Optimising process quality and cost for closed, automated and commercial scale manufacture of allogeneic stem cell therapy products

Embryonic stem cell (ESC) derived therapies offer huge potential for clinical and economic benefit; however, their development is currently restricted by the suitability of available equipment and limited scalability of common processing techniques. Although several current cell manufacturing technologies are hypothetically suitable, systems developed for more mature cell therapies such as mesenchymal stem cells are not scalable down to processing scales suitable for allogeneic ESC derived therapies, while small scale systems developed for autologous therapies such as chimeric antigen receptor T cells lack the processing capabilities required for adherent cells. This thesis explores the complexities faced in transferring allogeneic ESC derived cell therapy products (CTP) from manual processing, where such processes are typically developed, to closed, automated and commercial scale production systems.

This work first documents the intricacies of technology transfer in CTP production. The case example is a protocol to produce 5 x 108 mesencephalic dopaminergic (mesDA) progenitor cells on the CliniMACS Prodigy cell processing platform system from Miltenyi Biotec to Loughborough University. Numerous complex device and process robustness interactions are identified, with TrypLE and DTI contamination of above 3.31% for H9 cells and 1.95% for RC17 cells during cell seeding as a result of insufficient purification established as a key point of failure. This work highlights how a cell with a high level of environmental sensitivity compounds typical issues in multi-site protocol transfer and verification processes. Downstream processing techniques compatible with closed and automated processing are then explored, including the effects of physical manipulation on cell harvesting, the efficiency of closed and automated purification techniques, as well as the effects on cryopreservation of increased batch sizes and closed cryopreservation containers, with improved techniques determined in each case.

Based upon the knowledge obtained as a result of the technology transfer and downstream processing investigation, a process economics model is presented establishing baseline costs of £12,960 for manual processing and £20,106 for closed and automated processing against which process improvements may be compared. The TrypLE and DTI contamination issue highlighted during the technology transfer is explored in depth, with the cost and quality impacts of potential process improvements calculated and an optimal improvement plan proposed, providing potential savings of up to £444 for a H9 based process and £330 for an RC17 based process. This investigation highlights the possibility of successfully performing the protocol on the Prodigy and highlights a lack of data driven process change performed by protocol developers as part of their troubleshooting process. Finally, the complexity of CTP manufacturing processes is discussed and the importance of process robustness and fit for purpose process verification is underlined.