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...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2021-08, Vol.93 (32), p.11200-11207
Main Authors: Misiura, Anastasiia, Shen, Hao, Tauzin, Lawrence, Dutta, Chayan, Bishop, Logan D. C, Carrejo, Nicole C, Zepeda O, Jorge, Ramezani, Shahryar, Moringo, Nicholas A, Marciel, Amanda B, Rossky, Peter J, Landes, Christy F
Format: Article
Language:English
Subjects:
Antibodies
Chemistry
Chromatography
Circular dichroism
Dichroism
Elution
IgG antibody
Immunoglobulin G
Ion exchange
Ion-exchange chromatography
Spectroscopy
Stationary phase
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.1c01799