Zalutumumab

Validation of epitopes and paratopes for antibodies and antigens is crucial for understanding antibody binding and is of interest in the pharmaceutical field. Traditionally, high-resolution techniques like X-ray and NMR are used for delineating protein–protein interactions, but they are prone to sample preparation problems and size limitations. Mass spectrometry (MS)-based structural analysis, like H/D exchange (HDX), covalent labeling, and cross-linking, contributes unique information and advantages relative to traditional methods. As such, it is desirable to compare all three techniques side by side and assess how complementary their results are for defining epitopes on the targets of interest. In this study, we probed interaction sites of epidermal growth factor receptor (EGFR) with cetuximab and zalutumumab using HDX, covalent labeling, and cross-linking techniques. For covalent labeling experiments, sulfo-NHS acetate and DEPC were used as the labeling reagents. In cross-linking experiments, multiple cross-linkers, including BS3, EDC, DSSO, and ADH/DMTMM, were used for a comprehensive search of protein–protein interactions. The results from cross-linking and covalent labeling were compared to HDX as well as known epitopes. Covalent labeling, cross-linking, and HDX data showed consistent results for EGFR-Cetuximab epitopes. For the EGFR-Zalutumumab complex, no intermolecular cross-links within reasonable distances were identified, but the epitopes observed from covalent labeling agree with HDX. The results are consistent with reported crystal structures. Overall results with DEPC and sulfo-NHS labeling are comparable to HDX with a faster cycle time, making them amenable for rapid screening of many molecules. DEPC labeling has more residue coverage compared to sulfo-NHS acetate but faces unique challenges like faster hydrolysis and more complex modifications. We further extended the use of covalent labeling experiments in a cellular environment with the same model system and with results consistent with purified proteins, making covalent labeling a suitable method to screen epitopes for complex targets and receptors directly from cells.