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Single-Molecule Dynamics Reflect IgG Conformational Changes Associated with Ion-Exchange Chromatography.
Anastasiia Misiura1, Hao Shen2, Lawrence Tauzin1
1Department of Chemistry, Rice University, 6100 Main Street, Houston, Texas 77005, United States.
Analytical Chemistry
|August 4, 2021
Summary
Antibody unfolding during ion-exchange chromatography (IEX) is influenced by salt conditions. Lower salt concentrations promote unfolding and alter protein motion, impacting elution profiles in pharmaceutical separations.
Area of Science:
- Biochemistry
- Chromatography
- Protein Science
Background:
- Conformational changes in biologics like antibodies can compromise pharmaceutical separation efficiency.
- Understanding antibody-stationary phase interactions in ion-exchange chromatography (IEX) is crucial for optimizing these processes.
Purpose of the Study:
- To investigate the impact of conformational changes on antibody motion and elution profiles during IEX.
- To elucidate the mechanistic details of antibody-stationary phase interactions under varying salt conditions.
Main Methods:
- Utilized ion-exchange chromatography (IEX) coupled with three-dimensional single-protein tracking.
- Employed circular dichroism spectroscopy to monitor conformational changes in a model antibody (immunoglobulin G, IgG).
Main Results:
- Absence of salt enhanced electrostatic attraction between IgG and the stationary phase.
- Low salt conditions promoted surface-induced unfolding of IgG, slowing protein motion.
- Altered IgG motion and unfolding significantly decreased elution from the IEX column.
Conclusions:
- Revealed previously unreported details of antibody structural changes during IEX.
- Demonstrated how IgG conformational alterations influence macroscale elution profiles.
- Provided critical insights into antibody-stationary phase interactions for improved pharmaceutical separations.
