Serum protects protein-free competent Chinese hamster ovary cells against apoptosis induced by nutrient deprivation in batch culture

The development of serum- and protein-free Chinese hamster ovary (CHO) cell cultures is a high priority for the production of biopharmaceuticals. Prot ein-free competent CHO cells lines have been previously constructed by two different methods-metabolic engineering with cell-cycle regulatory proteins and long-term selective adaptation. Apoptosis was present in both cell lin es during protein-free, static-batch culture as a result of nutrient depriv ation, and glucose deprivation alone was a potent inducer of apoptosis comp ared to the depletion of other nutrients such as amino acids. By adding bac k serum to the cultures during batch growth or nutrient deprivation, it was shown that unidentified survival factors in serum can greatly reduce apopt osis in protein-competent cell lines in all phases of the culture. Both obs ervations contrast to previous reports for hybridoma cells, in which amino acids were the key determinants of apoptosis and serum had no additional an ti-apoptotic effect. Serum's protective effect against CHO cell death in ba tch culture was multifaceted and complex: (1) 10% FBS increased cell viabil ity to >99% during exponential growth from roughly 75-90%, (2) 5-10% fetal bovine serum (FBS) reduced specific glucose consumption rates in both cell lines by 40%, thereby delaying the onset of apoptosis caused by glucose dep rivation, and (3) 5% FBS reduced the specific cell death rate by 65% during a 3-d lactate-consumption phase characterized by substantial abortive prol iferation, in which the cells both proliferated and died at a constant rate . The benefit of serum on cell production over the various phases of batch growth was combined into a single parameter by integrating the viable cell concentration vs, time profile (termed here as cumulative volumetric viable cell-time, VCTvol). Despite the ability of both cell lines to grow indefin itely without any exogenous growth factors, the addition of serum resulted in a 2.3-fold increase in the VCTvol. Thus, it is clear that there is much room for improvement of protein-free CHO cell lines despite their adequate growth competence, and new strategies different from those successfully use d for hybridomas may be necessary to combat CHO cell apoptosis. (C) 1999 Jo hn Wiley & Sons, Inc.