Abstract
MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize the safety of biotherapeutics by driving the selection of the best candidates concerning their immunogenicity risk. However, there is little knowledge about the impact of bead material on the recovery of MHC-II MS-eluted ligands in MAPPs assays. Here, we firstly describe a robust MAPPs protocol by implementing streptavidin magnetic beads for the isolation of these peptides instead of commonly used NHS-activated beads. Moreover, we assessed the impact of the cell medium used for cell cultures on the morphology and recovery of the in vitro-generated APCs, and its potential implications in the amount of MHC-II isolated peptides. We also described an example of a MAPPs assay application to investigate drug-induced immunogenicity of two bispecific antibodies and compared them with monospecific trastuzumab IgG1 control. This work highlighted the importance of MAPPs in the preclinical in vitro strategy to mitigate the immunogenicity risk of biotherapeutics.