PROJECT SUMMARY/ABSTRACT Monoclonal antibodies (mAbs) represent an important class of biologic therapeutics that can treat COVID-19, cancer and other infectious diseases. Despite their promising potential, pro- cessing, storage and/or administration of mAbs into patients is challenging because the presence of hydrophobic interfaces during processing and administration (air entrapment in the IV bags or the oil-water interface at the interior of syringes) may promote mAb adsorption to such hy- drophobic interfaces. If mAbs change their native (folded) higher order structures (HOS) upon adsorption to these interfaces, their quality, safety and efficacy will be affected, posing immuno- genicity risks to already susceptible patients. The first step in mitigating these risks is to evaluate the in situ HOS of mAbs (whether folded or unfolded) at hydrophobic interfaces. Determining the in situ structure of mAbs at such interfaces has been a major challenge due to limitations of bulk scale or scattering-based microstructural probing techniques. In this program, we will go beyond such limits and use a combination of a unique molecular probing technique based on NMR spec- troscopy and dynamic surface tensiometry to resolve the details of mAbs HOS and adsorption kinetics at hydrophobic interfaces. In particular, by using high-field spatially and spectrally re- solved NMR spectroscopy that is uniquely available to use through National High Magnetic Field Laboratory, we will assess dynamically 1) the in situ HOS of pure mAbs at hydrophobic interfaces, and 2) nature of their associations with surfactants at interfaces. We will perform tensiometry along with NMR spectroscopy on pure mAbs, isotopically labeled mAbs and mAbs/surfactant combinations at hydrophobic interfaces. We will measure a) dynamic surface tension, b) spa- tially localized chemical shifts in 1D 1H and 2D 1H-13C NMR spectra, c) diffusion coefficients of the mAbs, and d) T2 relaxation of mAbs in the bulk and at the interface under different conditions (e.g., various mAbs and surfactant concentrations, solution pH and ionic strengths). By compar- ing the results of the bulk and interface in terms of metrics (a-d), the team will determine if the native HOS of mAbs has been altered by adsorption to hydrophobic interfaces or their associa- tions with surfactants. The outcome of this study will provide the first mechanistic understanding of mAbs HOS at hydrophobic interfaces. Additionally, the knowledge gained from this research is essential in developing a framework to mitigate mAbs adsorption to hydrophobic interfaces, which can be subsequently utilized to improve efficacious mAb deployment for patients. 1