Heterogeneity in gene expression and phenotypic traits is an inherent feature within the "clonal" cell lines used for biopharmaceutical production. This feature can allow the selection of cell lines with improved phenotypes and adaptation to growth under preferential conditions to improve productivity or provide a platform to study the molecular basis of improved characteristics. A repeated process of extended batch culture of a recombinant antibody producing GS-NS0 myeloma cell line generated a stable variant cell line displaying increased resistance to both environmental stresses and chemical apoptosis inducers, and resulted in extended culture viability and increased antibody production. An interesting feature of the variant cell line was an altered metabolic state with consumption of lactate as the culture progressed. The variant cell line also showed altered expression of proteins associated with autophagy suggesting a role for this process in extending cell survival in culture. Targeted transcriptomic analysis was carried out on the parental and variant cell lines using a qRT-PCR array containing a panel of apoptosis-associated genes to elucidate both the predominant apoptotic pathways in the NS0 cell line with batch culture progression, and the altered gene expression contributing to increased survival in the variant line. Results indicated a balance between pro- and anti-apoptotic signaling is triggered with the onset of cell death in the NS0 cell line. Pro-survival pathways such as NF kappa B signaling and the unfolded protein response were implicated along with death receptor, endoplasmic reticulum stress and p53 mediated apoptotic pathways. The identification of altered gene expression in the variant cell line also provides several potential targets for cell engineering strategies to create improved cell lines for production. Biotechnol. Bioeng. 2011;108: 880-892. (C) 2010 Wiley Periodicals, Inc.