Characterization of Full-Length Antibody Complexes Through HDX-Mass Spectrometry Coupled With NMR Spectroscopy: New...

Recombinant biomacromolecules have significantly transformed pharmaceuticals, in particular monoclonal antibodies (mAbs) that target key areas in oncology, immunology, inflammation, and neurodegenerative diseases. Developing biotherapeutics requires a detailed understanding of their biological properties. This, in turn, brings about the importance of characterizing higher-order protein structures at each stage, from production to patient. The flexibility and size of the mAbs often prevent achieving atomic-level structural information in the pharmaceutical formulation by traditional methods. In this study, we introduce an integration of solid-state nuclear magnetic resonance (SSNMR) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to explore the interaction between Avelumab and the programmed cell death-ligand 1 (PD-L1). With this approach, we were able to obtain epitope (by both NMR and HDX) and paratope mapping (by HDX), together with a detailed characterization of the structural rearrangements arising upon binding. This methodology provides both mechanistic and structural insights important for the design and optimization of biotherapeutics.