This article describes a cell banking process for rBHK cell lines in 100-mL cryobags. As the use of larger volume cell banks requires greater cell numbers and longer preparation time, extensive characterization of key process parameters beyond the conventional ranges was performed to support a cGMP banking process. All experiments were conducted using two recombinant BHK2.1 cell lines, one of them cotransfected with Hsp70. The results show that the entire cell banking process for these BHK cell lines can be performed at room temperature. A DMSO exposure time up to 5 h either directly in a bioreactor or in shaker flasks did not result in any significant negative effect after cell thaw, when the cryocontainers were frozen immediately after filling. Extensive characterization did not indicate any significant apoptotic effects after thaw. However, the Hsp70 cotransfected cell line did show a slightly better protection from potential cryopreservation-induced apoptosis. Surprisingly, it was found that cells transferred into cryobags showed a low recovery rate after thaw if the incubation time exceeded 1.5 h before freezing. Additional experiments confirmed that the DMSO exposure time inside the cryocontainer in contrast to the DMSO exposure in a reactor or shaker flasks is much more critical. The cryobag cell banking process should therefore he performed within a 11/2-2 h window; a banking process for vials should not exceed 21/2 h. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1154-1163, 2010