Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growthassociated production of a foreign protein, beta-galactosidase

Protein synthesis in mammalian cells can be observed in two strikingly diff erent patterns: 1) production of monoclonal antibodies in hybridoma culture s is typically inverse growth associated and 2) production of most therapeu tic glycoproteins in recombinant mammalian cell cultures is found to be gro wth associated. Production of monoclonal antibodies has been easily maximiz ed by culturing hybridoma cells at very low growth rates in high cell densi ty fed-batch or perfusion bioreactors. Applying the same bioreactor techniq ues to recombinant mammalian cell cultures results in drastically reduced p roduction rates due to their growth associated production kinetics. Optimiz ation of such growth associated production requires high cell growth condit ions, such as in repeated batch cultures or chemostat cultures with attenda nt excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) ce lls is related to the S (DNA synthesis)phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis i s growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was f ound to be active primarily in the G1-phase and is expected to yield the de sirable inverse growth associated production behavior. Our results in repea ted batch cultures show that the protein synthesis kinetics in this resulti ng CHO cell line is indeed inverse growth associated. Results from continuo us and high cell density perfusion culture experiments also indicate a stro ng inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated pr oduction.