The role of Process & Analytical Development in Stem-Cell based therapies: from bench to bedside, exemplified in tree case studies

Stem-Cell based therapies are one of the promising platforms in the context of Advanced Therapy Medicinal Products (ATMPs). However, their clinical application requires robust manufacturing processes capable of preserving cellular identity, functionality and genetic stability while complying with current Good Manufacturing Practice (cGMP) requirements. Process & Analytical Development (PD/AD) plays a key role in translating the biological potential of stem cells into controlled and reproducible manufacturing processes. This thesis investigates the role of PD/AD through three case studies conducted within a GMP-compliant Contract Development and Manufacturing Organization (CDMO). Specifically, involving Human Pluripotent Stem Cells (hPSCs) and mesenchymal stem cells (MSCs) characterized by increasing regulatory complexity and proximity to clinical application of ATMP development. Cell culture approaches and analytical methods, including flow cytometry and molecular assays, were applied to support process optimization and cellular characterization. The first case study focused on the development of an R&D workflow for genetic modification and clonal selection of hPSCs, towards a GMP compliant one, in a typical Process Development activity. This work shows how systematic comparability of both culture conditions as well as the electroporation instruments used, influenced electroporation performance and overall process efficiency. The impact on the process was significant, despite the inclusion of a subsequent Fluorescence Activated Cell Sorting (FACS) sorting strategy. Additionally, reduced post-electroporation proliferation and limited clonal recovery highlighted critical steps requiring further optimization toward a robust and GMP-compliant workflow. The second case study showcased the role of both Analytical and Process Development in the context of a process involving expansion of a hPSC cell line in stirred-tank bioreactors, whereby all reagents were already GMP compliant. In the assessment of pluripotency, the unexpected upregulation of a gene pseudonymized as ENDO1 triggered a sequence of systematic investigations, highlighting the importance of process understanding during scale up, despite coming from an established R&D protocol. The third case study addressed an unexpected reduction in MSC proliferation during manufacturing for a first-in-human autologous Phase I/II clinical trial. This required an impromptu Analytical Development, to establish an assay to monitor the phenotype. The additional flow cytometry assay revealed a pronounced senescent phenotype, with over 90% senescent cells and reduced Ki-67 expression, supporting the biological investigation of the observed manufacturing behavior. Overall, these case studies demonstrate that PD/AD extends beyond early-stage process optimization and represents a cross-functional and dynamic activity throughout the ATMP lifecycle. Its integration with analytical development and GMP manufacturing supports process understanding, investigation of unexpected cellular behaviors, risk management, and continuous improvement, ultimately contributing to more robust and reproducible manufacturing processes and supporting the clinical translation of cell-based therapies.