Higher expression of fab antibody fragments in a CHO cell line at reduced temperature

Abstract

A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied.

Proliferation control strategies to improve productivity and survival during CHO based production culture

“…(I) Temperature shift to conditions of mild hypothermia Low culture temperature has been shown to reduce the growth rate, increase culture longevity and specific productivity in CHO cells expressing wide range of recombinant proteins Ohsuye 1998, 1999;Kaufmann et al 1999;Schatz et al 2003;Yoon et al 2003aYoon et al , b, 2006Fox et al 2004;Fogolin et al 2004, Bollati-Fogolin et al 2005Trummer et al 2006). In this laboratory reducing the temperature of suspension CHO-K1 cells from 37 to 31°C during the latter stages of exponential growth resulted in an immediate cessation of proliferation.…”

“…Reduced culture temperature has also been shown to increase levels of recombinant mRNA, either due to enhanced transcription of the recombinant gene of interest or increased mRNA stability (Hendrick et al 2001;Yoon et al 2003b;Fox et al 2004Fox et al , 2005. For the above reasons, many processes employ a biphasic culture process for recombinant protein production (Moore et al 1997;Kaufmann et al 1999;Yoon et al 2003b;Schatz et al 2003;Fox et al 2004;Fogolin et al 2004, Bollati-Fogolin et al 2005Trummer et al 2006). This culture process consists of two phases: an initial proliferation phase designed to maximize biomass at 37°C, followed by an extended production phase, at reduced temperature (28-33°C), during which, the cells remain growth arrested while the production of heterologous proteins is maintained at a high and stable level.…”

“…Therefore although the effects of low culture temperature on the cellular productivity of recombinant proteins are variable and depend on the temperature, cell line, clone, expression system and/or protein, the effect of reducing cell growth is a universal phenomenon Ohsuye 1998, 1999;Kaufmann et al 1999;Ryll et al 2000;Schatz et al 2003;Yoon et al 2003aYoon et al , b, 2004Yoon et al , 2006Fox et al 2004;Fogolin et al 2004, Bollati-Fogolin et al 2005Trummer et al 2006;Al-Fageeh et al 2006). …”

The cold-shock response in mammalian cells: investigating the HeLa cell cold-shock proteome

“…In recent years there has been an ever-growing number of reports that the sub-physiological temperature in vitro culturing (<37°C) of mammalian cells can lead to enhanced heterologous protein production (Beer et al 2003;Ducommun et al 2002;Fogolin et al 2004;Fox et al 2004;Fox et al 2005b;Schatz et al 2003;Yoon et al 2003a;Yoon et al 2003b). This effect is extremely variable however, and appears to be both cell line and expression system dependent .…”

Transient transfection of CHO cells using linear polyethylenimine is a simple and effective means of producing rainbow trout recombinant IFN-γ protein

“…Incubation temperature has also been shown to be an important factor affecting the expression of recombinant proteins in mammalian cells, with lower temperatures increasing the cell percentage in G0/G1 phase and decreasing in G2/M phase as well slowing down the cell growth (Galbraith et al 2006;Schatz et al 2003;Zhang 2009). Despite reduced protein synthesis rates at lower temperature, the increased stability of the mRNA encoding the recombinant protein leads to an increase in their cellular abundance (Galbraith et al 2006).…”