(505b) A Scalable Membrane Process for the Purification of Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-bound nanoparticles naturally released by animal and plant cells. Their ability to transfer their rich cargo between cells makes EVs key players in intercellular communication, with significant potential in disease diagnostics, drug delivery and regenerative medicine. However, the lack of suitable sources and isolation techniques limits their application on an industrial scale. To date, EVs are mostly isolated from mammalian cell culture by ultracentrifugation, which is an expensive, low-throughput process. Other isolation methods, such as sucrose gradient centrifugation, immunoaffinity chromatography, and polymer precipitation, have been tested but have proven difficult to balance yield and specificity. This study presents an efficient and scalable tangential flow filtration (TFF) method to isolate EVs from two different innovative, low-cost, and highly available sources: lemon juice (Citrus Limon) and milk whey from Parmesan cheese production. The research, conducted using hollow fiber modules, focused on identifying the optimal process, membrane type (ultrafiltration or microfiltration) and operating mode (concentration and diafiltration).

The resulting product streams were analyzed using size exclusion chromatography (SEC), which, in combination with other physical and biochemical techniques, allowed the recovery of EVs and the removal of impurities to be evaluated and the performance of TFF to be compared with that of traditional UC. The optimized TFF process was found to effectively produce a purer stream with a higher relative content of EVs, but at a slightly lower concentration. For example, the membrane process reduced total protein contamination in whey from 11.25 mg/mL to 3.29 mg/mL with UF and to less than 1 mg/mL with MF, the latter being more than five times lower than the minimum concentration achieved with UC (5.08 mg/mL), and produced a final product containing approximately 4 × 10¹³ EVs/mL with an average size of approximately 180 nm and a surface charge of -11.8 mV.

Overall, the continuous membrane process proved to be a promising alternative for EV isolation suitable for both plant and animal sources. It offers an efficient and scalable solution that could be integrated into existing recovery strategies, in particular to increase the value of by-products such as whey within a circular economy framework.