Improvement of the energy metabolism of recombinant CHO cells by cell sorting for reduced mitochondrial membrane potential

Abstract

One of the major problems in process performance of mammalian cell cultures is the production of lactic acid. Cell specific glucose uptake rates usually correlate to glucose concentration and approximately 80% of the metabolised glucose is converted into lactic acid. As the mitochondrial membrane potential was shown to correlate to cell specific glucose uptake rates, we used Rhodamine 123, a lipophilic cationic dye for cell sorting to improve the energy metabolism of existing production cell lines. Two recombinant CHO cell lines with known differences in lactic acid production rate were used to evaluate Rhodamine 123 staining as a descriptor for glucose uptake rates and to determine whether it is possible to isolate subclones with altered metabolic properties. Such subclones would exhibit an improved process performance, and in addition could be used as models for genomic and metabolic studies. From the cell line with high lactate production, a subclone sorted for reduced mitochondrial membrane potential was found to have a lower specific lactate formation rate compared to the parental cell line in batch cultures. In addition, the glucose consumption rate was also reduced, while both the growth rate and the final cell concentration were increased. A subclone sorted for high mitochondrial membrane potential, on the other hand, had a higher glucose consumption rate, a higher lactate production rate and reduced growth. The potential of using flow cytometric cell sorting methods based on physiological activity for cell line optimisation is discussed.

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…Biological processes identified to be overrepresented included mainly lipid biosynthetic processes and negative regulation of apoptosis. This independently confirms observations in earlier studies focussing on transcriptomic profiling of individual CHO cell lines with a highqP phenotype that identified ER, Golgi and intracellular transport processes, which are all membrane-associated, as contributing to productivity [7,19,20,50].…”

“…The latter can be caused by both genomic changes and epigenetic regulation [17][18][19]. This genomic and phenotypic heterogeneity has advantages and disadvantages: On the one hand it allows the effective selection of cells with different physiological and process relevant properties during screening [6,[20][21][22][23][24][25], on the other hand it limits the stability of these properties [21,26,27] and requires large numbers of subclones to be tested over prolonged periods of time to identify the few stable clones suited for production. The endogenous heterogeneity present in CHO cells is further enhanced by the process of recombination and gene amplification during cell line development, where the gene of interest and selection markers are integrated at a random site into the host cell genome, which may cause knockout of genes as well as changes in their expression levels either by separation of genes from their genetic control elements or by co-amplification with the recombinant gene.…”

Complete Knockout of the Lactate Dehydrogenase A Gene is Lethal in Pyruvate Dehydrogenase Kinase 1, 2, 3 Down-Regulated CHO Cells

“…In fed-batch culture, lactate can be a major waste product [3]. High levels of lactate can negatively impact cell growth and metabolism [4][5][6][7].…”

Correlation of antibody production rate with glucose and lactate metabolism in Chinese hamster ovary cells

“…Several parameters are generally used to evaluate cellular metabolism, such as the specific glucose uptake rate (q Gluc ) (Marchant et al 2008), specific lactate production rate (q Lac ) (Hinterkörner et al 2007), the stoichiometric ratio of lactate production to glucose consumption (DL/DG) (Gambhir et al 2003) and the specific combined consumption rate of glucose and lactate (q GL ) (Tsao et al 2005). The specific combined consumption rate of glucose and lactate, q GL , reflects the cell-specific pyruvate shuttling rate into TCA cycle, thus a higher q GL implying an increase in oxidative glucose catabolism (Ma et al 2009).…”