Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post‐translational modifications. A potential issue when utilizing CHO cells for therapeutic protein production is the selection of cell lines that do not retain stable protein expression during long‐term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS‐CHO cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this cell line, stability of expression was retained for 40–50 generations after which time a 40% loss in antibody production was detected. The instability observed within the cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative cell time values and an apparent increased sensitivity to cellular stress (exemplified by increased mRNA expression of the stress‐inducible gene GADD153). Changes were also noted in cellular metabolism during LTC (alterations to extracellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO cells during LTC ranging from instability of recombinant target production at a post‐mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning cellular phenotype changes require further detailed analysis at a molecular level. Biotechnol. Bioeng. 2012; 109:2093–2103. © 2012 Wiley Periodicals, Inc. Long‐term culture (LTC) of rCHO cells is associated with altered cellular stress perception that has consequences for recombinant protein yield. Expression of Growth Arrest and DNA Damage gene 153 (GADD153) exhibits earlier activation during batch culture indicating altered cellular perception or response to a constant culture environment. These data reflect part of a cellular phenotypic alteration that occurs during LTC, including metabolic changes and the capacity to support recombinant protein expression.