The aim of the study was the development of a robust and high-throughput method that would enable a comprehensive evaluation of extracellular vesicles (EVs) in complex samples. Our goal was to provide an insight into the heterogeneity of thtwo-dimensional liquid chromatography (2D-LC), in which we coupled the isolation of nanoparticles employing size exclusion chromatography (SEC), with their subsequent separation using anion-exchange chromatography (AEX).
The developed 2D SEC-AEX method takes 30 minutes. The result of the analysis is a detailed sample profile, displaying: 1) sample components resolved by their size, and 2) heterogeneity of particles, resolved by their charge.
The applicability of the proposed approach is demonstrated through the evaluation of various samples, including conditioned media from different cell cultures, and EV-enriched preparations. The obtained profiles display the differences in sample compositions e present EV- and non-EV particles while at the same time elucidating the background matrix. We designed a and indicate the possible strategies for further fractionation and purification of populations of interest.
