Costariol, Elena; (2021) Establishing a scalable manufacturing process for the clinical scale production of CAR-T cell therapies in stirred-tank bioreactors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

CAR-T immunotherapies present a novel therapeutic modality for the treatment of various blood tumours. However, the development of such immunotherapies requires the manufacture of large numbers of CAR-T cells (2-6 x 108 total viable CAR-T cells per dose), which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culture systems used for their production. Such systems are easy to use in a pre-clinical research settings, but are not efficient when a higher number of doses need to be produced. Furthermore, there is a general concern that primary T-cells are shear sensitive and do not grow in agitated systems, such as stirred-tank bioreactors. This doctoral thesis aims to demonstrate that primary human T-cells and CAR-T cells can be cultivated in stirred-tank bioreactors at different scales (15 ml, 250 ml, and 1 L), which can be used for both autologous and allogeneic products. Furthermore, data in this thesis shows that the growth of T-cells and transduced CAR-T cells was significantly better in stirred-tank bioreactors than in T-flask static culture. At agitation speeds of 200 rpm and greater (up to 500 rpm) in the ambr 250 stirred-tank bioreactor, the CAR-T cells were able to proliferate effectively, reaching viable cell densities of > 5 x106 cells ml-1 over 7 days. This is comparable with current expansion systems and significantly better than static expansion platforms (T-flasks and gas-permeable culture bags). Importantly, the cell quality and potency was assessed at the end of the expansion and was equivalent to the one presented by the cells grown under static conditions as control. It was demonstrated that higher agitation rates, corresponding to higher power inputs lead to a better proliferation. This improvement is likely due to the inability at the lower agitation rates to effectively suspend the Dynabeads used to activate T-cells. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated.

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