title: Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis.
abstract: The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking analysis (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multi-angle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle number is obtained with a lower limit of detection (LLOD) of 1.7×107part/mL and lower limit of quantification (LLOQ) of 3.4×108part/mL. SE-HPLC with UV detection was calibrated with standards characterized by NTA, and a LLOD of 6.9×109part/mL and LLOQ of 2.1×1010part/mL were found. SE-HPLC with MALS does not require a pre-calibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approximation, the particle concentration can be directly deduced from the scattered light. A LLOD of 4.8×108part/mL and LLOQ of 2.1×109part/mL were measured and substantially lower compared to the UV method. The absolute particle concentration measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Steppert
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Burgstaller
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
authors.forename: Miriam
authors.initials: M
authors.lastname: Klausberger
authors.affiliation: Icosagen AS, Tartumaa, Estonia.
authors.forename: Andres
authors.initials: A
authors.lastname: Tover
authors.affiliation: ACIB GmbH, Vienna, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Berger
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria; ACIB GmbH, Vienna, Austria. Electronic address: alois.jungbauer@boku.ac.at.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D008027:Light; D057230:Limit of Detection; D053758:Nanoparticles / Q000032:analysis* / Q000737:chemistry; D010316:Particle Size; D014773:Virology / Q000379:methods*; D014780:Viruses / Q000302:isolation & purification*
keywords: Enveloped VLP; HPLC; MALS; Vaccine