Abstract

Bovine milk-derived extracellular vesicles (BmEVs) have emerged as promising, biocompatible nanocarriers for drug delivery and therapeutic interventions. However, the lack of standardized and scalable isolation protocols remains a significant bottleneck for their industrial and clinical translation. In this study, we performed a multi-dimensional head-to-head comparison between traditional ultracentrifugation (UC) and tangential flow filtration (TFF) systems to evaluate their potential for large-scale BmEV manufacturing. We first evaluated the impact of TFF membrane pore sizes (300 kDa and 750 kDa) and flow rates on purification dynamics. The results suggest that TFF achieved isolation yields and quality comparable to UC while maintaining the essential physicochemical properties, including size, zeta potential, and morphology. Subsequent label-free data-independent acquisition (DIA) quantitative proteomics revealed that the TFF-750 kDa approach closely replicated the global proteomic landscape of the gold-standard UC. Notably, the TFF-750 kDa system showed enhanced preservation of transmembrane EV markers compared to UC, whereas the TFF-300 kDa membrane led to substantial co-retention of milk protein impurities. Furthermore, in vitro biosafety assessments indicated that TFF-750 kDa isolates exhibited efficient HepG2 cellular uptake, stability under simulated gastric fluid conditions, and favorable hemocompatibility with no significant cytotoxicity. These findings support the potential of the TFF-750 kDa method as a promising and scalable alternative to UC for producing biocompatible and functional BmEV formulations. Graphical abstract The alternative text for this image may have been generated using AI.