Quantitation of baculovirus particles by flow cytometry.

A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.