(M1130-08-50) A Multicomponent Media Analysis Workflow using Triple Quadrupole Mass Spectrometry during Human Primary T cell Culture

Purpose: In Chimeric antigen receptor (CAR) T cell gene therapy, a gene is introduced to the primary T cell to make chimeric antigen receptor proteins that attach only to the cancer cells and attack cancer cells specifically. CART cell therapy has proven to be an effective approach for cancer and is especially successful in treating leukemias and lymphomas. A CART cell therapy consists of T cell extraction, enrichment and activation, transduction, expansion, isolation, and administration steps. The composition of the cell culture media used during the expansion phase can have a significant impact on the final quality of the product CART cells and the therapeutic efficacy. Therefore, obtaining comprehensive real-time media composition results to monitor cell consumption and secretion prior to harvest will be beneficial. The data provides insight on the progress of the cell growth and allow the researchers to intervene, if modification is necessary, to increase the T cell’s and CART cell’s quality and quantity.

Methods: Primary human T cell (to mimic the CAR T cell expansion process), T cell expansion medium, human CD3/CD28 T cell activator, cytokine, and IL-2 were incubated in experimental growing flasks at 37 °C with 5% CO₂. T cell culture media samples of flask A (without media exchange throughout the whole culture period) and flask B (with fresh media exchange at day 3, 5, and 7) from day 0 to 9 were collected and analyzed. Media samples were collected every two hours in the first day and every six hours afterwards. The result obtained from the T-cell culture should be applicable to the CAR T-cell expansion workflow. A 17-minute method that analyzes 144 cell growth related compounds was developed on a triple quadrupole mass spectrometer for T cell/CART cell culture media analysis. Cell culture related compounds including amino acids, nucleic acids, metabolites, sugars, and vitamins were simultaneously analyzed by MRM in both ESI positive and negative modes at each experimental time point. Mobile phases were water and acetonitrile with 0.1% formic acid.

Results: T cells from experimental flask B (with media exchange) at the end of experimental period obtained over 3 times higher live cell count than flask A (without media exchange). Glutamine (m/z 145.2 >127.2; Rt: 1.33 min), serine (m/z 105.9 >60.1; Rt: 1.25 min), 5-glutamylcysteine (m/z 251.1 >122.1; Rt: 2.97 min), and pyruvic acid (m/z 86.9 >43.0; Rt: 1.74 min) had significantly higher concentrations in flask B by the end of the experiment than in flask A. The differentiating compounds were identified by a volcano plot. Time course area trends for all compounds were obtained. Potential T cell metabolomic mechanisms that correlates to the results were identified. For example, time course area trends of glutamine decreased as o-phosphoethanolamine increased during T cell culture experiment. The result may indicate that glutamine starvation led to the accumulation of o-phosphoethanolamine by downregulation of the PCYT2 enzyme. The consumption of serine throughout the T cell culture experiment was observed with a decreasing trend. Serine provides the crucial biosynthetic precursors, glycine and one-carbon units, for purine production. Therefore, extracellular serine intake was expected as T cell expansion process progressed. An increase in glycolysis over oxidative phosphorylation after T cell activation was also expected to support rapid cell growth. This phenomenon was observed with the increasing trend of lactic acid and the decreasing trend of glucose.

Conclusion: A multicomponent T cell/CART cell culture media analysis workflow is demonstrated in this poster. Time course trends for all components during human primary T cell culture were obtained. Differentiating compounds from the two experimental flasks were identified. The real time data obtains from this workflow allows the scientist to monitor the progress of the cell growth during the expansion process by the media environment changes and intervene to increase titer.