2D 1HN, 15N Correlated NMR Methods at Natural Abundance for Obtaining Structural Maps and Statistical Comparability of Monoclonal Antibodies

High-resolution nuclear magnetic resonance spectroscopy (NMR) provides a robust approach for producing unique spectral signatures of protein higher order structure at atomic resolution. Such signatures can be used as a tool to establish consistency of protein folding for the assessment of monoclonal antibody (mAb) drug quality and comparability. Using the NIST monoclonal antibody (NISTmAb) and a commercial-sourced polyclonal antibody, both IgG1 kappa isotype, we apply 2D NMR methods at natural abundance for the acquisition and unbiased statistical analysis of H-1(N) -N-15 correlated spectra of intact antibody (Ab) and protease-cleaved Fab and Fc fragments. The study demonstrates the feasibility of applying 2D NMR techniques to Abs and the precision with which these methods can be used to map structure and establish comparability between samples at atomic resolution. The statistical analyses suggests that, within the limit of detection, no significant structural differences are observed between the Fab and Fc domains of each respective intact Ab and its corresponding fragments. Discrimination between dissimilar species, such as between the Fab domains of both Abs or between the glycosylated and deglycosylated Fc domains, was further demonstrated. As such, these methods should find general utility for the assessment of mAb higher order structure.