Process development and optimization for increase population growth and protein productivity in mammalian cell culture have been studied for many years. In this study, the behavior of hybridoma cells was investigated using six-well micro-titer plate systems with a working volume of 4 ml. Mouse hybridoma cell lines D2 and 2C83G2 were seeded in serum-free and serum-containing media and cultured for 8 days. alpha-Ketoglutarate is an integral component of the tricarboxylic acid (TCA) cycle and is produced from glutamine via glutamate. To study its effect on cell growth, metabolism, and monoclonal antibody (mAb) production, 2 mM alpha-ketoglutarate (pH 7.2) was added in both media at the beginning of the cultivation and in another set after 72 h. High cell density was observed in D2 cell culturing in serum-free medium, while 2C83G2 cell line showed high cell density in serum-containing medium. However, both cell lines cultured in serum-free medium gave viability above 70% when grown for 8 days. The supplement of 2 mM alpha-ketoglutarate supported cell growth and mAb production of both hybridoma cell lines in serum-free and serum-containing medium. The addition of alpha-ketoglutarate at the beginning of the batch cultivation gave better result in cell growth and mAb production as compared to alpha-ketoglutarate supplementation after 72 h. However, addition after 72 h was better than no addition at all. This indicates that alpha-ketoglutarate have a positive effect on production and release of antibody.