A Real-Time Mass Spectrometry Platform for In-Process Monitoring and Cell State Analysis

PROJECT SUMMARY/ABSTRACT Advanced cell therapies offer life-saving benefits to patients; however, they are limited by variable quality and efficacy that results in inconsistent treatment outcomes. These challenges arise from difficulties in identifying and controlling key factors that influence the final product’s therapeutic potential, coupled with the complexity of how the final product will interact within each patient’s unique biological landscape. Current cell manufacturing processes often rely on off-line, endpoint assays for cell state analysis and release criteria, which provide limited real-time, in-process insights into critical process parameters (CPPs) and quality attributes (CQAs) predictive of final quality and efficacy. In part, this is due to a void in existing process analytical technologies (PATs) capable of real-time discovery and measurement of CPPs/CQAs. PATs with this capability could facilitate in-process adjustment and allow for deeper, temporal insights into biological processes. In addition, current assays focus on basic quality metrics of the cell product (e.g., viability, proliferation, surface markers, etc.) and neglect the role of the patient’s unique biology on efficacy and outcomes. Tools and assays that can improve understanding of product quality and patient efficacy could drastically improve biotherapeutics manufacturing. Mass spectrometry (MS), in particular electrospray ionization MS (ESI-MS), is a promising PAT for real-time measurement of CPPs/CQAs due to a unique combination of picomolar sensitivity and untargeted, multi-analyte specificity while still preserving biochemical structure. However, ESI-MS for biological specimens is limited by the need for extensive off-line sample preparation before MS and time-consuming, complex data processing after MS, precluding real-time monitoring. Therefore, this work aims to develop an innovative platform tool that integrates with any ESI-MS system for at-line, real-time measurement of media biomolecules (e.g., cell secretome and media additives) for CPP/CQA discovery and monitoring. Our previously developed microfluidic chip provides simplified sample preparation, while an in-development software interface generates critical process insight from ESI-MS data within minutes, addressing both sample preparation and data analytics challenges. During Phase I, a prototype device with integrated components (incubator, liquid handler, and the microfluidic sample preparation device) will be fabricated and evaluated using proof-of-concept MSC-based test beds for cell manufacturing in-process monitoring and for rapid, patient-specific donor cell screening. This will demonstrate real-time monitoring of cell media biomolecules, giving previously unattainable temporal insights into cell state throughout every stage of development. Phase II will expand this real-time monitoring technology for scalable applications (i.e., bioreactors, non-adherent cells) and develop a commercial-grade software interface for automated real-time data extraction and process feedback control. Successful development of this tool will pave the way for new progress in drug discovery and process development, including improved CPP/CQA discovery and monitoring, ultimately improving cell therapy manufacturing and patient outcomes.