Tunable purification of plasma extracellular vesicles by multimodal exclusion chromatography for bioanalysis

Purification of extracellular vesicles (EVs) from plasma remains a major challenge for downstream bioanalytical applications because abundant soluble proteins and lipoproteins are readily co-isolated with EVs. Conventional approaches such as ultracentrifugation (UC) and size-exclusion chromatography (SEC) reduce this background but do not eliminate plasma-derived contaminants that can interfere with quantitative and comparative EV analyses. In this study, multimodal exclusion chromatography was evaluated as a post-isolation purification strategy for plasma-derived EV preparations. UC-enriched EVs from multiple donors were further purified using SEC, dual-mode multimodal exclusion chromatography (mEC-2) or tri-mode multimodal exclusion chromatography (mEC-3). Column performance was assessed using nanoparticle tracking analysis, protein quantification, Western blotting and negative-stain transmission electron microscopy. Increasing chromatographic stringency was associated with lower relative albumin and apolipoprotein signals, higher particle-to-protein ratios and reduced non-vesicular background. These improvements were most consistent for mEC-3 but were accompanied by reduced particle recovery compared with SEC. Multimodal exclusion chromatography provides a reproducible and adaptable post-isolation approach for improving the analytical cleanliness of plasma EV preparations, enabling selection of purification stringency according to downstream application requirements.