20; the total number of virus particles is 1.3E+15 ; Cell yield is about 2.45E+4.

Embodiment 3

[0080] This example is similar to the method for continuous large-scale production of recombinant adeno-associated virus described in Example 1, the difference is that:

[0081] The carrier filling number in the cell culture bag in step 2 and step 3 is 40g / L;

[0082] The number of cells inoculated in step 2 and step 3 is 4E+5 / ml;

[0083] In step 2, the daily sugar consumption of the cells in the 3L cell culture bag is 9.0±0.5g. When the cells are expanded for culture, some of them are kept in the 3L cell culture bag for culture, and some are inoculated into the 30L cell culture bag for scale-up culture;

[0084] In step 3, when the cells in the 30L cell culture bag are inoculated with adenovirus, the daily sugar consumption is 90.0±5g, the total number of cells is 1.26E+11; the MOI of the virus infected host cells is 20; the total number of virus particles is 2.65E+15 ; Cell yield is about 2.10E+4.

[0085] The purity of the recombinant adenovirus products obtained in Exam...

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Abstract

The invention discloses a method for continuously massively producing recombinant adenoviruses. The method comprises the following steps: 1, cell culture; 2, cell culture bag culture; 3, large-scale amplification of the adenoviruses; and 4, purification, wherein cell culture bags in step 2 and step 3 are placed on a shaking table, and culture in a thermotank is carried out; and the culture conditions of the cell culture bags are as follows: the temperature is 37 DEG C, the pH value is 7.2, the shaking speed is 10-20 rpm, and the angle of the shaking table is 7-9 DEG. The cell growth environment is improved, damages of ventilation to cells are reduced, and the dissolved oxygen effect is increased, so the problems of difficult digestion, separation and transfer of cells in traditional technologies are solved, thereby the enlargement culture problem and the continuous production problem of adherent cells are solved, and the problem of difficult freeze-thaw cracking of the cells is solved;and the adherent cells are cultured in the cell culture bags filled with a sheet carrier and the shaking bed, so the damages of a mechanical shearing force to the cells are reduced, and the growth density of the cells is improved, thereby the total cell yield of the cells is improved, and the yield of the adenoviruses is increased.

Description

technical field [0001] The invention relates to the technical field of producing recombinant adenovirus and adeno-associated virus, in particular to a method for continuous large-scale production of recombinant adenovirus. Background technique [0002] Adenovirus vectors have high transgene efficiency, and in vitro experiments are usually close to 100% transduction efficiency; different types of human tissue cells can be transduced, regardless of whether the target cells are dividing cells; The titer of the recombinant virus in the culture can reach (10E+11) / ml; it enters the cell and does not integrate into the host cell genome, but is only expressed transiently, with high safety. Therefore, adenoviral vectors have been used more and more in clinical trials of gene therapy, and have become the most widely used and most promising viral vectors after retroviral vectors. [0003] At present, in the large-scale production of adenovirus, cell factories, microcarriers, cell tank...

Claims

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Application Information

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IPC IPC(8): C12M1/00C12M1/10C12N7/01
CPCC12M23/14C12M25/00C12M27/16C12N7/00C12N2710/10051
Inventor 甄宝贵李驰王树华吴菲菲
Owner WUHAN CEKG TECH CO LTD
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