A parallel SEC Column configuration strategy for comprehensive characterization of HPV type 16 virus-like particles

Characterisation of human papillomavirus-like particles (HPV VLPs) is challenging due to their large size and inherent structural heterogeneity. Monitoring oligomerisation of L1 proteins during VLP assembly is particularly difficult, as conventional large-pore-size exclusion chromatography (SEC) columns lack the resolving power to distinguish between L1 monomers and higher-order oligomers. This study presents an analytical approach utilizing two SEC columns with distinct pore sizes arranged in parallel, facilitating the separation of a wide range of molecular species within a single workflow. Hyphenation with multi-angle light scattering (MALS) provides the absolute molecular weight of the resolved species. Using the proposed parallel SEC column configuration, five distinct peaks were successfully resolved, compared to only two peaks with the 2000 Å column and three peaks with the 300 Å column, highlighting the enhanced separation capability for VLP species in 30 min, reducing both analysis time and sample consumption by up to 50% compared with performing two separate single-column analyses. This approach also simplifies the analytical workflow and lowers operational costs relative to more complex platforms such as 2D-LC systems. The proposed method was employed to characterise aggregates, monomeric VLPs, partially assembled VLPs and free L1 proteins in crude, purified and reassembled VLP samples. The proposed approach was validated using commercially available Gardasil HPV-VLP. Overall, the workflow enhances resolution across the size spectrum, provides accurate quantification of intact VLPs, reduces runtime and improves overall analytical throughput for VLP characterisation.