Extracellular vesicles (EVs) are nanoparticles with high diagnostic and therapeutic potential, but their large-scale application remains hampered by challenges related to the availability of suitable sources and isolation techniques. In this study, we investigated sweet whey, the main byproduct of the dairy industry, as an innovative source of EVs. Whey was pretreated by centrifugation, followed by purification and concentration using a two-stage tangential flow filtration (TFF) process, the first operating in diafiltration mode and the second in concentration mode. Two TFF hollow fiber membrane modules were tested: a microfiltration membrane with a pore size of 0.15 μm and an ultrafiltration membrane with a molecular weight cutoff of 750 kDa. Process performance was evaluated using several analytical techniques and parameters, with impurity removal, EV loss, and relative EV content in the final product calculated from HPLC size exclusion chromatography results as the primary metrics. TFF performance was compared with a standard ultracentrifugation protocol.TFF microfiltration outperformed ultracentrifugation in terms of both final product purity (>99%), relative EV content (>57%), EVloss (<6%), and process throughput. However, TFF ultrafiltration completely prevented EV loss (0.04%), but it was not as efficient at removing impurities (<98%) when compared to microfiltration. This study demonstrates for the first time that sweet whey is a low-cost, sustainable, and widely available source of EVs. By combining these properties with a highly efficient, continuous, and easily scalable TFF process, we have addressed the barriers that limit the industrial-scale therapeutic application of EVs, further capitalizing on the use of a byproduct rather than consuming a primary resource.
Continuous Tangential Flow Filtration to Isolate Extracellular Vesicles from Dairy By-Products
Piunti, Caterina;Cimetta, Elisa;
2025
Abstract
Extracellular vesicles (EVs) are nanoparticles with high diagnostic and therapeutic potential, but their large-scale application remains hampered by challenges related to the availability of suitable sources and isolation techniques. In this study, we investigated sweet whey, the main byproduct of the dairy industry, as an innovative source of EVs. Whey was pretreated by centrifugation, followed by purification and concentration using a two-stage tangential flow filtration (TFF) process, the first operating in diafiltration mode and the second in concentration mode. Two TFF hollow fiber membrane modules were tested: a microfiltration membrane with a pore size of 0.15 μm and an ultrafiltration membrane with a molecular weight cutoff of 750 kDa. Process performance was evaluated using several analytical techniques and parameters, with impurity removal, EV loss, and relative EV content in the final product calculated from HPLC size exclusion chromatography results as the primary metrics. TFF performance was compared with a standard ultracentrifugation protocol.TFF microfiltration outperformed ultracentrifugation in terms of both final product purity (>99%), relative EV content (>57%), EVloss (<6%), and process throughput. However, TFF ultrafiltration completely prevented EV loss (0.04%), but it was not as efficient at removing impurities (<98%) when compared to microfiltration. This study demonstrates for the first time that sweet whey is a low-cost, sustainable, and widely available source of EVs. By combining these properties with a highly efficient, continuous, and easily scalable TFF process, we have addressed the barriers that limit the industrial-scale therapeutic application of EVs, further capitalizing on the use of a byproduct rather than consuming a primary resource.
Pubblicazioni consigliate
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
