The dominant cost of manufacturing biologically-based pharmaceuticals is associated with purification, possibly caused by a lack in the molecular-scale understanding of heterogeneous binding dynamics obscured in ensemble techniques.  Single molecule fluorescence microscopic methods were developed to study the dynamics of the protein a-lactalbumin at a clustered-charge anion-exchange chromatographic adsorbent surface, as it has been hypothesized that traditional ion-exchange separations are dominated by adsorption at stochastically clustered sites. Dissociation and association kinetics suggest clustering of adsorbents is required for proteins to be retained in any way other than non-specific interactions with the stationary phase support.  Clustering of adsorbent charges introduced two favorable, long-time retentions not present at isolated single-charged peptide interfaces, and specific interactions were mapped at a super-resolution level of ~40 nm.  Equilibrium constants were calculated from the kinetics and compared directly to ensemble results showing bulk measurements may not be taking advantage of the strongest electrostatic interactions possible.

Super-resolution single molecule adsorption kinetics demonstrate that charge-clustering dominates ion-exchange separations mechanisms

245th National Meeting (2013)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.