Mapping conformational changes on bispecific antigen-binding biotherapeutic by covalent labeling and mass spectrometry.

Biotherapeutic's higher order structure (HOS) is a critical determinant of its functional properties and conformational relevance. Here, we evaluated two covalent labeling methods: diethylpyrocarbonate (DEPC)-labeling and fast photooxidation of proteins (FPOP), in conjunction with mass spectrometry (MS), to investigate structural modifications for the new class of immuno-oncological therapy known as bispecific antigen-binding biotherapeutics (BABB). The evaluated techniques unveiled subtle structural changes occurring at the amino acid residue level within the antigen-binding domain under both native and thermal stress conditions, which cannot be detected by conventional biophysical techniques, e.g., near-ultraviolet circular dichroism (NUV-CD). The determined variations in labeling uptake under native and stress conditions, corroborated by binding assays, shed light on the binding effect, and highlighted the potential of covalent-labeling methods to effectively monitor conformational changes that ultimately influence the product quality. Our study provides a foundation for implementing the developed techniques in elucidating the inherent structural characteristics of novel therapeutics and their conformational stability.