1. Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy
- Author
-
Yao Xu, Li-Xing Gu, Yong Zhou, Xing-Hua Liao, Tong-Cun Zhang, and Qu-Lai Tang
- Subjects
0301 basic medicine ,CAR-T cell therapy ,Virus Cultivation ,T-Lymphocytes ,Cell ,Genetic Vectors ,Bioengineering ,lentiviral vectors ,HEK293T cells ,Gene delivery ,Applied Microbiology and Biotechnology ,Immunotherapy, Adoptive ,Cryopreservation ,Viral vector ,03 medical and health sciences ,0302 clinical medicine ,Bioreactors ,Bioreactor ,medicine ,Humans ,Functional ,Chemistry ,HEK 293 cells ,Lentivirus ,General Medicine ,Chimeric antigen receptor ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,HEK293 Cells ,030220 oncology & carcinogenesis ,stirred bioreactors ,CAR T-cell therapy ,TP248.13-248.65 ,Biotechnology ,Research Article ,Research Paper - Abstract
As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.
- Published
- 2021