Year-end Sale % OFF 
Abstract
Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy- and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold.
Highlights
Mammalian cell lines, such as Chinese hamster ovary (CHO) cells, are the industry standard for production of recombinant therapeutic proteins, including monoclonal antibodies, hormones, blood factors, and enzymes [1]
To determine if mild hypothermia alters the innate differential expression of this antibody pair, CHO-S cells were transiently transfected with Ala or Gly double gene vectors (DGV, described in Section 3.2) and immediately incubated at 37 °C or 32 °C for five days
Closer analysis of reported data shows that mild hypothermia (31–33 °C) is successful at increasing specific and/or volumetric productivity in CHO cells, but the degree of enhancement varies with the protein being expressed
Summary
Mammalian cell lines, such as Chinese hamster ovary (CHO) cells, are the industry standard for production of recombinant therapeutic proteins, including monoclonal antibodies, hormones, blood factors, and enzymes [1]. The Smales’ group showed that cap-dependent mRNA translation is not severely attenuated in CHOK1 cells when the culture temperature is shifted from 37 °C to 32 °C [7]. They suggested the increase in protein expression was due to a combination of increased mRNA half-life and improved fidelity of protein folding and post-translational events at 32 °C. We determined the effect of culture temperature on the biophysical properties of the secreted antibody products in attempt to elucidate how reduced culture temperature can improve recombinant protein expression and identify the cellular mechanisms involved in this enhancement
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
