Optimized Tangential Flow Filtration for Efficient Isolation of Highly Pure and Bioactive sEVs

Small extracellular vesicles (sEVs) have demonstrated significant therapeutic potential. However, the lack of efficient and scalable methods for sEV separation remains a critical barrier to their large-scale production as therapeutic agents. Tangential flow filtration (TFF) has emerged as a promising technique for large-scale sEV separation, but the absence of comprehensive and systematic studies makes it challenging to determine optimal TFF parameters for sEV isolation. In this study, we investigated the effects of key TFF parameters, including shear stress, molecular weight cutoff (MWCO), transmembrane pressure (TMP), and washing-filtration cycles, on sEVs separation and impurity removal. We evaluated particle concentration, size distribution, morphology, chemical properties, fluorescence, and protein content to assess the impact of these parameters. The results revealed that excessive shear stress (>9.05 Pa) during TFF led to protein aggregation and sEV damage. Using a filter membrane with an MWCO of 750 kDa and maintaining TMP between 3 and 5 psi prevented sEV leakage and achieved moderately high filtration efficiency. Based on these findings, optimal TFF parameters were identified. sEVs isolated using optimized TFF conditions (TFF-sEVs) were then compared with sEVs isolated via ultracentrifugation (UC-sEVs). TFF-sEVs exhibited comparable physical properties and purity to UC-sEVs but demonstrated superior proliferation-promoting activity and a 3-fold higher recovery rate. Quantitative proteomic analysis revealed that TFF-sEVs were enriched with extracellular vesicle membrane proteins and corona proteins, contributing to their enhanced bioactivity. In summary, optimizing TFF parameters enables efficient purification of sEVs, balancing purity, recovery, and bioactivity, paving the way for scalable production of sEVs and accelerating the clinical translation of sEV-based therapeutics.