Developing a process control strategy for the consistent and scalable manufacture of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03· 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.