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Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size‐ and charge‐based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size‐based separation coisolated protein contaminants, whereas size‐based TFF with charged‐based HPAEC dramatically improved purity of BEVs produced by probiotic Gram‐negativeEscherichia coliand Gram‐positive lactic acid bacteria (LAB).Escherichia coliBEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti‐inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large‐scale biomanufacturing of therapeutic BEV products.

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been explored in clinical trials for treatment of diseases with complex pathophysiologies. However, production of MSC EVs is currently hampered by donor‐specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self‐renewing source for obtaining differentiated iPSC‐derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti‐inflammatory bioactivity is superior to donor‐matched iMSC EVs in cell‐based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro‐vascularization and anti‐inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.