ArticleAnalytical methods : advancing methods and applications2026
Repeatability and reproducibility of chromatographic parameters and resulting figures of merit on polyester C-CP fiber columns for the routine isolation and analysis of HEK293-derived extracellular vesicles.
Article in Analytical methods : advancing methods and applications, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
An exponential growth in the interest in extracellular vesicles (EVs) has been observed in recent years, as evidenced by the increasing number of research publications and the growing number of potential EV-based therapeutics in the development pipeline. A current bottleneck in the field, however, remains the need for efficient and reproducible methods for the routine and scalable isolation of EVs. Here, the suitability of polyester capillary-channeled polymer (C-CP) fiber columns operated in hydrophobic interaction chromatographic mode is demonstrated for the routine analysis of extracellular vesicles, using retention time, peak area, and column backpressure as performance metrics. Run-to-run repeatability (single column) and consistency in the fiber column assembly process (column-to-column reproducibility) were demonstrated on 10 analytical columns from 5 assembly batches. Both repeatability and reproducibility showed high consistency, with low relative standard deviations for retention time (<0.6% RSD) and peak area (<4% RSD). Pressure measurements showed greater variability with <7% RSD for run-to-run repeatability and <17% RSD for column-to-column reproducibility. The chromatographic profiles were highly reproducible across all five batches. A previously prepared column (six months old) showed very similar performance to newly made columns, confirming column stability during storage. Method robustness was confirmed over five days on two columns, with inter-day variability of 0.3% RSD for retention time and 1.2% RSD for peak area. A four-minute, "fast" method was developed for scouting analysis conditions and for in-process monitoring of EV levels during cell culture. Finally, the optimal injection volume of the clarified culture medium is established when targeting separation efficiency for subpopulation analysis (4 µL) and optimum injection volume (12 µL) for isolation of bulk EV populations.
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