We present the direct super-resolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. Chromatographic protein separations, immunoassay, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is only available via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that functional adsorbent sites can be mapped with a resolution of 30 nm, that clusters of charges are necessary to create detectable adsorption sites, and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that for ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the separations process only accidentally.

Unified super-resolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

247th National Meeting (2014)

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