Purification of virus-like particles and separation of other extracellular particles

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes.

Researchers at the University of Natural Resources and Life Sciences Vienna developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction.

Transmission electron microscopy from HIV-1 gag VLPs

exosome rna

HIV-1 gag VLPs purified by (A) sucrose density gradient centrifugation and anion-exchange chromatography eluted at low (B) intermediate (C) or high salt concentration (D). The scale bar corresponds to 500 nm in (A) and to 100 nm in (B) to (D).

Steppert P, Burgstaller D, Klausberger M, Berger E, Aguilar PP, Schneider TA, Kramberger P Tover A, Nöbauer K, Razzazi-Fazeli E, Jungbauer A. (2016) Purification of HIV-1 gag virus-like particles and separation of other extracellular particles. J Chromatogr A 1455:93-101. [article]

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