Understanding the effect of temperature downshift onCHOcell growth, antibody titer and product quality by intracellular metabolite profiling and in vivo monitoring of redox state

Abstract

The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell‐specific production rate (qp) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature‐induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature‐induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high‐producing (HP) and low‐producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed‐batch culture. Although the results showed that low‐temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p < 0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift led to a higher cellular viability and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p < 0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra‐ and intra‐cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated tricarboxylic acid (TCA) cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress‐alleviating strategies. To experimentally address this, we developed two high‐performance fluorescent biosensors, denoted SoNar and iNap1, for real‐time monitoring of intracellular nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide + hydrogen (NAD+/NADH) ratio and nicotinamide adenine dinucleotide phosphate (NADPH) amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD+/NADH ratio, which might be ascribed to the re‐consumption of lactate, and increased the intracellular NADPH amount (p < 0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high‐level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real‐time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells

“…Under low temperatures, pH4.5 leads to the most unstable structure of the antibody and a greater degree of fragmentation (Zheng et al 2017). Culture temperature has been extensively studied and applied to CHO cell-based RTPs production (Zhu et al 2023). Subphysiological temperature slows down translation by affecting a number of cellular responses, including phosphorylation of translation extension factor eEF2, leading to a slowing of peptide elongation.…”

Exploring metabolic responses and pathway changes in CHO‐K1 cells under varied aeration conditions and copper supplementations using 1H NMR‐based metabolomics

“…[4,5] Extensive efforts have been made to enhance mAb production through the optimization of culture media, bioprocess operational conditions, and cell lines. [6][7][8][9] Copper, recognized as a critical material attribute (CMA), emerges as a micronutrient of pivotal importance for CHO cells. Prior literature attests to copper's facilitation of the oxidative phosphorylation pathway which means a more effective energy metabolism and results in improvement in cell growth and mAb production.…”

“…[ 4,5 ] Extensive efforts have been made to enhance mAb production through the optimization of culture media, bioprocess operational conditions, and cell lines. [ 6–9 ]…”