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A kind of method for preparing adenovirus vector vaccine by perfusion culture technique

A vector vaccine and culture method technology, applied in the field of biological products, can solve the problems of low cell density yield, limited growth, low cell density, etc., and achieve the effects of increasing single cell yield and virus titer.

Active Publication Date: 2021-08-24
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

293 cells are human kidney epithelial cell lines, and there are many derivatives, such as HEK293, 293T / 17, etc.; 293 cells generally grow adherently, but there are also cell lines adapted to suspension culture
[0007] In the prior art, the production of adenovirus by 293 cells is limited by the low cell density and low cell toxin production, resulting in low yield. The main reason for the low cell density is that it is difficult for 293 cells to perform high-density production on 293 cells due to the influence of nutrient supply, oxygen, and metabolism in cell culture. nourish
The competition between infected cells and normal cell growth, nutrients, and the state of infected cells leads to low toxin production
Although regular supplementation with fresh medium, used to maintain a nutrient environment, can enhance cell growth, this growth is limited

Method used

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  • A kind of method for preparing adenovirus vector vaccine by perfusion culture technique
  • A kind of method for preparing adenovirus vector vaccine by perfusion culture technique
  • A kind of method for preparing adenovirus vector vaccine by perfusion culture technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Confirmation of technical parameters for culturing 293 cells by perfusion method.

[0074] Perfusion process 1: After recovery, 293 cells were expanded and inoculated into the fermenter. When the cell density reached 1×10 6 Perfusion was started after cells / mL, and the perfusion rate was 2VVD. Continue to grow the cell density to 5×10 6 cells / mL, the perfusion rate was adjusted to 3VVD.

[0075] Perfusion process 2: After recovery, 293 cells were expanded and inoculated into the fermenter. When the cell density reached 1×10 6 / mL or 5×10 6 cells / mL and start perfusion. Throughout the culture process, the perfusion rate was maintained constant at 1VVD or 3VVD.

[0076] Perfusion process 3: After 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1×10 6 / mL or 5×10 6 cells / mL and start perfusion. Throughout the culture process, the perfusion rate was maintained constant at 2VVD or 4VVD.

[0077]...

Embodiment 2

[0089] Example 2: Comparison of cell density and viability between the perfusion culture process and the batch process culture.

[0090] Experiment 1: Culture was carried out by adopting the conventional batch culture technology in this field.

[0091] Experiment 2: Using the perfusion process 1 of Example 1, that is, after the 293 cells were recovered, they were expanded and inoculated into the fermenter. When the cell density reached 1×10 6 Perfusion was started after cells / mL, and the perfusion rate was 2VVD. Continue to grow the cell density to 5×10 6 cells / mL, the perfusion rate was adjusted to 3VVD.

[0092] In experiments 1 and 2, other parameters (such as culture temperature, pH dissolved oxygen concentration, stirring speed, etc.) were basically the same during the culture process. The cell density and viability after the culture were detected, and the results are shown in Table 4.

[0093] Table 4 The viability and density of 293 cells cultured by batch process an...

Embodiment 3

[0096] Example 3: Effect of glutamine on cell production in a perfusion process.

[0097] Experiment 1: After recovery of 293 cells, they were expanded and inoculated into fermenters. When the cell density reached 1×10 6 Perfusion was started after cells / mL, and the perfusion rate was 2VVD. Continue to grow the cell density to 5×10 6 cells / mL, the perfusion rate was adjusted to 3VVD. Glutamine was not added during the perfusion process.

[0098] Experiment 2: After recovery of 293 cells, they were expanded and inoculated into fermenters. When the cell density reached 1×10 6 Perfusion was started after cells / mL, and the perfusion rate was 2VVD. Continue to grow the cell density to 5×10 6 cells / mL, the perfusion rate was adjusted to 3VVD. Monitor the concentration of glutamine in the perfusion process, add glutamine, and maintain the concentration of glutamine to 2mM.

[0099] Experiment 3: After recovery, 293 cells were expanded and inoculated into fermenters, when the cel...

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Abstract

The invention discloses a method for preparing an adenovirus vector vaccine through a perfusion culture technique. Wherein, the method includes the step of culturing the adenovirus host cells, especially the step of adjusting the perfusion rate according to the cell density (for example, adjusting the perfusion rate through at least two stages). The method realizes the high-density growth of the adenovirus host cells, and at the same time improves the single-cell yield of the infected virus and the specific activity of the virus harvest liquid.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a method for culturing an adenovirus host cell, a method for producing an adenovirus and a method for preparing an adenovirus vector vaccine, in particular to a method for preparing an adenovirus vector vaccine through a perfusion culture process. Background technique [0002] Adenovirus is a non-enveloped DNA virus, which has the characteristics of easy infection, wide host range, low toxicity, safe use, large capacity, non-integration, low pathogenicity to humans, and no induction of cancer. Become one of the most promising gene carriers in gene transfer. [0003] The modified adenovirus removes the E1 / E3 gene expression cassette, prevents the transcription of functional proteins dependent on the E1 region and the E3 region, and the subsequent replication of viral DNA and the production of viral coat proteins. The E1 / E3-deleted adenovirus can provide E1 / E3 region ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/861A61K39/00A61K48/00
CPCC12N5/0603C12N5/0686C12N15/86A61K39/00A61K48/0008C12N2510/00C12N2710/10343C12N2710/10352C12N2800/107A61K2039/53A61K2039/5256Y02A50/30C12N2710/10351C12N2710/10334C07K14/005C12N2770/20034C12N2770/20022C12N7/00A61K39/215A61P31/12C12M29/10C12N2500/32C12N2710/10362
Inventor 肖猛刘云杰朱涛许允立徐灿李军强巢守柏
Owner CANSINO BIOLOGICS INC
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