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

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...

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.

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