Scalable membrane-based processes to isolate extracellular vesicles from lemon juice

• A continuous membrane UF/DF process for the isolation of EVs from lemons is presented. • The two-step membrane process is compared to UC and SEC for isolating lemon EVs. • The UF/DF process outperforms conventional purification methods. • Membranes provide a robust foundation for the industrial translation of plant EVs. Plant-derived extracellular vesicles (EVs) are attracting increasing interest for their potential as carriers of bioactive molecules for therapeutic applications. In this study, we focus on the isolation of EVs from Citrus limon juice using membrane-based separation techniques. After a pretreatment to obtain clarified lemon juice, we initially used a batch diafiltration approach (staged UF/DF) for preliminary membrane screening and EV purification, including a concentration step. The 200 kDa polyethersulfone (PES) membrane was identified as the best performing membrane, as determined by size exclusion chromatography (SEC) analysis of the diafiltration streams. However, due to limited EV recovery, we transitioned to a continuous tangential flow diafiltration (continuous UF/DF) process using 750 kDa PES hollow fiber modules. Although both staged and continuous UF/DF were effective in removing 99.7 % of impurities, staged UF/DF required longer processing times and resulted in lower EV recovery with respect to continuous UF/DF. The final process was also compared with traditional purification methods such as differential ultracentrifugation (UC) and preparative SEC. Continuous tangential flow diafiltration was found to be the most efficient method for removing impurities and recovering EVs, providing a scalable and effective alternative to traditional UC-based processes. This study highlights the potential of lemon juice as a sustainable and cost-effective source of EVs for therapeutic applications, coupled with scalable and efficient membrane-based purification, opening new opportunities for the use of plant-derived EVs in clinical and industrial settings.