Extracellular vesicles (EVs) hold great promise in the fields of diagnostics and therapeutics. However, the heterogeneity of these membrane-enclosed messengers and the complexity of the biological samples in which they occur pose significant research challenges. The aim of this study was to improve the reliability of size-exclusion chromatography (SEC) and immunolabelling as common approaches in the EV field, and to provide a comprehensive characterisation tool for diverse EV preparations. Profiling of SEC-separated sample components was conducted through light absorbance, fluorescence, and light scattering, providing insights into particle content, size, protein content, and specific markers. Key considerations for assay robustness, including SEC mobile phase composition and immunostaining parameters, were addressed. Respecting the importance of controlled immunolabelling and preanalytical factors, the method efficiently reveals changes in the sample profile with respect to particles and small impurities. The detailed analytical capabilities and method adaptability offer a practical way to enhance the efficiency of EV research and applications.

Mesenchymal stem cell‐derived extracellular vesicles (MSC‐EVs) therapeutics hold the potential to harness the regenerative power of their parental cells, promoting proliferation and reducing inflammation. There is a need for the production and isolation process that can be easily scaled and deliver the desired quality and quantity of EVs. We present a chromatographic method development based on preferential exclusion principle that can be used for EV isolation from adherent and microcarrier‐based upstream. The method employs monolithic hydroxyl columns to purify and concentrate MSC‐EVs. The heterogeneity in EV size and composition poses challenges in upstream and downstream processes; therefore, process development was guided by PATfix biochromatography analytics, tracking EV‐specific surface antigens. The developed purification method enables direct concentration of MSC‐EVs from conditioned media while removing 97% of protein impurities and 95% of double‐stranded deoxyribonucleic acid (dsDNA)‐related impurities in the process.