Bioprocess considerations for T‐cell therapy: Investigating the impact of agitation, dissolved oxygen, and pH on T‐cell expansion and differentiation

Abstract

Adoptive T‐cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single‐use 24‐well microbioreactor (micro‐Matrix) was assessed for its use as a high‐throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion‐mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 106 cells/ml. The distribution of CD8+ central memory T cells (TCM), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T‐cell manufacturing process.

High-density microbioreactor process designed for automated point-of-care manufacturing of CAR T cells

“…Apart from being potential future manufacturing platforms, scaled-down microbioreactors could also be valuable process development tools. There is a general lack of process understanding in cell therapy manufacturing 17,52,53 , partly due to the lack of suitable manufacturing technologies at the appropriate scale. This microbioreactor, with four pods per system, and on-board environmental measurements that allow greater process control and predictability, could facilitate future high-throughput process characterization under controlled conditions.…”

“…This microbioreactor, with four pods per system, and on-board environmental measurements that allow greater process control and predictability, could facilitate future high-throughput process characterization under controlled conditions. Probing a larger process design space, such as through Design of Experiments (DoE) or systems modeling methodologies according to Quality by Design (QbD) principles, could lead to a better understanding of how multivariate process parameters and biological factors intersect and impact upon CAR T cell quality attributes 13,52 . This would aid in process optimization studies, such as for different cell therapy products, which could have unique metabolic characteristics 54 , or for different donors, for which considerable biological heterogeneity is well recognized 55,56 .…”

Progress on Electro‐Enhancement of Cell Manufacturing

“…Oxygen (O 2 ) levels are equally vital to optimizing proliferation, [ 49b,64 ] especially for therapeutic cell expansion in a bioreactor. [ 65 ] Most animal cells grow well in 21% atmospheric O 2 levels and manufacturers monitor the levels of dissolved oxygen (dO 2 ) as larger vessels in up‐scaling projects are harder to maintain.…”

“…commented that a controlled shear stress can mimic physiological conditions and mechanotransduction processes in the body. [ 64b ]…”

Modulation of TCR Signaling by Tyrosine Phosphatases: From Autoimmunity to Immunotherapy

“…Other Challenges for Improving Adoptive T Cell Therapy Ex vivo manufacturing of T cells on a commercial scale remains a challenge. Problems include the variability of the starting material between patients and limited understanding of the parameters that are necessary to produce high-quality T cells in sufficient numbers for the transfer (Amini et al, 2020). There is some evidence from animal models suggesting that multiple doses of adoptively transferred T cells are superior to a single infusion, and therefore, expanding as many cells as possible would be an advantage.…”