Rapid Characterization of Structural and Behavioral Changes of Therapeutic Proteins by Relaxation and Diffusion ¹ H-SOFAST NMR Experiments

Biologic drugs have emerged as a rapidly expanding and important modality, offering promising therapeutic solutions by interacting with previously "undruggable" targets, thus significantly expanding the range of modern pharmaceutical applications. However, the inherent complexity of these drugs also introduces liabilities and poses challenges in their development, necessitating efficient screening methods to evaluate the structural stability and behavior. Although nuclear magnetic resonance (NMR) spectroscopy is well-suited for detecting weak interactions, changes in dynamics, high-order structure, and association states of macromolecules in fully formulated samples, the inherent low sensitivity limits its utility as a fast screening and characterization tool. In this study, we present two fast pulsing NMR experiments, namely the band-Selective Optimized Flip-Angle Internally encoded Relaxation (SOFAIR) and the band-Selective Optimized Flip-angle Internally encoded Translational diffusion (SOFIT)), which enable rapid and reliable measurements of transverse relaxation rates and diffusion coefficients with more than 10-fold higher sensitivity compared to commonly used methods, like Carr-Purcell-Meiboom-Gill and diffusion-ordered spectroscopy, allowing the rapid assessment of biologics even at low concentrations. We demonstrated the effectiveness and versatility of these experiments by evaluating several examples, including thermally stressed proteins, proteins at different concentrations, and a therapeutic protein in various formulations. We anticipate that these novel approaches will greatly facilitate the analysis and characterization of biologics during drug discovery.