Francesca Sacco, Fabio Baroni, Angela Capolupo, Sofia Petrocchi, Jonathan Zöller, Linda Cerofolini, Pasquale Russomanno, Rebecca Calamandrei, Bianca Susini, Siyu Lin, Fabio D'Amici, Enrico Ravera, Julian D. Langer, Marco Fragai
ABSTRACT 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.