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Methods for producing recombinant adenovirus and drug preparations of recombinant adenovirus with serum-free suspension cells

A technology of recombinant adenovirus and suspension cells, applied in biochemical equipment and methods, viruses/bacteriophages, microorganisms, etc., can solve the problems that cannot meet the requirements of large-scale industrialization of gene therapy products, and achieve the effect of large-scale industrialization

Pending Publication Date: 2014-06-25
深圳市赛百诺基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional laboratory gene therapy product preparation methods can no longer meet the requirements of GMP large-scale industrialization of gene therapy products

Method used

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  • Methods for producing recombinant adenovirus and drug preparations of recombinant adenovirus with serum-free suspension cells
  • Methods for producing recombinant adenovirus and drug preparations of recombinant adenovirus with serum-free suspension cells
  • Methods for producing recombinant adenovirus and drug preparations of recombinant adenovirus with serum-free suspension cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0048] Example 2, 293F cells (#11625-019) were purchased from Invitrogen in the United States. 293F cells were cultured and expanded in suspension in spinner bottles with serum-free CD293 medium. After obtaining a certain amount of cells, the cells are frozen. The cell freezing liquid adopts CD293+10%DMSO, the cell concentration is 1-5x10 7 / ml. Cells were frozen using a programmable cell freezer. Then store it in the upper vapor layer of the liquid nitrogen tank for later use.

[0049]In order to ensure the purity and stability of the cells, the cells were cloned and screened by the terminal dilution method. The resulting cell clone was named SBN-293SFS cells. SBN-293SFS cells were suspended and expanded in spinner bottles with serum-free CD293 medium. After obtaining a certain amount of cells, the cells are frozen. The cell freezing liquid adopts CD293+10%DMSO, the cell concentration is 1-5x10 7 / ml. Cells were frozen using a programmable cell freezer. Then store i...

Embodiment 3

[0057] Example 3: Large-scale high-density culture of SBN-293SFS cells and infection of recombinant adenovirus using Wave-20 perfusion bioreactor.

[0058] In order to avoid the complexity of centrifuging a large amount of cell culture medium under sterile conditions, we adopted a method of diluting high-density SBN-293SFS cells with fresh CD293 medium, and the dilution factor was about ten times. Provide enough fresh medium for the cells. We used the Wave-20 perfusion bioreactor to culture SBN-293SFS cells at high density on a large scale. Cell concentration can reach or exceed 1x10 7 / ml. When the cell concentration reaches 1x10 7 / ml, add fresh CD293 medium to dilute the cells to 1x10 6 / ml, and at the same time transfer the cell culture medium to another bioreactor with larger volume for the infection and production of recombinant adenovirus.

[0059] Thaw a tube of SBN-293SFS cells and inoculate into a spinner bottle with 100 ml of CD293 medium. At 37°C, 10% CO 2 C...

Embodiment 4

[0064] Example 4: Large-scale high-density culture of SBN-293SFS cells and infection and production of 100 liters of recombinant adenovirus and separation and purification by ion exchange column chromatography using Wave-20 perfusion bioreactor.

[0065] After confirming the feasibility of large-scale high-density culture of SBN-293SFS cells and diluted cells for recombinant adenovirus infection in Wave-20 perfusion bioreactor, all cells cultured in Wave-20 perfusion bioreactor were treated with recombinant adenovirus. The virus was infected with a total volume of 100 liters. The multiplicity of infection was 50 virus particles / cell. The pH of the bioreactor is controlled between 6.5-8.2, the DO is controlled between 15%-75%, the temperature of the culture medium is controlled between 36-37°C, the swing speed is controlled between 15-20rpm, and the swing angle is controlled between Between 8-15 degrees. After culturing for 4 days, Tween-20 was added to the cell culture mediu...

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Abstract

The invention provides a method for producing a recombinant adenovirus with serum-free suspension cells. The method comprises the following steps: (1) cell expansion; (2) inoculation into a bioreactor, culture solution perfusion culture and cell re-expansion: inoculating cells harvested from spinner bottles into the bioreactor to be cultured, wherein the pH value and the dissolved oxygen (DO) value of the culture solution are automatically controlled by a controller of the bioreactor via the pH value and DO probes, the pH value is controlled between 6.5 and 8.2, DO is controlled between 15% and 75%, the temperature of the culture solution is automatically controlled between 36 DEG C and 37 DEG C by an electric heater in a platform of the bioreactor, the swing speed is controlled between 15rpm and 20rpm, and the swing angle is controlled between 8 degrees and 15 degrees; (3) cell dilution and cell transfer to the bioreactor with big volume; (4) recombinant adenovirus infection and expansion. The method can achieve large-scale industrialization and can be used for preparing recombinant adenovirus products safely, economically and effectively according to GMP (good manufacture practice).

Description

【Technical field】 [0001] The invention belongs to the field of biopharmaceutical technology, in particular to a method and process for large-scale production of recombinant adenovirus gene therapy product GMP. 【Background technique】 [0002] After decades of research and development, the field of gene therapy has made great progress and achieved product breakthroughs. Shenzhen Saibainuo Gene Technology Co., Ltd.'s recombinant adenovirus p53 product and UniQure's Glybera recombinant adeno-associated virus (AAV) product have been approved by the government's pharmaceutical regulatory agency for marketing. Jinyousheng products are tumor gene therapy products recombined with replication-defective type 5 adenovirus vector and human p53 gene. Studies have shown that the p53 gene guides the synthesis of p53 protein. In normal tissues, the expression level of p53 protein is very low. When stimulated by DNA damage and other stimuli, the expression level of p53 protein increases, pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K48/00A61P35/00
Inventor 张书元
Owner 深圳市赛百诺基因技术有限公司
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