An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca(2+) and Mg(2+) for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl(2) and MgCl(2) concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca(2+)/Mg(2+) feeding to 117 h with Ca(2+)/Mg(2+) feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 x 10(6) and 9.12 x 10(6) cells ml(-1), respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.