High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy
High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy
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DOI：
10.1002/bit.28522
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发表时间：
2023-08-09
影响因子：
3.8
通讯作者：
Jay，Steven M.
中科院分区：
文献类型：
--
作者：
Pirolli，Nicholas H.;Reus，Laura Samantha C.;Jay，Steven M.
细菌细胞外囊泡（BEV），包括外膜囊泡，已经成为治疗癌症和炎性疾病以及其他应用的有前景的新一类疫苗和治疗剂。然而，BEV的临床转化受到当前缺乏可扩展且有效的纯化方法的阻碍。在这里，我们通过开发一种使用切向流过滤（TFF）与高效阴离子交换色谱（HPAEC）串联的正交基于大小和电荷的BEV富集方法来解决下游BEV生物制造的限制。数据显示，基于尺寸的分离共分离蛋白质污染物，而基于尺寸的TFF与基于电荷的HPAEC显著提高了由益生菌革兰氏阴性大肠杆菌和革兰氏阳性乳酸菌（LAB）产生的BEV的纯度。使用建立的生物化学标记物定量大肠杆菌BEV纯度，而通过观察到的抗炎生物活性增强来评估提高的LAB BEV纯度。总的来说，这项工作建立了正交TFF + HPAEC作为BEV纯化的可扩展和有效的方法，为治疗性BEV产品的未来大规模生物制造带来了希望。
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‐negativeEscherichia coliand Gram‐positive lactic acid bacteria (LAB).Escherichia coliBEV 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.