Given increasing applications of recombinant adenoviruses for gene therapy and vaccination, there is a need for highly robust and fast purification platforms for their large scale manufacture. Traditional chromatographic methods using resins as matrices have several limitations such as high-pressure drops and slow processing rates due to pore diffusion and channelling of the feed through the bed. In contrast, membrane adsorbers offer the advantage of fast, gentle, and effective isolation. Furthermore, membranes are easy to use, no column packing is needed and, when used as disposables, no cleaning validation is necessary, representing a substantial advantage to meet cGMP requirements. In this work, a strategy for purification of adenovirus vectors from cell-culture bulks fully based on membrane devices is presented. Ultrafiltration membranes with molecular weight cutoffs of 300, 500, and 750 kDa were tested for the concentration of cell-culture supernatant after an initial clarification step. The results show that the use of ultrafiltration/diafiltration membranes not only concentrates the virus but also leads to the removal of 90% of host cell DNA and proteins in the retentate. Two membrane adsorbers (Sartobind Q and Sartobind anion direct) were evaluated for adenovirus vectors capture and purification. To define the best operating conditions, the effect of pH, conductivity, and recirculation of load bulk on the recovery yield of infectious adenoviruses were evaluated. Sartobind anion direct allows for higher recovery yields (up to 62%) of infectious adenoviruses than Sartobind Q; identical ratios between total and infectious adenoviruses (TP/IP) were achieved for both membrane adsorbers. The overall recovery yield of the process is $52%; this work credits membrane technology as an alternative for the concentration and purification of adenoviruses and as a promising solution for downstream processing of other viral vectors.

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in Wiley InterScience (www.interscience.wiley.com).As the market requirements for adenovirus vectors (AdV) increase, the maximisation 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 Â 10 6 cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 Â 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 synchronised 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 C during 67 h and restoring it to 37 C during 72 h. By using this strategy we were able to synchronise 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. V