Optimization of protein separation and purification is an empirical process that lacks predictability and is known to be the bottleneck in downstream processing of protein-based drugs, also called biologics. One of the most widely used methods of protein purification and separation is chromatography. Despite the wide industrial usage and intensive development, there is still no detailed molecular-scale picture of protein dynamics during chromatographic separation. The lack of a predictive chromatographic theory is rooted in the absence of an in-depth understanding of interactions occurring inside a chromatographic column. To advance the understanding of underlying phenomena responsible for successful chromatographic separation of biologics, a microscopic, single-molecule picture of the elution process is necessary. The macroscopic picture of chromatography is dictated by processes at the single-molecule level. However, ensemble methods inherently average underlying heterogeneity and, therefore, are not able to yield a full understanding of chromatography on a microscopic level. This work focuses on ion-exchange chromatography (IEX) which separates proteins based on their net surface charge. By using both ensemble and single-molecule techniques we want to develop a mechanistic understanding of IEX. We use Circular Dichroism (CD) to show that negatively charged ligands in cation exchange column cause surface-induced unfolding of the proteins. Using 3D single-molecule fluorescence microscopy we observe increased linear velocities of protein molecules at increased ionic strengths. From our chromatographic profiles, single-molecule results and CD spectra we conclude that lower salt concentrations of a mobile phase lead to stronger surface-induced protein unfolding and lower linear velocity of the protein molecules inside chromatographic column. This work allows us to resolve the details of protein dynamics inside chromatographic column, providing a molecular-scale picture of protein chromatography.

Convergence of ensemble and single-molecule techniques to understand protein liquid chromatography

2019 Fall National Meeting

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