Suspension cell-based CAR-CD19 lentivirus preparation process

Abstract

The invention discloses a suspension cell-based CAR-CD19 lentivirus preparation process, which comprises the following steps: S1, recovering HEK293 cells, and carrying out adaptive domestication culture in a domestic culture medium; S2, domesticating the well cultured HEK293 cells to establish a cell bank; S3, amplifying the HEK293 cells until the density of the HEK293 cells reaches the required density; S4, in a four-plasmid system, carrying out transfection on the HEK293 cells; and S5, after transfecting the CAR-CD19 lentivirus plasmid for 48 hours, harvesting a cell culture supernatant, thereby obtaining a lentivirus harvesting solution with the cell viability of 90% or more and the titer of 5*10<6> TU/mL or more. According to the CAR-CD19 lentivirus preparation process, the viability of the cultured lentivirus cells reaches 90% or above, the lentivirus titer of the obtained supernatant reaches 5*10<6> TU/mL or above, and impurities released by cell death can be greatly reduced, so that the pressure of downstream purification is relieved.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more particularly to a CAR-CD19 lentiviral preparation process based on suspension cells. Background technique [0002] Slow viruses are commonly used in chimeric antigens receptor T-Cellimmunotherapy, Car-T), which has high transduction efficiency, integrated T cell genome and continuously expresses target proteins. Nowadays, CAR-T produces most slow viral vectors-mediated CAR gene transfer. Currently, most of the process of producing a slow virus is based on an adherent cellular process (such as HEK293T), which has a slow virus, and a small amount of hardware input. However, it is dependent on pool serum (FBS), which is not appropriate to the current regulations, so it is necessary to strictly audit FBS manufacturers, and the removal is required to evaluate and detect. Second, the batch of fetal bovine serum (FBS) and the difference in origin may also result in a difference in objection to t...

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention not only solves the shortcomings of adherent cells that rely on FBS and is difficult to scale up production, but also avoids the shortcomings of high cost and complicated operation of lentiviral commercial expression systems, and provides a suspension cell-based CAR that can solve the aforementioned problems - CD19 lentivirus preparation process

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