Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

Abstract

Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds improve recombinant antibody yields from a model GS-CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing.

Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients

“…Alternative fates for carbon have also been suggested since glucose can also be converted into glycerol along with oxidation of NADH to NAD + , as well as sorbitol and threitol – in both cases, accompanied by the oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP + ). These compounds are formed from glycolytic intermediates and accumulate both intracellularly and extracellularly in the transition of the exponential to stationary phase of culture, after the addition of feed containing depleted nutrients (Table ) . Additionally, G6P is shunted away from glycolysis to enter the pentose phosphate pathway (PPP) where the sugar precursors are required for the synthesis of nucleotides, NADPH, and glycolytic intermediates are produced.…”

“…Intermediates of the TCA cycle (citrate, succinate, fumarate and malate) accumulate during culture phases, which indicates a bottleneck (Table ) (reviewed by Dickson). These intermediates were observed to build up in the cell culture medium after the addition of a feed, which contains pyruvate and amino acids (aspartate, asparagine, and glutamate), and has been linked to growth limitation …”

“…Changes in amino acid concentrations at defined culture phases have been correlated to cell growth inhibition and cell death. For example, asparagine consumption has a negative effect in cell growth, while alanine production inhibits TCA cycle and also represents a source of ammonium . In another study, lysine was supplied in excess in media formulations (considering a relatively low cell density) and accumulates during the death phase .…”

“…A number of lipids have been shown to deplete or build up over time in CHO cell culture (Table ). In particular, glycerolipids have been seen in multiple studies to affect growth in a variety of ways . Choline phosphate (PCHO), glycero‐3‐phosphocholine (G3PC), and glycerol‐3‐phosphate (G3P) have all been shown to build up in culture over time, which has been seen to lead to growth limitation in both fed‐batch and batch cultures .…”

Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

“…At the same time, understanding the biology of cellular processes during CHO cell protein production is being actively pursued . Recently, molecular profiling‐based genomics, proteomics, and metabolomics are being applied to bioreactor processes to study cellular production . Ultimately, the combination of multiple ‘Omics methods will lead to an improved understanding of the manufacturing and cellular processes, resulting in improvements in bioreactor productivity , production of specific glycoforms , and potential identification of biomarkers for process assessment and control .…”