Fluorescence recovery after photobleaching investigation of protein transport and exchange in chromatographic media.

A fully-mechanistic understanding of protein transport and sorption in chromatographic materials has remained elusive despite the application of modern continuum and molecular observation techniques. While measuring overall uptake rates in proteins in chromatographic media is relatively straightforward, quantifying mechanistic contributions is much more challenging. Further, at equilibrium in fully-loaded particles, measuring rates of kinetic exchange and diffusion can be very challenging. As models of multicomponent separations rely on accurate depictions of protein displacement and elution, a straightforward method is desired to measure the mobility of bound protein in chromatographic media. We have adapted fluorescence recovery after photobleaching (FRAP) methods to study transport and exchange of protein at equilibrium in a single particle. Further, we have developed a mathematical model to capture diffusion and desorption rates governing fluorescence recovery and investigate how these rates vary as a function of protein size, binding strength and media type. An emphasis is placed on explaining differences between polymer-modified and traditional media, which in the former case is characterized by rapid uptake, slow displacement and large elution pools, differences that have been postulated to result from steric and kinetic limitations. Finally, good qualitative agreement is achieved predicting flow confocal displacement profiles in polymer-modified materials, based solely on estimates of kinetic and diffusion parameters from FRAP observations.