An HPLC-SEC-based rapid quantification method for vesicular stomatitis virus particles to facilitate process development.

ABSTRACT

Virus particle (VP) quantification plays a pivotal role in the development of production processes of VPs for virus-based therapies. The yield based on total VP count serves as a process performance indicator for evaluating process efficiency and consistency. Here, a label-free particle quantification method for enveloped VPs was developed, with potential applications in oncolytic virotherapy, vaccine development, and gene therapy. The method comprises size-exclusion chromatography (SEC) separation using high-performance liquid chromatography (HPLC) instruments. Ultraviolet (UV) was used for particle quantification and multi-angle light scattering (MALS) for particle characterization. Consistent recoveries of over 97% in the SEC were achieved upon mobile phase screenings and addition of bovine serum albumin (BSA) as sample stabilizer. A calibration curve was generated, and the method's performance and applicability to in-process samples were characterized. The assay's repeatability variation was <1% and its intermediate precision variation was <3%. The linear range of the method spans from 7.08 × 108 to 1.72 × 1011 VP/mL, with a limit of detection (LOD) of 7.72 × 107 VP/mL and a lower limit of quantification (LLOQ) of 4.20 × 108 VP/mL. The method, characterized by its high precision, requires minimal hands-on time and provides same-day results, making it efficient for process development.