Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis

- Orthogonal quantification methods for enveloped virus-like particles.
- Multiple options for virus-like particle detection.
- HPLC-method for accurate quantification.
- HPLC method correlates with single particle tracking analysis.

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 × 107 part/mL and lower limit of quantification (LLOQ) of 3.4 × 108 part/mL. SE-HPLC with UV detection was calibrated with standards characterized by NTA, and a LLOD of 6.9 × 109 part/mL and LLOQ of 2.1 × 1010 part/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 × 108 part/mL and LLOQ of 2.1 × 109 part/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.