SUMMARY

Real-time, advanced diagnostics of the biochemical state of cells remains a significant challenge for the development, production, and application of cell-based therapies. The development of new process analytical technologies is critical not only for characterization of the cell state in research settings, but also monitoring and control of the complex and dynamic biomanufacturing workflows needed to produce these therapies. The proposed research aims to develop and apply a new process analytical technology to enable rapid, in situ, intracellular analysis from small numbers of cells removed directly from bioprocessing systems. Preliminary research has focused on parallel development of two sample preparation workflows. The first is an integrated microfluidic platform for in-line analysis of a small number of cells via direct infusion nano-electrospray ionization mass spectrometry (ESI-MS). Central to this platform is a microfabricated cell processing device that prepares cells from limited sample volumes removed directly from cell culture systems. The second workflow is a liquid-liquid extraction scheme leveraging temperature induced phase separation of aqueous-acetonitrile solutions in thermoelectrically cooled microchannels. Following this binary chromatographic separation scheme, both hydrophobic and hydrophilic analytes display enhanced detection from complex, biochemically relevant solutions. The integration of these sample preparation technologies will result in a sample-to-analysis workflow designed to overcome the labor intensive, time-consuming, and destructive nature of existing mass spectrometry approaches for sensitive and specific analysis of cells. This new process analytical technology will be applied for dynamic, in situ monitoring of cell culture systems, representing a critical step toward in-process control of cell therapies.