Chromatographic protein separations at both laboratory and industrial scales are often optimized empirically. Given the increasing importance of protein separations to pharmaceutical applications, access to mechanistic details could allow us to improve separations more efficiently rather than relying on time-intensive, iterative column optimizations. We demonstrate that it is possible to combine super-resolution experimental methods and kinetic analysis with a rigorous statistical theory to provide mechanistic insight on a model protein separations system. We report direct measurement of super-resolved single a-lactalbumin adsorption/desorption at single ligands on an agarose support, allowing the first direct test of a molecular-scale statistical theory of protein adsorption that was developed over 50 years ago. Together with a molecular-scale comparison of engineered vs. accidentally clustered ligands, the resulting simulated chromatographic elution profiles demonstrate that engineered ligand clusters should be pursued for next-generation stationary phases for ion-exchange protein purification.

Super-resolution methods for understanding adsorptive protein separations

248th National Meeting (2014)

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