Novel technology for producing recombinant adeno associated viruses in pilot test manner

A virus and virus packaging technology, applied in the direction of viruses/phages, microorganisms, sensory diseases, etc., can solve the problems of low virus production titer, low transfection efficiency, low degree of automation, etc., and achieve the effect of improving culture efficiency

Inactive Publication Date: 2016-04-13
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Spinner bottle culture cells proliferate slowly, and it is difficult to monitor and replenish the cell culture conditions such as pH, aerated dissolved oxygen, and culture medium components during the production process, and cannot provide optimal cell proliferation conditions, which is labor-intensive and low in automation; manual cell culture Transfection is prone to unstable transfection, and low transfection efficiency is prone to errors
Coupled with the uncontrollable culture conditions during spinner bottle culture, resulting in large differences in cell viability and instability among batches, it is easy to cause low titer or no virus production during the virus packaging production process
In addition, the use of spinner bottle culture and manual cell transfection is not suitable for mass production of viral vectors. The only way to achieve mass production of viruses is to increase the number of spinner bottles, resulting in a large investment in workshop scale, equipment, and personnel.

Method used

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  • Novel technology for producing recombinant adeno associated viruses in pilot test manner
  • Novel technology for producing recombinant adeno associated viruses in pilot test manner
  • Novel technology for producing recombinant adeno associated viruses in pilot test manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Pilot production of recombinant adeno-associated virus by microcarrier culture of 293 series virus packaging cells using WAVE wave bioreactor

[0029] 1.293TN cell culture

[0030] 1.1 Microcarrier pretreatment: refer to the "Microcarrier Cell Culture: Principles and Methods" (18-1140-62) manual provided by GE Healthcare to perform microcarrier pretreatment.

[0031] 1.2 Cell recovery culture: preheat DMEM medium, take out the cell cryopreservation tube from the liquid nitrogen tank, quickly put it into a water bath filled with 37°C water, and shake it from time to time, thaw as soon as possible and move to the ultra-clean workbench; aseptic operation Aspirate the cell suspension and add it to 3ml of culture medium, then add 5ml of culture medium to dilute, blow it off gently; collect cells by centrifugation at 1000rpm for 3min; resuspend cells in appropriate amount of culture medium for inoculation.

[0032]1.3 Microcarrier culture: use EDTA-trypsin-treated...

experiment example 1

[0040] Experimental Example 1. Suitable Microcarrier Screening Experiment

[0041] Under the premise of fixed inoculation with the same number of 293TN cells, three groups of experiments were set up to investigate the effects of microcarriers Cytodex1, Cytodex2 and Cytodex3 on cell proliferation. Three parallel experiments were set up for each group of investigation experiments, and the initial cell seeding density of each experiment was 3×10 5 Each / mL, and the amount of added microcarriers Cytodex1, Cytodex2 and Cytodex3 were all 3g / L.

[0042] The experimental results are shown in Table 1. It can be seen from the table that after 24h and 48h of microcarrier culture, the cell density has increased. However, the 293TN cells cultured with different microcarriers had some differences in their cell density changes, especially when the 293TN cells were cultured with the microcarrier Cytodex2, the density of the cells did not increase significantly; , 48h later, the cell density...

experiment example 2

[0045] Experimental example 2. Microcarrier dosage optimization experiment

[0046] Taking microcarrier Cytodex1 and microcarrier Cytodex3 as optimization objects, the dosage is set to 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, and the initial cell seeding density is 3 ×10 5 cells / mL, cultured to 48h and sampled for cell counting.

[0047] The experimental results are shown in Table 2. It can be seen from the table that with the increase of the amount of microcarriers, the cell density after 48 hours of culture gradually increased. 6 pcs / ml~2.7×10 6 cells / ml, if the amount of microcarriers continues to increase, the cell density will show a downward trend. Therefore, the present invention determines that the fixed initial seeding cell density is 3×10 5 In the case of cells / mL, the optimal dosage of microcarriers Cytodex1 and Cytodex3 for 293TN cell culture is 2g / L-5g / L.

[0048] Table 2. Effect of microcarrier dosage on cell growth

[0049]

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PUM

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Abstract

The invention discloses a novel technology for producing recombinant adeno associated viruses by means of 293-series cell microcarrier cultivation and virus packaging by the aid of WAVE type bioreactors in a pilot test manner. The novel technology includes (1), cultivating cells, to be more specific, inoculating the 293-series cells onto microcarriers in cell cultivation bags, adding cultivation media into the cell cultivation bags and shaking the cultivation bags; (2), packaging viruses, to be more specific, settling the microcarriers, adding fresh cultivation media, transfection reagents and three-plasmid systems for packaging the viruses to the cultivation bags, continuing to cultivate the cells after co-transfection is carried out and packaging the viruses; (3), separating and purifying the viruses, to be more specific, collecting supernatant by means of centrifuging or suction filtration or filtration after the cells are completely cultivated, and separating and purifying the viruses by the aid of AKTA protein and nucleic acid chromatographic systems; (4), determining titer of the viruses by the aid of Q-PCR (quantitative-polymerase chain reaction) instruments. The novel technology has the advantages that the cells can be automatically cultivated on a large scale and can be transfected, the viruses can be packaged, the recombinant viruses can be produced in a pilot test manner, requirements of the majority of scientific research users can be met, supply and demand contradiction on virus and carrier markets can be relieved, and the labor cost input can be reduced.

Description

technical field [0001] The patent of the present invention relates to a new technology for pilot-scale production of recombinant adeno-associated virus, especially a technology for using WAVE wave bioreactor to carry out 293 series cell microcarrier culture and packaging of recombinant adeno-associated virus to realize the pilot-scale production of virus , belonging to the field of biotechnology. Background technique [0002] Adeno-associated virus (AAV) is currently recognized as a safe and reliable gene delivery carrier in the world, and has been further witnessed in many clinical researches and applications, including Parkinson's disease and Alzheimer's disease Disease, hemophilia, muscular dystrophy, heart failure breast cancer, lung cancer and other tumors and congenital blindness are well treated. [0003] In the past two years, the National Natural Science Foundation of China has increased the proportion of research on AAV among the technical methods involving gene t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/02A61K48/00A61P35/00A61P9/00A61P27/02
Inventor 胡惠忠陈国泽杨兴林蔡永超贾翠英潘讴东祖勇夏清梅殷珊
Owner OBIO TECH SHANGHAI CORP LTD
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