Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy

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.

Keywords: CAR-T cell therapy; Functional; HEK293T cells; lentiviral vectors; stirred bioreactors.

Graphical abstract

Figure 1.

293 T suspension cells grown in shake flasks in F medium during cell adaptation. (a) the percentage of cell growth and viability. (b) cell morphology without shaking was observed using a 100× bright field microscope and images were captured using a cell phone camera

Figure 2.

Growth results over 8 d of adapted suspension 293 T cells with cell densities of 1.0 × 10⁵ cells/mL (group A), 2.0 × 10⁵ cells/mL (group B), and 4.0 × 10⁵ cells/mL (group C), respectively. (a) live cell growth curve; (b) cell viability percentages. data are means (SD) of 3 experiments

Figure 3.

Verification of virus packaging capability of 293 T suspension cells. (a) observation of GFP expression in 293 T suspension cells after infection using 100 µm for green field microscopy. (b) 100 µL cell supernatant was collected, treated at 72 h post-transduction, and tested using an HIV p24 Antigen Rapid Test Cassette. the test shows positive results (2 horizontal lines). (c, d) inoculated 293 T cells with 6 increasing amounts (0–64 µL) of the supernatant were detected using a flow cytometer

Figure 4.

Effect of thawing on 293 T suspension cells. (a) Viabilities and densities of 293 T suspension cells grown after freezing in 4 solutions at the first passage and then thawing. **P < 0.01. (b) the titer of the resuscitated adherent and suspension 293 T cells packing the LV. data are means (SD) of 3 experiments

Figure 5.

Verification of the virus packaging capability of 293 T suspension cells in a 5.5 L working volume bioreactor. (a) the percentage of cell growth curve and viability during fermentation; (b) titer of the 293 T suspension cells packing the LV data are means (SD) of 3 experiments. (c) observation of GFP expression in T cells after transduction using 100 µm for green field microscopy. (d) inoculated T cells at a multiplicity of transduction (MOI) of 0, 0.2, 2, 20, and 200 viruses were detected using a flow cytometer. data are means (SD) of 3 experiments. **P < 0.01, ^NSP ˃ 0.05