Volume 5 Supplement 8
Analysis of glycolytic flux as a rapid screen to identify low lactate producing CHO cell lines with desirable monoclonal antibody yield and glycan profile
© Legmann et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
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.
Material and methods
The illustration demonstrates how the XF96 measures changes in the microenvironment (3μl) surrounding live cells in a 96-well microplate. The O2 and proton concentration is determined by a quenching reaction of a fluorophore specific for O2 (blue) or protons (red) that is embedded into a single polymer martix on the biosensor cartridge.
In this study, we developed and validated a rapid assay, employing the XF96 analyzer, to screen for low ECAR producing cells to predict low lactic acid production. The experiments show that there is a good agreement between ECAR and lactic acid and therefore ECAR can be rapidly measured as an index of glycolytic activity and serve as an accurate surrogate of lactate production. The data show that alternating carbon sugars during the production phase ,such as galactose and fructose, can help to control glycolysis and, therefore, reduce the lactate accumulation in mammalian cell cultures without compromising on recombinant glycoprotein productivity and desired glycoform profile.
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