Single-Molecule Dynamics Reflect IgG Conformational Changes Associated with Ion-Exchange Chromatography

Conformational changes of antibodies and other biologics can decrease the effectiveness of pharmaceutical separations. Hence, a detailed mechanistic picture of antibody-stationary phase interactions that occur during ion-exchange chromatography (IEX) can provide critical insights. This work examines antibody conformational changes and how they perturb antibody motion and affect ensemble elution profiles. We combine IEX, three-dimensional single-protein tracking, and circular dichroism spectroscopy to investigate conformational changes of a model antibody, immunoglobulin G (IgG), as it interacts with the stationary phase as a function of salt conditions. The results indicate that the absence of salt enhances electrostatic attraction between IgG and the stationary phase, promotes surface-induced unfolding, slows IgG motion, and decreases elution from the column. Our results reveal previously unreported details of antibody structural changes and their influence on macroscale elution profiles.

Automated summary

This study investigated the conformational changes of antibodies during ion-exchange chromatography, revealing that absence of salt enhances electrostatic attraction, promotes surface-induced unfolding, slows antibody motion, and decreases elution rate. These findings provide new insights into the influence of antibody structural changes on macroscopic elution profiles.