Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27

We constructed stable Chinese hamster ovary (CHO) cell lines which conditio nally and coordinately express the model product gene secreted alkaline pho sphatase (SEAP) and one of the cytostatic genes p21, p27, and p53175P, a p5 3 mutant deficient in apoptotic but not cell-cycle arrest function. The use of dicistronic expression technology allowed the conditional expression of the model product gene and the cytostatic gene in a coordinated fashion fr om a single expression unit under the control of the tetracycline-responsiv e promoter PhCMV*-1. Due to the presence of a cytostatic gene in the multic istronic expression unit, the growth behavior of the engineered CHO cell li nes could be controlled by the addition or withdrawal of the exogenous agen t tetracycline to or from the cell culture medium. Withdrawal of tetracycli ne resulted in sustained growth arrest of the stable cell lines for a prolo nged period. The growth arrest of such cell lines was found to be accompani ed by a 10-15-fold increase in their production of SEAP per cell. This cont rolled proliferation technology allows the design of a novel two-stage prod uction process which consists of a proliferation phase leading to the desir ed cell density, followed by an extended production phase during which the cells remain growth-arrested and increase cell-specific production of a het erologous protein.