Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase, improves adenovirus production

► In this study, the singular and combined effects of osmolality on the phases of cell growth and adenovirus production were evaluated in culture media with osmolalities ranging from 250 to 410 mOsm using a factorial design of experiments. ► Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. ► However growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410 mOsm during the cell growth phase. ► Optimal virus productivity was achieved by growing cells in media with an osmolality of 370 mOsm or greater, followed by a virus production phase at an osmolality of 290 mOsm. ► This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media demonstrating potential industrial high yield production of adenoviral vectors and vaccines.