Rapid Characterization of Structural and Behavioral\nChanges of Therapeutic Proteins by Relaxation and Diffusion <sup>1</sup>H‑SOFAST NMR Experiments

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