Anion exchange HPLC purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

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. Researchers at the University of Maryland 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.

Anion exchange chromatography (AEC) can be used for BEV separation

(a) Zeta potential of TFF-isolated BEVs from EcN, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus and Lacticaseibacillus casei obtained by phase analysis light scattering (n = 3 technical replicates), (b) schematic depicting benchtop AEC BEV purification process. (c, d) Nanoparticle tracking analysis of EcN (c) and L. casei (d) BEV samples following TFF-only purification or TFF + AEC purification (n = 3 technical replicates). (e) Transmission electron micrographs of L. casei BEVs purified by TFF and AEC, (f) TNF-α levels from RAW264.7 mouse macrophage conditioned media following treatment with dexamethasone (Dex) or equal protein doses of BEVs purified by TFF-only, TFF + SEC, or TFF + HPAEC and subsequent LPS-stimulation (n = 3). Data analyzed by one-way ANOVA with Holm-Šidák post hoc test. (g) EcN BEV purity analyzed using relative levels of OmpA and flagellin to produce an OmpA:flagellin ratio (right) indicative of relative BEV purity of TFF-only and TFF + HPAEC purification. ANOVA, analysis of variance formula; BEV, Bacterial extracellular vesicles; EcN, Escherichia coli Nissle 1917; HPAEC, high performance anion exchange chromatography; SEC, size exclusion chromatography; TEM, transmission electron microscopy; TFF, tangential flow filtration; TNF-α, tumor necrosis factor-α.