Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1588, Issue -, Pages 77-84Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2018.12.035
Keywords
HIV-1 gag VLP; Exosomes; Capto Heparin; Capto Core 700; HEK 293
Funding
- Federal Ministry for Digital and Economic Affairs (bmwd)
- Federal Ministry for Transport, Innovation and Technology (bmvit)
- Styrian Business Promotion Agency SFG
- Standortagentur Tirol
- ZIT -Technology Agency of the City of Vienna through the COMET-Funding Program
- Austrian Science Fund (project BioToP) [FWF W1224]
- Government of Lower Austria
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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 (R) 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. (C) 2018 The Authors. Published by Elsevier B.V.
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