Abstract
Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) are pivotal for the curative effects of mesenchymal stromal cells, but their translation into clinical products is hindered by the technical challenges of scaled production and purification. Ultrafiltration, a pressure-driven membrane separation method, is well known as an efficient, scalable, and cost-effective approach for bioseparation. However, there has been little study so far that comprehensively evaluates the potential application of ultrafiltration for scaled sEV isolation and purification. In this study, the feasibility and effectiveness of ultrafiltration for MSC-sEV isolation and purification are studied, and the effects of key process design and operational parameters, including the membrane pore size, transmembrane pressure (TMP), stirring speed (shear rate), feed concentration, are quantified using a stirred cell setup. Results revealed that 500 kDa molecular weight cut-off (MWCO) polyethersulfone membrane demonstrated superior suitability for MSC-sEV separation, yielding higher purity and productivity compared to 100 and 300 kDa MWCO membranes of the same material. The MSC-sEV productivity and purity could also be improved by applying a moderate stirring speed and lower operational pressure, respectively. Isovolumetric diafiltration was incorporated to enhance the purity of MSC-sEVs, successfully removing about 99% of protein contaminants by six diafiltration volumes (DVs). Subsequently, a fed-batch ultra-diafiltration (UF/DF) process with optimised filtration parameters was developed and compared with the currently most used ultracentrifugation (UC) method, showing exceptional effectiveness and performance in the isolation of MSC-sEVs: it increased the recovery of MSC-sEV from 20.59% to 60.88% (about three folds increase) and nearly doubled the purity, while also reducing processing time from over 4 h to 3.5 h, with a potential further reduction to less than 2.5 h through automation. The study concludes that ultrafiltration could be a promising method for both lab-scale preparation and industrial-scale manufacture of MSC-sEVs, offering advantages of high recovery, scalability, fast, and cost-effectiveness.