To investigate the stability of a BHK-21 A cell line transfected with human EPO (huEPO) and the human α2,6-sialyltransferase ST6N (huST6N), the cell line was cultivated over a time period of 18 days in a 2.5 L perfused bioreactor in protein- and serum-free medium and under physiological stress after addition of ammonium. The stability of gene expression and the activity of the newly introduced huST6N and the endogenous α2,3-sialyltransferase (α2,3-ST) were analyzed during the different cultivation conditions by RT-PCR and in vitro assays. Furthermore, the purified secreted huEPO was analyzed by western blot and the liberated N-glycans were characterized by HPAEC-PAD analysis. The results confirmed the stability of the cell line with respect to the expression of the newly introduced human proteins even under physiological stress. The elevated ammonium concentration led to reversible changes on the EPO N-glycans.