Austrian Centre of Industrial Biotechnology, Vienna, Austria.
Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
J Chromatogr A. 2019 Mar 15;1588:77-84. doi: 10.1016/j.chroma.2018.12.035. Epub 2018 Dec 18.
Separation of enveloped virus-like particles from other extracellular vesicles is a challenging separation problem due to the similarity of these bionanoparticles. Without simple and scalable methods for purification and analytics, it is difficult to gain deeper insight into their biological function. A two-step chromatographic purification method was developed. In the first step, virus-like particles and extracellular vesicles were collected and separated from smaller impurities in a flow-through mode. Benzonase treated HEK 293 cell culture supernatant was directly loaded onto a column packed with core-shell beads. The collected flow-through was further purified using heparin affinity chromatography. In heparin affinity chromatography 54% of the total particle load were found in the flow-through, and 15% of the particles were eluted during the salt linear gradient. The particle characterization, especially particle size distribution and mass spectrometry data, suggests that extracellular vesicles dominate the flow-through fraction and HIV-1 gag VLPs are enriched in the elution peak. This is in part in contradiction to other protocols where the extracellular vesicles are recovered by binding to heparin affinity chromatography. The developed method is easily scalable to pilot and process scale and allows a fast accomplishment of this separation within one day.
由于这些生物纳米颗粒的相似性，从其他细胞外囊泡中分离包膜病毒样颗粒是一个具有挑战性的分离问题。如果没有简单且可扩展的纯化和分析方法，就很难更深入地了解它们的生物学功能。本研究开发了一种两步色谱纯化方法。在第一步中，采用流穿模式从较小的杂质中收集和分离病毒样颗粒和细胞外囊泡。用苯甲酸钠处理的 HEK 293 细胞培养上清液直接加载到填充有核壳珠的柱子上。收集的流穿液进一步通过肝素亲和层析进行纯化。在肝素亲和层析中，54%的总颗粒负载量存在于流穿液中，而在盐线性梯度洗脱时，有 15%的颗粒被洗脱。颗粒特性，特别是粒径分布和质谱数据表明，细胞外囊泡主要存在于流穿液部分，HIV-1 gag VLPs 则在洗脱峰中富集。这与其他方案有些矛盾，因为其他方案中，细胞外囊泡通过结合肝素亲和层析来回收。所开发的方法易于扩展到中试和工艺规模，并允许在一天内快速完成这种分离。
