293 cell cycle synchronisation adenovirus vector production.

As the market requirements for adenovirus vectors (AdV) increase, the maximization of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 x 10(6) cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 x 10(6) cell/mL at infection time has usually been utilized due to the so called "cell density effect". In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronized at each phase of the cell cycle; a 2.6-fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31 degrees C during 67 h and restoring it to 37 degrees C during 72 h. By using this strategy we were able to synchronize 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3-fold on AdV cell specific titer after infection.