Kinetics and stability of GM-CSF production by recombinant yeast cells immobilized in a fibrous-bed bioreactor

The continuous production of murine granulocyte-macrophage colony- stimulating factor (GM-CSF) by recombinant yeast cells immobilized in a fibrous-bed bioreactor was studied. A high cell density of ~68 g/L and a GM- CSF productivity of ~3.5 mg/L·h were attained in the fibrous-bed bioreactor fed with a rich (nonselective, pH 6.7) medium at a dilution rate of 0.16 h^{- 1}. The GM-CSF production was stable even though the fraction of plasmid- carrying cells in the reactor effluent gradually dropped below 5% over a period of 2 weeks. At the end of that period, the immobilized cells in the fibrous matrix still had a high fraction, ~26%, of plasmid-carrying cells. Similar results were obtained with reactors operated at 0.05 h^-1 dilution rate and pH 4.0. Although the GM-CSF production was lower at pH 4, the reactor was stably operated for over 4 weeks without contamination or significant loss of productivity. The stable long-term GM-CSF production from the fibrous-bed bioreactor was attributed to the effect of cell immobilization on plasmid stability. Because GM-CSF production was growth- associated, as was found in batch fermentation with free cells, this stabilization effect cannot be attributed solely to the reduced cell growth in the immobilized cell environment. Plasmid-carrying cells were preferentially retained in the fibrous matrix, perhaps because their abilities to adhere to the fiber surface and to form cell aggregates were higher than those of plasmid-free cells.