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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.