Compartment-specific metabolome labeling enables the identification of subcellular fluxes that may serve as promising metabolic engineering targets in CHO cells

Abstract

Abstract13C labeling data are used to calculate quantitative intracellular flux patterns reflecting in vivo conditions. Given that approaches for compartment-specific metabolomics exist, the benefits they offer compared to conventional non-compartmented 13C flux studies remain to be determined. Using compartment-specific labeling information of IgG1-producing Chinese hamster ovary cells, this study investigated differences of flux patterns exploiting and ignoring metabolic labeling data of cytosol and mitochondria. Although cellular analysis provided good estimates for the majority of intracellular fluxes, half of the mitochondrial transporters, and NADH and ATP balances, severe differences were found for some reactions. Accurate flux estimations of almost all iso-enzymes heavily depended on the sub-cellular labeling information. Furthermore, key discrepancies were found for the mitochondrial carriers vAGC1 (Aspartate/Glutamate antiporter), vDIC (Malate/H+ symporter), and vOGC (α-ketoglutarate/malate antiporter). Special emphasis is given to the flux of cytosolic malic enzyme (vME): it could not be estimated without the compartment-specific malate labeling information. Interesting enough, cytosolic malic enzyme is an important metabolic engineering target for improving cell-specific IgG1 productivity. Hence, compartment-specific 13C labeling analysis serves as prerequisite for related metabolic engineering studies.

Monitoring Intracellular Metabolite Dynamics in Saccharomyces cerevisiae during Industrially Relevant Famine Stimuli

“…Targeted metabolites were detected with high selectivity in multiple reaction monitoring (MRM) mode using established and preoptimized precursor-to-product transitions and MS/MS parameters with a mass resolution of 0.1 u. Intracellular metabolite pools were absolutely quantified by a threefold standard addition of defined amounts of reference standard mixes (internal calibration). Applied amounts were adjusted according to previously estimated concentration levels and linear dynamic ranges of the targeted metabolites [ 109 ]. The absolute concentration levels of the AxP species were normalized to results from a reference method [ 54 ] to compensate for known HILIC-specific peak tailing effects in iron-based LC systems [ 110 ].…”

Metabolic Profiling of CHO Cells during the Production of Biotherapeutics

“…Numerous studies report that during the exponential phase, glucose is mainly oxidized via glycolysis, which results in the formation of pyruvate. A significant portion of the pyruvate formed is converted into lactate, which is secreted and acidifies the medium, resulting in growth inhibition, with the rest being used to supply the TCA cycle [ 11 , 16 , 19 , 23 , 24 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. This metabolic state was previously recognized as the Warburg effect and characterizes cancer cells that consume high levels of glucose.…”

“…At the late exponential phase, a decrease by one-third in terms of glucose consumption and glycolytic activity was measured in favor of lactate uptake [ 18 , 37 ]. Interestingly, it was possible to prolong the exponential phase by the adding pyruvate and amino acids to the medium [ 16 ].…”

Advancements in CHO metabolomics: techniques, current state and evolving methodologies

“…As the growth rate decreases (stationary-phase), the metabolic-shift to utilize glucose in the TCA cycle increases to achieve high viable cell mass and antibody titres ( Dean and Reddy, 2013 ). A significant portion of the pyruvate formed from the glucose is converted into lactate, which is secreted and acidifies the medium, with the rest being used to supply the TCA cycle thus channelising the cells into the stationary phase indicating the shift from lactate producers (glucose consumers) to lactate consumer cells ( Selvarasu et al, 2012 ; Wijaya et al, 2021 ). However, contrary to glucose consumption, glutamine is also reported to be utilized more efficiently than glucose for anaplerotic replenishment of TCA intermediates and contributed more significantly to lactate production during the exponential phase ( Templeton et al, 2013 ).…”

“…Their accumulation in the culture medium during the growth phase mediates the transition of exponential to stationary phase. These metabolites inhibit the pyruvate kinase which produces ammonia through pyruvate transamination during late stages of culture ( Wijaya et al, 2021 ; Pereira et al, 2018 ). The amino acids, glutamine and asparagine present inside the cell milieu has often been correlated with the productivity and healthiness of the cells in culture ( Wishart et al, 2009 ; Mohmad-Saberi et al, 2013 ).…”