Development of a novel cold inducible gene expression system for increased recombinant protein production in CHO cells

Research has shown that the production of recombinant proteins by mammalian cells exposed to mild hypothermic conditions (30-32oC) is a viable strategy to increase product yield during fermentation. During mild hypothermia cell cultures have been shown to exhibit prolonged cell viability, reduced nutrient consumption and an increased specific production rate of some proteins. The potential significance of this approach is clear given the increasing number of therapeutics currently under development and the relatively high dose requirement for typical therapeutic functions. The objective of this project was to analyse the processes involved during mild hypothermia in CHO-Mab cells in order to elucidate the actual mechanisms involved during mild hypothermia and the implications of this strategy in the production of therapeutic proteins, in particular monoclonal antibodies (Mabs). CHO-Mab cells exposed to mild hypothermia (32oC) showed that mild hypothermia may be composed of two phases, an adaptive phase and a growth phase. Mild hypothermia also resulted in culture longevity, decreased metabolism, decreased protein turnover, as well as an increase in the specific Mab production (qP) in CHO-Mab cells possibly through mRNA stabilisation. Increased induction of the two main mammalian cold shock genes, CIRP and RBM3, was also observed. In addition, overexpression of CIRP resulted in a decreased growth rate of CHO-AA8 cells and did not increase the transient production of recombinant SEAP protein. During mild hypothermia, the cellular protein translation machinery as measured by the ratios of eIF4EP to total eIF4E/P and eIF2aP to total eIF2a/P in CHO-Mab cells was altered and the observed changes occurred within the first 72 h post mild hypothermia, indicating that changes relating to protein production during mild hypothermia may be transient. Thus, while mild hypothermia has been shown to effect culture positively for some proteins, this effect is more likely to be a transient event in the cells occurring within 72 h post mild hypothermia while adapting to mild hypothermic conditions. Thus, signalling pathways have to be examined in order to get a clear picture of the underlying processes. Nevertheless, the development of cold inducible gene expression systems utilising the cold shock elements or promoters that induce the overexpression of the two main mammalian cold shock genes, CIRP and RBM3 during mild hypothermia could allow for the increased production of recombinant proteins in the biotechnology industry. Finally, we propose a model for the coordinated series of events during mild hypothermia in mammalian cells that could help explain the molecular events