Mass Spectrometry in Allogeneic CAR-T Cell Manufacturing: From Cellular Starting Materials to Multi-Attribute Quality...

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable clinical efficacy in hematological malignancies, yet their broader application is constrained by manufacturing complexity and variability, particularly in autologous settings. The development of allogeneic CAR T-cell therapies offers a promising alternative by enabling scalable, "off-the-shelf" production; however, these approaches introduce additional challenges related to donor variability, genome editing, and product consistency. Robust analytical strategies are therefore required to ensure safety, efficacy, and batch-to-batch reproducibility. Conventional analytical methods, such as flow cytometry and enzyme-linked immunosorbent assays, provide targeted, high-confidence measurements of predefined cellular and soluble markers but are inherently limited in their ability to capture the full molecular and functional complexity of CAR T-cell products. In current manufacturing paradigms, these assays are typically deployed as isolated quality control readouts rather than as components of an integrated control strategy that links donor variability, gene-editing material quality, in-process metabolic state, final product critical quality attributes, and clinical biomarker responses. In this context, mass spectrometry (MS) has emerged as a powerful platform for high-dimensional molecular characterization, enabling analysis of gene-editing reagents, proteins, metabolites, lipids, and both culture and spent-media composition across the CAR T-cell manufacturing workflow. In this review, we examine the various applications and tools of MS across key stages of the allogeneic CAR T-cell workflow, including donor characterization, analysis of gene-editing materials, in-process culture monitoring, drug product quality assessment, and post-infusion biomarker evaluation. Collectively, these approaches demonstrate the potential of MS-driven analytics to address current limitations in CAR T-cell manufacturing by improving process understanding, enabling comprehensive quality assessment, and supporting regulatory decision-making. The integration of MS into CAR T-cell workflows may ultimately facilitate the development of more consistent, scalable, and effective cell therapies.