Probing short time diffusion of NISTmAb to predict long-term therapeutic stability of monoclonal antibodies

Hydrogen deuterium exchange, (HDX) is of increasing interest for characterization of protein dynamics in solution, at equilibrium state of different conformations. The natural tendency for proton exchange of surface labile hydrogen atoms for deuterium can indicate which portions of the protein have been exposed to the solvent. Though this differs from the more traditional methods for approximating protein dynamics, like nuclear magnetic resonance and cryo-transmission electron microscopy, quantifying HDX with mass spectrometry allows for identification of site-specific deuterium incorporation. From this, it can be inferred that these sites have been exposed to the solvent, via the protein’s cyclical occupation of alternate conformational states. A different approach of measuring HDX is possible with small angle neutron scattering, (SANS), which is extremely sensitive to hydrogen exchange due to the large scattering cross section of hydrogen. Unlike HDX-MS, SANS allows for the continual measurement of HDX over time. By using SANS to study temperature probed HDX of monoclonal antibody, NISTmAb RM 8670, over the course of numerous days, the effect of different buffer conditions, temperature dependency, and time dependency, on HDX, can be extracted. The rate and amount of HDX, (as reported in the attached figure) measured with HDX-SANS correlated with the expected stability of different NISTmAb formulations containing Hofmeister salts, where more destabilizing Hofmesiter salt excipients yielded enhanced deuteron incorporation. These results are compared with complimentary findings of short time diffusion measured by neutron spin echo to examine how the stability information obtained from HDX-SANS is related to the internal domain motions of NISTmAb. Data analysis techniques for HDX-SANS and comparison with neutron spin echo results will be discussed to reveal novel information about protien dynamics, of critical relevance to the biopharmaceutical industry.