Scalable Workflow for Extracellular Vesicle Isolation, Purification and Storage Guided by Subtype-Specific Features

Extracellular vesicles (EV) are promising drug delivery platforms; however, clinical translation is limited by challenges in scalable production and storage. This study evaluates how isolation, purification, and preservation strategies influence the physicochemical properties, yield, surface composition, and functional cargo of large EV (lEV) and small EV (sEV), using erythropoietin (EPO) as a model therapeutic cargo. EV were isolated by ultracentrifugation (UC) alone or combined with tangential flow filtration (TFF), and stored under - 80°C (reference condition), refrigerated, or lyophilized using a standard (Std Lyo) or a proprietary optimized cycle (Opt Lyo) designed to minimize structural damage without lyoprotectants. Refrigeration reduced particle recovery and altered surface charge in both EV subtypes, indicating decreased stability. Std Lyo induced structural alterations and fragmentation, particularly in sEV, only partially mitigated by trehalose addition. In contrast, Opt Lyo better preserved size distribution and physicochemical stability across subtypes. Both lyophilization cycles altered surface marker detectability, reflecting membrane reorganization. TFF markedly enhanced yield and purity, especially in sEV, but also modified EV composition and their response to Opt Lyo, influencing downstream stability. EPO cargo in lEV showed no significant differences across conditions, with lower mean values after lyophilization, whereas sEV were more sensitive. Uptake assays revealed subtype- and process-dependent differences in cellular internalization, with limited involvement of heparin-sensitive pathways under the tested conditions. Collectively, these findings support a scalable workflow in which TFF-based purification followed by UC and Opt Lyo maximize overall performance, while revealing subtype-specific vulnerabilities that define the final EV product and inform tailored optimization.