Giorgio Carta (Universidade da Virginia, dos Estados Unidos da América) é o convidado do Seminário que tem como tema “Dynamics of Adsorbed Proteins – Conformational Change, Unfolding, and Aggregate Formation during Chromatography".
O evento está agendado para a Faculdade de Ciências (Anfiteatro das Sessões Solenes), com início às 15h00.
A sessão pode ser acompanhada online, através da plataforma Zoom (Link | ID da reunião: 922 1339 2832 | Senha de acesso: 674588).
Abstrat
Conformational change, unfolding, and on-column aggregate formation have been observed for a variety of proteins adsorbed on chromatographic media including ion exchangers and hydrophobic interaction matrices. In these cases, multiple peaks are observed to elute from chromatographic columns even though only a pure protein is loaded. The most common interpretation of on-column aggregate formation is based on the assumption that proteins are destabilized by partial unfolding while bound to the chromatographic surface. When the destabilized molecules are desorbed in a mobile phase with increasing eluting strength, they form aggregates in solution that are detected at the column outlet. In this work, we have studied the behavior of hydrophilic and multimodal anion exchangers using BSA as a model protein. While with hydrophilic resins, loading BSA in pure monomer form and eluting with a salt gradient result in a single peak, multiple peaks with increasing molecular mass are eluted from multimodal resin columns for identical conditions. In this case, the percentage of the protein eluted in higher molecular mass forms increases with the time elapsed between load and elution indicating that the on-column aggregate formation is kinetically limited. Further, if elution is conducted with a multistep gradient, peaks containing individual aggregated species (i.e., dimer, trimer, tetramer, etc.) are recovered at the column outlet. Once recovered, these species are stable and do not revert back to monomer or lower molecular weight species. The two mechanisms below are considered to explain these phenomena. Using a variety of chromatographic and biophysical measurements and modeling we conclude that mechanism 2 is likely responsible for the observed behavior. From a practical viewpoint, this study suggests that optimizing resin surface and load conditions should be considered to reduce aggregate formation rather than attempting to optimize elution conditions.
