Bench-top NMR of water signals: A non-destructive tool for biomacromolecule characterization

Bench-top NMR spectroscopy has emerged as an accessible, non-destructive tool for analyzing biomacromolecules in solution. This review focuses on how water proton NMR (wNMR) provides sensitive readouts of protein states in solution. We highlight representative studies on monoclonal antibodies, insulin, and vaccines under diverse stresses, where wNMR measurements consistently correlate with aggregation and stability outcomes. In parallel, we revisit the fundamental principles of wNMR, focusing on the molecular origins of water relaxation and the roles of hydration dynamics, proton exchange, and water confinement near protein surfaces. To illustrate these mechanisms, we present simple illustrative experiments using bench-top wNMR and ATR-FTIR spectroscopy with aqueous salts and polymers, revealing how non-protein solutes modulate hydrogen-bond networks and water dynamics. Finally, we discuss emerging perspectives that extend the scope of bench-top NMR beyond protein formulations. Together, these developments position bench-top NMR not only as a practical tool for stability assessment but also as a promising framework for probing water-mediated phenomena across biopharmaceutical and soft-matter systems. • Bench-top NMR enables nondestructive evaluation of protein formulations • Water proton relaxation monitors protein aggregation and stability changes in solution • Control studies with salts and polymers clarify water signal determinants • Potential applications include vaccines, viral capsids, and nucleic acid therapeutics