Characterization of antigen presentation capability for neoantigen-based products using targeted LC-MS/MS method

The generation of an immune response in neoantigen-based products relies on antigen presentation, which is closely analyzed by bioassays for T-cell functions such as tetramer or cytokine release. Mass spectrometry (MS) has the potential to directly assess the antigen-presenting capability of antigen-presenting cells (APCs), offering advantages such as speed, multi-target analysis, robustness, and ease of transferability. However, it has not been used for quality control of these products due to challenges in sensitivity, including the number of cells and peptide diversity. In this study, we describe the development and validation of an improved targeted LC-MS/MS method with high sensitivity for characterizing antigen presentation, which could be applied in the quality control of neoantigen-based products. The parameters for the extraction were carefully optimized by different short peptides. Highly sensitive targeted triple quadrupole mass spectrometry combined with ultra-high performance liquid chromatography (UHPLC) was employed using a selective ion monitoring mode (Multiple Reaction Monitoring, MRM). Besides, we successfully implemented robust quality control peptides to ensure the reliability and consistency of this method, which proved invaluable for different APCs. With reference to the guidelines from ICH Q2 (R2), M10, as well as considering the specific attributes of the product itself, we validated the method for selectivity, specificity, sensitivity, limit of detection (LOD), recovery rate, matrix effect, repeatability, and application in dendritic cells (DCs) associated with neoantigen-based products. The validation process yields satisfactory results. Combining this approach with T cell assays will comprehensively assess cell product quality attributes from physicochemical and biological perspectives.