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

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.

Harnessing Bacterial Extracellular Vesicle Immune Effects for Cancer Therapy

“…Given the breadth of possible bEV sources and engineering modifications, it is likely that some of these will have to be determined on a case-by-case basis. Still, one could envision certain common criteria (for example, endotoxin activity or lack of high-molecular-weight, non-vesicular component contamination, such as gram-negative bacterial fimbriae [ 149 ] or the Factor H binding protein from Neisseria ) [ 150 ]. Some of these criteria may need to comprise part of even early preclinical development to avoid investment of resources in candidate therapeutic bEVs that do not have beneficial properties.…”

“…It remains to be seen whether these strategies will retain the desired functionality of purified bEVs. Regarding downstream purification, most development involves employing tangential flow filtration, followed by size exclusion or affinity chromatography [ 149 , 159 ]. Recently, Won et al piloted a modified large-scale manufacturing process by combining metal precipitation and size flexclusion chromatography, which succeeded in cost-effectively mass-producing E. coli bEVs [ 99 ].…”