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

化学 三级四极质谱仪 选择性反应监测 关键质量属性 色谱法 抗原 质谱法 分析灵敏度 重复性 串联质谱法 免疫学 病理 物理化学 生物 医学 粒径 替代医学
作者
Nan Xiang,Kehua Zhang,Yinghua Zhao,Chong‐Feng Xu,Xiuqing Zhang,Shufang Meng
出处
期刊:Journal of Pharmaceutical and Biomedical Analysis [Elsevier BV]
卷期号:240: 115886-115886
标识
DOI:10.1016/j.jpba.2023.115886
摘要

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.
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