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Background In CHO cell lines currently selected for the production of recombinant antibody, approximately 80% of the metabolized glucose is converted into lactic acid. These cells with a glycolytic phenotype exhibit significantly higher rates of proton production (extracellular acidification rate, ECAR) from lactate production than cells using oxidative phosphorylation (oxygen consumption rate, OCR). Therefore, shifts in the cell’s metabolism can be detected conveniently and dynamically through simultaneous detection of ECAR and OCR. Such measurements can characterize the metabolic programming of individual cell types and forecast the quality potential of their produced glycoproteins. In this study, we utilized an XF96 analyzer to measure glycolysis and mitochondrial respiration simultaneously, and in real-time. This allows one to determine the response of these two pathways to ATP demand, and indirectly, biosynthetic needs. A rapid screen was performed to determine the desired lactic acid production by exposing the cells to alternate sources of substrates, such as galactose or fructose. Specific metrics of the study included cell growth, product yield, and glycan profile. Higher titer, viable cell density, and viability along with glycol-similarity were observed for galactose and glutamine feeding strategies during the production phase. We believe this rapid, cell based metabolic screen that is label-free and non-invasive can be used to identify low lactic acid CHO mAb cell producers in both batch and fed-batch systems. This selection is accomplished without compromising clone productivity and product quality.

Cancer cells, unlike normal cells, show selective dependency on certain nutrient substrates. These cells exhibit an addiction to glucose or glutamine, and a lack of flexibility in using a full spectrum of nutrient substrates. This dependency varies widely among different cancer cell types and depends on specific mutations in oncogenes and tumor suppressors, the cellular context in which these mutations occur and epigenetic factors. These wide variations in metabolic states have implications for cancer cell responses to therapy. To date, methods to analyze cellular substrate metabolism have required either the use of radioactive or stable isotope labeled substrate and/or large quantity of cells, may be labor intensive and have low throughput. Stable isotope labeling coupled with Mass Spectrum and NMR analysis has provided highly detailed information about substrate metabolism, but is relatively inaccessible techniques for a majority of researchers and they require special skills and expertise. We have devised several alternative easier and more rapid experimental methods to analyze substrate flux and metabolic states of cancer cells in a microplate. They have allowed us to analyze substrate flux including glucose, glutamine, and fatty acids, and to interrogate their bioenergetic machineries, glyclolysis and mitochondrial oxidative phosphorylation. These methods measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells, and can determine their dynamic responses to pathway perturbations in real-time allowing substrate flux analysis. Essentially, a large number of cell types (lines) can be analyzed using only small quantities of materials and over a short time periods (1-3 hrs) in a microplate using the XF Extracelllar Flux analyzer. In proof-of-principle experiments, we investigated substrate flux and the metabolic state of a syngenic pair of human glioblastoma cells, SF188s (slowly dividing) and SF188f (fast dividing) cells. We found that SF188f cells exhibited a substrate shift, away from glycolysis toward glutamine and fatty acid oxidation, in contrast to SF188s cells. The mitochondrial respiratory capacity and basal respiration of SF188f cells were markedly increased, but the ATP production-coupled respiration was greatly reduced. Further, the leaked respiration was also increased compared with the SF188s cells. That is, a large fraction of SF188f cells’ mitochondrial respiration was not devoted to ATP production. We suggest that cancer cells can decouple the TCA cycle from oxidative phosphorylation on an “on demand” basis. This decoupling provides a mechanism by which cancer cells can overcome the metabolic control imposed on the TCA cycle by oxidative phosphorylation, enabling them to meet their extra demand for building block synthesis in addition to energy. This also provides an explanation for the excessive rate of oxygen consumption observed in SF188f cells. In these studies, we also demonstrated that the metabolic state of SF188s and SF188f cells, e.g., glycolysis relative to glutamine oxidation, correlates with their selective susceptibility to inhibitors of glycolysis, 2-deoxyglucose, and glutamine oxidation, aminooxyacetate respectively. Therefore, these methods provide valuable tools for rapid analysis of substrate flux and bioeneregetic machineries in cancer or non-cancer cells. They can also enhance understanding the genetic and epigenetic regulation of cancer metabolism in cancer development.