Intracellular CHO cell metabolite profiling and in vivo monitoring of
redox state unravel the effect of temperature downshift on cell growth,
antibody titer and product quality
Abstract
The strategy of temperature downshift has been widely used in the
biopharmaceutical industry to improve antibody production and
cell-specific production rate (q p) 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 leaded 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
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 NAD
+/NADH ratio and 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.