Insights into the Conformation and Self-Association of a Concentrated Monoclonal Antibody using Isothermal Chemical Denaturation and Nuclear Magnetic Resonance

The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. In this case study, we compared results from isothermal chemical denaturation (ICD) and NMR with standard methods to relate conformational states of a model monoclonal antibody (mAb1) with protein-protein interactions (PPI) that lead to self - association in concentrated solutions. The increase in aggregation rate and relative viscosity for mAb1 was found to be both concentration and pH dependent. The free energy of unfolding (Delta G degrees) from ICD and thermal analysis in dilute solutions indicated that although the native state predominated between pH 4 - pH 7, it was disrupted at the CH2 and unfolded noncooperatively under acidic conditions. One-dimensional (1D) H-1 NMR and two-dimensional (2D) C-13-H-1 NMR performed, in concentrated solutions, confirmed that PPI between pH 4-7 occurred while mAb1 was in the native state. NMR corroborated that mAb1 maintained a dominant native state at formulation-relevant conditions at the tested pH range, had increased global molecular tumbling dynamics at lower pH and confirmed increased PPI at higher pH conditions. This report aligns and compares typical characterization of an IgG1 with assessment of structure by NMR and provided a more precise assessment and deeper insight into the conformation of an IgG1 in concentrated solutions. (C) 2021 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association.

Automated summary

This investigation highlighted the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. Comparing results from isothermal chemical denaturation (ICD) and NMR with standard methods provided insights into the conformational states of a model monoclonal antibody (mAb1) and its protein-protein interactions (PPI) in concentrated solutions at different pH levels. NMR analysis confirmed the dominant native state of mAb1 under formulation-relevant conditions and provided a deeper understanding of its conformation in concentrated solutions.