Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags.In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr ® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality.Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg −1 ), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 10 6 viable cells ml −1 obtained at the highest P/M of 74 × 10 −4 W kg −1 in comparison with 0.91 ± 0.07 × 10 6 viable cells ml −1 at the lowest P/M of 3.1 × 10 −4 W kg −1 . We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads ® , which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. 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. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads ® in an automated stirred-tank bioreactor system such as the ambr ® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications. K E Y W O R D Sbioprocessing, immunotherapy, manufacture, scale-up, stirred-tank bioreactor, T-cell

Human mesenchymal stem/stromal cells (hMSCs) present a key therapeutic cellular intervention for use in cell and gene therapy (CGT) applications due to their immunomodulatory properties and multi-differentiation capability. Some of the indications where hMSCs have demonstrated pre-clinical or clinical efficacy to improve outcomes are cartilage repair, acute myocardial infarction, graft versus host disease, Crohn's disease and arthritis. The current engineering challenge is to produce hMSCs at an affordable price and at a commercially-relevant scale whilst minimising process variability and manual, human operations. By employing bioreactors and microcarriers (due to the adherent nature of hMSCs), it is expected that production costs would decrease due to improved process monitoring and control leading to better consistency and process efficiency, and enabling economies of scale. This approach will result in off the shelf (allogeneic) hMSC-based products becoming more accessible and affordable. Importantly, cell quality, including potency, must be maintained during the bioreactor manufacturing process. This review aims to examine the various factors to be considered when developing a hMSC manufacturing process using microcarriers and bioreactors and their potential impact on the final product. As concluding remarks, gaps in the current literature and potential future areas of research are also discussed.

Chimeric antigen receptor T‐cell (CAR‐T) therapies have proven clinical efficacy for the treatment of hematological malignancies. However, CAR‐T cell therapies are prohibitively expensive to manufacture. The authors demonstrate the manufacture of human CAR‐T cells from multiple donors in an automated stirred‐tank bioreactor. The authors successfully produced functional human CAR‐T cells from multiple donors under dynamic conditions in a stirred‐tank bioreactor, resulting in overall cell yields which were significantly better than in static T‐flask culture. At agitation speeds of 200 rpm and greater (up to 500 rpm), the CAR‐T cells are able to proliferate effectively, reaching viable cell densities of >5 × 106 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, engineered T‐cells post‐expansion retained expression of the CAR gene and retained their cytolytic function even when grown at the highest agitation intensity. This proves that power inputs used in this study do not affect cell efficacy to target and kill the leukemia cells. This is the first demonstration of human CAR‐T cell manufacture in stirred‐tank bioreactors and the findings present significant implications and opportunities for larger‐scale allogeneic CAR‐T production.