A Multi-Omics Approach to Metabolically Optimize a Xeno Free, Serum Free Medium for T Cell Expansion

Background & Aim The manufacture of T cells with high viability and consistent performance has been challenging in the CAR T cell space. Current manufacturing processes use un-defined, serum containing media to grow and expand T cells for CAR T cell therapy which can be a source of consistency challenges. Use of chemically defined (CD) media to expand T cells faces a different set of challenges in terms of providing the optimal combination of nutrients to replace serum, and a poor understanding of the metabolic requirements of cells in any given media. Thus, we have utilized a multi-omics approach to fully characterize the proteomic and metabolomic signatures of T cells expanded in a serum free, xeno-free medium at different phases of growth. Methods, Results & Conclusion We observed significant variation in cell viability in the early phase of expansion that consistently recovers during later stages of expansion. We therefore sampled cells at days 3, 5, and 7 for label free proteomics and untargeted extra and intracellular metabolomics analysis in order to identify metabolic signatures that could be corrected with media supplementation. We identified over 4,500 proteins and 500 metabolites. Using an in-house bioinformatics strategy to analyze the multi-omics data, we highlighted several biological pathways that were significantly different at days 5 and 7 compared to day 3. Media supplements were rationally designed to address the interpreted metabolic needs of T cells at specific times in the expansion phase. Supplementation of media resulted in a significant 24% increase in viability and 3.6 fold increase in population doublings compared to un-supplemented media. In addition, high viability was maintained throughout the culture and performance of individual donors was normalized across multiple platforms such as static plates/bags, G-Rex®, and bioreactors. No impact on cellular phenotype was observed in supplemented versus un-supplemented media. We have incorporated the current media development strategy into designing a metabolic media matrix to address the metabolic needs of diverse donor pools. These data demonstrate the utility of multi-omics approaches paired with rational media design in comparison to traditional approaches, as well as our ability to develop robust CD media that consistently produces high quality T cells at serum-grade performance or better.