Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

Increasing cell-specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5'-deoxy-5'-(methylthio)adenosine (MTA), a chemical degradation product of S-(5'-adenosyl)-ʟ-methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1-producing CHO cells by 50%. Compartment-specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell-cycle arrest and increased cell volumes. Carbon fluxes into the pentose-phosphate pathway increased 22 fold in MTA-treated cells compared to that in non-MTA-treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α-ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA-treated cells improved three fold compared to that in non-MTA-treated cells, which can be regarded as a major factor for explaining the boosted CSPs.

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

13C 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.

Methylthioadenosine (MTA) boosts cell-specific productivities of Chinese hamster ovary cultures: dosage effects on proliferation, cell cycle and gene expression.

A major goal for process and cell engineering in the biopharmaceutical industry is enhancing production through increasing volumetric and cell-specific productivities (CSP). Here, we present 5'-deoxy-5'-(methylthio)adenosine (MTA), the degradation product of S-(5'-adenosyl)-L-methionine (SAM), as a highly attractive native additive which can boost CSP by 79% when added to exponentially growing cells at a concentration of 250-300 µm. Notably, cell viability and cell size remain higher than in non-treated cultures. In addition, cell cycle arrests first in S-, then in G2-phase before levelling out compared to non-treated cultivations. Intensive differential gene analysis reveals that expression of genes for cytoskeleton mediated proteins and vesicle transport is amplified by treatment. Furthermore, the interaction of MTA with cell proliferation additionally stimulated recombinant protein formation. The results may serve as a promising starting point for further developments in process and cell engineering to boost productivity.

Comparison of l-tyrosine containing dipeptides reveals maximum ATP availability for l-prolyl-l-tyrosine in CHO cells.

Increasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l-tyrosine is added as dipeptide to deal with its poor solubility at neutral pH. Showcasing IgG1 production with CHO cells, we investigated the supplementation of three l-tyrosine (TYR, Y) containing dipeptides: glycyl-l-tyrosine (GY), l-tyrosyl-l-valine (YV), and l-prolyl-l-tyrosine (PY). While GY and YV led to almost no phenotypic and metabolic differences compared to reference samples, PY significantly amplified TYR uptake thus maximizing related catabolic activity. Consequently, ATP formation was roughly four times higher upon PY application than in reference samples.

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells.

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.