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

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.

Adaptation for survival: phenotype and transcriptome response of CHO cells to elevated stress induced by agitation and sparging.

In this work, the response and adaption of CHO cells to hydrodynamic stress in laboratory scale bioreactors originating from agitation, sparging and their combination is studied experimentally. First, the maximum hydrodynamic stress, τ(max), is characterized over a broad range of operating conditions using a shear sensitive particulate system. Separate stress regimes are determined, where τ(max) is controlled either by sparging, agitation, or their combination. Such conditions are consequently applied during cultivations of an industrial CHO cell line to determine the cellular responses to corresponding stresses. Our results suggest that the studied CHO cell line has different threshold values and response mechanisms for hydrodynamic stress resulting from agitation or sparging, respectively. For agitation, a characteristic local minimum in viability was found after stress induction followed by viability recovery, while at highest sparging stress a monotonic decrease in viability was observed. If both stresses were combined, also both characteristic stress responses could be observed, amplifying each other. On the other hand, cellular metabolism, productivity and product quality did not change significantly. Transcriptome analysis using mRNA microarrays confirmed that separate adaptation mechanisms are activated in the different stress situations studied, allowing identification of these stresses using a transcriptome fingerprinting approach. Functional analysis of the transcripts was consequently used to improve our understanding of the molecular mechanisms of shear stress response and adaptation.