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bioRxiv · 10.1101/2023.05.01.538917

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

Pirolli, N. H.·Reus, L. S. C.·Mamczarz, Z.·Khan, S.·Bentley, W. E.·Jay, S. M.
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Abstract

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles (OMVs), 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 co-isolated 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). E. 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.

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Pirolli, N. H., Reus, L. S. C., Mamczarz, Z., Khan, S., Bentley, W. E., Jay, S. M.. 2023-05-01. High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy. https://doi.org/10.1101/2023.05.01.538917

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