A 13-day fed-batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell-specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1519-1527, 2013