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

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 par-
ticle 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 x 10${}^8$ to 1.72 x10${}^{11}$ VP/mL, with a limit of detection (LOD) of 7.72 x 10${}^7$ VP/mL and a lower limit of
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quantification (LLOQ) of 4.20 x 10${}^8$ 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.