Kinetics of continuous GM-CSF production by recombinant Saccharomyces cerevisiae in an airlift bioreactor

Continuous production of murine GM-CSF by recombinant Saccharomyces cerevisiae in an airlift bioreactor was studied at three different dilution rates. The reactor was initially fed with a selective medium to increase cell concentration, and then was fed with a rich, nonselective medium for GM-CSF production. Ethanol was used as the main carbon source to provoke GM-CSF expression. In continuous culture, GM-CSF production was maintained for over 150 h, even though the fraction of plasmid-carrying cells continuously dropped to lower than 20%. The stable GM-CSF production during the later phase of the continuous culture was attributed to increased specific cell productivity possibly resulting from an increase in the plasmid copy number in plasmid-carrying cells. This also indicated the possibility of natural selection of high-copy number cells in continuous culture. Plasmid stability was found to be growth rate (dilution rate) dependent; it increased with the dilution rate. Reactor productivity and specific productivity also increased with the dilution rate. A two-parameter kinetic equation was used to model the plasmid stability kinetics. The growth rate ratio between plasmid-carrying and plasmid-free cells increased from 0.996 to 0.998 while the segregational instability or the probability of plasmid loss from each cell division increased from 1.1% to 16% as the dilution rate decreased from 0.11 h(-1) to 0.05 h(-1). Oscillation of the dilution rate between 0.05 h(-1) and 0.11 h(-1) stabilized the plasmids and gave a higher productivity than that achieved without oscillation.