Exploring Size Exclusion Chromatography Columns 20 and 35 nm Pore Size Effect for Isolation of Extracellular Vesicles

Abstract Extracellular vesicles (EVs) have shown great promise as minimally invasive biomarkers for a variety of diseases. However, challenges persist regarding EV isolation, particularly in their co-isolation with impurities such as soluble proteins and lipoproteins. Among the methods available for EV isolation, size-exclusion chromatography (SEC) is widely used, as it is reproducible and amenable to high-throughput with a rapid turnaround time. However, its size-based separation leads to the co-isolation of EVs with impurities of similar size. This study, for the first time to our knowledge, compares SEC columns with different pore sizes, 20 and 35 nm, to evaluate their efficacy in non-EV contaminant removal and EV recovery from pancreatic EndoC-ßH1 cell culture media and human plasma. To assess EV purity and yield, we compare EV particle concentration, the presence of unintended co-isolates, and RNA EV cargo. This study demonstrates that smaller pore size SEC columns enhance EV yield and purity, making them ideal for biomarker studies involving limited biological samples or downstream analysis sensitive to contaminants. Competing Interest Statement SD is a founding member and has equity in Thryv Therapeutics and Switch Therapeutics. He also consults for Thryv Therapeutics. None of these are relevant for this project. RNK is on the Scientific Advisory Boards for Novo Nordisk, Biomea and REDD Pharmaceuticals. The rest of the authors declare no competing interests.