Atomic Level Characterization of Excipient Interactions with Monoclonal Antibodies

Monoclonal antibodies (mAb) are the primary therapeutic platform for biologic drug development due to their high specificity and binding affinity to a clinical target. Protein therapeutics such as mAbs must retain their higher order structure (HOS) to be safe and effective, making HOS a critical quality attribute (CQA) of mAb drugs. Correspondingly, once a mAb candidate is selected for further clinical development, formulation of the drug substance is undertaken to find drug compositions that support long term stability and effective drug delivery. Suitable excipient composition will help avoid high viscosity and self-aggregation of mAbs and ensure the maintenance of proper HOS. Currently, the standard practice for formulation characterization screening employs low and moderate resolution analytical technologies, such as CD, DLS, FTIR. These techniques, however, are not sufficient, as it is becoming increasingly clear that description of the molecular structure of mAbs must take into account conformational fluctuations, internal dynamics, and intermolecular interactions including excipient-induced changes to mAb HOS. With the recent publication of the nuclear magnetic resonance (NMR) backbone assignments of the Fab domain derived from a model IgGκ platform therapeutic NISTmAb , biophysical studies are now possible to investigate Fab HOS at atomic level using high resolution NMR. Here, based on a review of all commercial mAb formulations, we performed an in-depth NMR analyses with common excipients, including sodium chloride, sucrose, and polysorbate 80. 1H,15N relaxation series show that excipients can induce HOS conformational changes, especially on the addition of sodium chloride and sucrose. Conversely, polysorbate 80 seems to have very little effect. The analysis of order parameter S2 calculated using d’ Auvergne protocol (DIFF_MODEL = “ellipsoid”), suggests a breathing conformation of NIST-Fab in the presence of salt or sucrose. Based on our findings, we provide a basis for the stabilizing of NIST-Fab by excipients based on structure and dynamics. Our investigation will help to elucidate the biophysical understanding of mAb-excipient interactions and help guide antibody formulation during clinical development.