BmEVs naturally carry bioactive molecules, making them ideal candidates for therapeutic delivery systems. Their industrial application, however, hinges on robust, standardized, and scalable manufacturing processes. While UC has long been the gold standard for EV isolation, it suffers from low throughput, significant casein contamination, and potential vesicle damage under high centrifugal forces (>100,000 × g). These drawbacks hinder clinical translation and large-scale biomanufacturing, creating an urgent need for improved methodologies.
The research team conducted a head-to-head comparison between UC and two TFF configurations using 300 kDa and 750 kDa hollow fiber membranes. TFF operates via a gentle, continuous cross-flow mechanism that minimizes shear stress and enables volume reduction and buffer exchange in a single step. Results showed that while both TFF systems achieved high particle recovery, the 750 kDa configuration significantly outperformed the 300 kDa in purity by effectively removing abundant milk proteins such as β-casein (CSN2) and α-lactalbumin (LALBA). Proteomic analysis revealed that TFF-750 kDa not only matched UC in overall yield but also better preserved transmembrane exosome markers like CD9, CD63, and TSG101, suggesting superior structural integrity.
Functionally, BmEVs isolated via TFF-750 kDa demonstrated excellent cellular uptake in HepG2 liver cells, stability under simulated gastric conditions, and no cytotoxicity or hemolysis (hemolysis rates <5%), confirming their biosafety profile. Unlike UC, which is limited by rotor capacity and batch processing, TFF can be easily scaled up by increasing membrane surface area, enabling continuous production suitable for industrial applications. This work establishes TFF-750 kDa as a viable, scalable platform for producing high-quality BmEVs, paving the way for their use in biomanufacturing and future clinical development of milk-derived nanotherapeutics.
The work entitled “Comparative profiling of tangential flow filtration and ultracentrifugation for the scalable production of bovine milk extracellular vesicles” was published on Systems Microbiology and Biomanufacturing (published on May 13, 2026).
DOI：

10.1007/s43393-026-00499-9