(319e) Volume and Frequency Selective NMR Spectroscopy of Monoclonal Antibodies at Water-Oil Interfaces

Monoclonal antibodies (mAb) represent an important class of biologic therapeutics that can treat a variety of diseases including cancer. Despite many advantages, their processing, storage and/or administration remains challenging because of either the high flow environment in processing or presence of hydrophobic interfaces during administration and storage promote mAb aggregation. In this work, a spatially resolved Point RESolved Spectroscopy (PRESS) sequence was modified to acquire relaxation-enhanced (RE) ¹H NMR spectra at 21.1 T (¹H Larmor frequency = 900 MHz) at the National High Magnetic Field Laboratory. With or without additional water suppression via a VAPOR scheme, this sequence (REPRESS) imparts both spatial and frequency selectivity on the analysis of localized voxels (minimum dimension of 115 μm). Using localizer MRI scans for placement and verification of the REPRESS voxels, the structure of a model mAb was evaluated in the bulk (mAb in sodium phosphate buffer) as well as in incrementally closer voxels (375x1500x1500 μm) that approach a model water-oil interface, as well as voxels at the interface and within the oil. Experiments were performed on a model mAb that consists of a maltose-based protein with a molecular weight of 155 kDa at different concentrations and temperatures. Both short echo time and diffusion-weighted REPRESS acquisitions were acquired from nine adjacent voxel locations in 375 μm steps, with six voxels in the bulk mAb above the interface, one voxel centered at the water-oil interface and two voxels below the interface in the oil. Results indicate that REPRESS spectra of mAb approaching the interface differ dramatically from the bulk mAb. REPRESS mAb spectra in the bulk yield highly resolved lineshapes (FWHM ≈ 30 Hz) and multiple resonances while spectra nearing the interface display significant spectral broadening, indicative of a shortened T₂/T₂^* relaxation that is likely due to conformational alteration of mAb molecular structure and potentially aggregate formation. Through a self-similarity analysis, this study provides the first direct evidence of mAb higher order structure changes at water-oil interfaces.