High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti_inflammatory efficacy

Abstract

Abstract 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_negative Escherichia coli and Gram_positive lactic acid bacteria (LAB). Escherichia coli BEV 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.