A method for continuous large-scale production of recombinant adenovirus

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 small shear force

Active Publication Date: 2022-07-05
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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  • A method for continuous large-scale production of recombinant adenovirus
  • A method for continuous large-scale production of recombinant adenovirus
  • A method for continuous large-scale production of recombinant adenovirus

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 HEK293 cell cryopreservation tube and put it in warm water at 37°C for instant dissolution, and take it out immediately when only a small ice core remains, wipe the surface of the cryopreservation tube with a 70%-75% alcohol cotton ball After disinfection, open the cap of the tube on the ultra-clean workbench, aspirate the cell suspension with a 5ml pipette and gently pour it into a sterile centrifuge tube containing DMEM medium, and mix gently; freeze the cells with a 5ml pipette. The stored solution was sucked into a 15ml centrifuge tube, centrifuged (800 rpm for 4 minutes), the supernatant was discarded, and DMEM medium was added to resuspend the cells; the resuspended cells were added to the square flask (sampling, counting) , cultured in an incubator, incubator conditions...

Embodiment 2

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

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

[0076] In steps 2 and 3, the cell seeding density 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 and cultured, part of the cells are left in the 3L cell culture bag for culture, and part is inoculated into the 30L cell culture bag for enlarged 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, and the total number of cells is 5.3E+10; the MOI of virus-infected host cells is 20; the total number of virus particles is 1.3E+15 ; The 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, except that:

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

[0082] In step 2 and step 3, the number of cell inoculations 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 and cultured, part of the cells are left in the 3L cell culture bag for culture, and part is inoculated into the 30L cell culture bag for enlarged 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, and the total number of cells is 1.26E+11; the MOI of virus-infected host cells is 20; the total number of virus particles is 2.65E+15 ; The 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 continuous large-scale production of recombinant adenovirus, comprising the following steps: step 1, cell culture; step 2, cell culture bag culture; step 3, large-scale amplification of adenovirus; step 4, purification; wherein , the cell culture bags in step 2 and step 3 are placed in a shaker and cultivated in an incubator; the cell culture bag culture conditions are temperature 37°C, pH 7.2, rocking speed 10-20rpm, shaking angle 7-9° . By improving the cell growth environment, reducing the damage of aeration to the cells, and increasing the effect of dissolved oxygen, the invention solves the problems of difficulty in cell digestion and separation and transfer in the traditional process, further solves the problems of enlarged culture and continuous production of adherent cells, and solves the problem of cell freezing. It is difficult to melt and lyse the adherent cells, and the adherent cells are cultured in a cell culture bag filled with a sheet carrier and a shaker to reduce the damage to the cells by mechanical shearing force, increase the growth density of the cells, and thereby increase the total cell yield, thereby increasing the glandular capacity. virus yield.

Description

technical field [0001] The present 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 transgenic efficiency, and in vitro experiments are usually close to 100% transduction efficiency; they can transduce different types of human tissue cells, regardless of whether the target cells are dividing cells; The titer of the recombinant virus in the culture can reach (10E+11) / ml; it does not integrate into the host cell genome when it enters the cell, and is only expressed instantaneously, with high safety. Therefore, adenoviral vectors have more and more applications in gene therapy clinical trials, and become the most widely used and most promising viral vectors after retroviral vectors. [0003] At present, in the large-scale production of adenovirus, cell factory, microcarrier,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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