The production of hGM-CSF was investigated in both a flask and a 5-1 bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g1(-1) and 6.5 mug 1(-1), respectively, those in the bioreactor were 15.6 g1(-1) and 7.6 mug 1(-1). No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day(-1)). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 mug accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day(-1), respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g1(-1) of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day(-1), respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes. (C) 2004 Elsevier Inc. All rights reserved.