High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Biotechnol Bioeng. 2023 Nov;120(11):3368-3380. doi: 10.1002/bit.28522. Epub 2023 Aug 9.


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.

Keywords: bacterial therapeutics; outer membrane vesicles; probiotics; purification.