4.1 Conclusions
Current advanced therapy manufacturing faces severe limitations in process control because existing PATs provide analytical outputs based on bulk measurements that do not represent the true state of the product. The Dynamic Sampling Platform (DSP) ESI-MS is a sensitive analytical platform for probing heterogeneities in a bioreactor. DSP incorporates highly localized sampling direct from culture, inline sample treatment, and real-time analysis that can be adapted for a range of cell cultures (e.g., 3D bioreactors) and sensing techniques such as ESI-MS (and others, e.g. Raman, NMR). For this study, DSP was coupled to a spatially resolved sampling inlet for sampling directly from a 2D cell culture bioreactor. The minimal dead volume DSP system enabled inline preparation of a sample for direct ESI-MS analysis via a significantly improved and modified DMSP (Dynamic Mass Spectrometry Probe).19 DSP with real-time, untargeted, MS sensing capability aims to capture and detect low concentration biomolecules secreted by cells. These biomarkers in the microenvironment correlate to cell states relevant for commercial cell production (e.g. differentiation, proliferation, confluence). Capturing and characterizing these CQAs in real-time is critical to enabling feedback control necessary to scaleup and scale out advanced therapeutics.
Application of the DSP enabled in situ ESI-MS analytics to preosteoblast MC3T3 cells revealed that localized sampling was requisite for detection of differences between the cells in an undifferentiated vs a differentiated state. The mass spectrometer in this study was a time-of-flight (TOF) style, which provides fast analysis and accurate mass of biomolecules but cannot fragment these molecules for database matching. Exploratory work on matching the PCA based m/z values to tentative chemical IDs from offline HPLC-MSn of the same samples resulted in multiple matches, which suggests that DSP may have utility as a discovery tool. Future work aims to incorporate DSP into workflows with tandem mass spectrometry capabilities, allowing for real-time identification of detected biomolecules with both accurate mass and fragmentation patterns, facilitating the complete identification of CQAs in real-time. Importantly, in its current design, DSP can be used in conjunction with traditional approaches such as HPLC-MS to bring an important new dimension of dynamic monitoring to clinically relevant workflows. Ultimately, DSP should allow for better understanding of how secreted biomolecules correlate with cell state and growth trajectory (e.g., MSC differentiation or T-Cell CD4 vs CD8 sub-type concentration) and, when developed further, enable the real-time monitoring of CQAs for improved manufacturing within all cell-based workflows.33-35