High correlation of HPSEC-MALLS-based viral particle quantification to infectivity enables quality-by-design for recombinant vesicular stomatitis virus process development.

Traditional viral titer methods (e.g., TCID₅₀) are activity-based and time-consuming, limiting rapid process development and quality by design (QbD) for viral vector production. Here, using recombinant vesicular stomatitis virus (rVSV) as a model, we established a method based on high performance size exclusion chromatography coupled with multi angle laser light scattering (HPSEC -MALLS) to simultaneously monitor viral particle concentration and purity. Importantly, the physical particle concentration measured by HPSEC-MALLS correlated strongly with biological infectivity (TCID₅₀), yielding an R² of 0.975. Leveraging this correlation, we achieved real-time quality control across downstream and upstream processes. During the upstream culture stage, a multiplicity of infection (MOI) of 0.1 maximized particle production. For purification, Capto Core 700 multimodal chromatography achieved 83.53% particle recovery while removing >99% of host cell proteins and nucleic acids. In formulation studies, sucrose preserved 95.20% particle stability after five weeks at 4 °C, whereas trehalose yielded 94.92% retention following lyophilization. This HPSEC-MALLS platform enables rapid, label-free, multi parameter quality control throughout the entire viral vector process, accelerating both process optimization and clinical translation.