Application of 13C flux analysis to determine impacts of media alterations on industrial CHO cell metabolism

Industrial bioprocesses place extraordinary demands on the metabolism of host cells to meet the biosynthetic requirements for maximal growth and protein production. Identifying host cell metabolic phenotypes that promote high recombinant protein titer is a major goal of the biotech industry. 13C metabolic flux analysis (MFA) provides a rigorous approach to quantify these metabolic phenotypes by applying stable isotope tracers to map the flow of carbon through intracellular metabolic pathways. We have conducted a series of 13C MFA studies to examine the metabolic impacts of altering the composition of a proprietary chemically defined growth medium on CHO cell metabolism. CHO cell cultures characteristically produce excess ammonia and lactate as byproducts, both of which are toxic at high concentrations. Whereas lactate is often consumed during stationary growth phase in CHO cell cultures, ammonia continues to accumulate in the extracellular media throughout the course of cell growth due mainly to glutamine catabolism. For CHO cells that utilize glutamine, rational media design can alleviate ammonia stress from the cell culture. However, manipulating carbon sources in the growth medium can also have negative effects on cellular metabolism such as decreased culture growth, viability, recombinant protein productivity, or longevity. This study highlights a rationally engineered cell culture medium that successfully reduces culture ammonia levels by 40% while maintaining the original metabolic phenotype. First, the basal media developed in-house by Sanofi was chemically altered to cause CHO cells to produce significantly less ammonia byproduct. This low ammonia-producing media variant was experimentally developed by altering the ratio of carbon sources in the media to strategically reduce flux through metabolic pathways that result in ammonia production while supplementing complementary, non-ammonia producing pathways to balance metabolism. This altered media variant successfully decreased the ammonia concentration in industrial CHO while maintaining culture growth, viability, and specific productivity. Parallel 13C MFA studies were performed on IgG-producing CHO cells grown identically in three media variants: the basal control media, the low-ammonia media, and the low-ammonia media supplemented with basal ammonia levels. The latter media was used to control for any direct effects of changing ammonia concentrations on cellular metabolism. 13C labeling studies utilizing [U-13C5]glutamine and [1,213C2]glucose were carried out in parallel for each condition. From the comparison of the 13C flux analysis across the three media types, we have concluded that the media alterations did not have a significant impact on the intracellular metabolism of CHO cultures. This suggests that Sanofi can use their newly developed media formulation to decrease toxic ammonia buildup in IgG-producing CHO cell lines without significantly altering host metabolic phenotype or productivit

Dynamic regulation of mitochondrial metabolism as a strategy to maximize mAb production in industrial CHO cell cultures

Please click Additional Files below to see the full abstract

Metabolic engineering of high-productivity CHO host lines for biomanufacturing

Please click Additional Files below to see the full abstract

Modeling Method for Increased Precision and Scope of Directly Measurable Fluxes at a Genome-Scale

Metabolic flux analysis (MFA) is considered to be the gold standard for determining the intracellular flux distribution of biological systems. The majority of work using MFA has been limited to core models of metabolism due to challenges in implementing genome-scale MFA and the undesirable trade-off between increased scope and decreased precision in flux estimations. This work presents a tunable workflow for expanding the scope of MFA to the genome-scale without trade-offs in flux precision. The genome-scale MFA model presented here, iDM2014, accounts for 537 net reactions, which includes the core pathways of traditional MFA models and also covers the additional pathways of purine, pyrimidine, isoprenoid, methionine, riboflavin, coenzyme A, and folate, as well as other biosynthetic pathways. When evaluating the iDM2014 using a set of measured intracellular intermediate and cofactor mass isotopomer distributions (MIDs), it was found that a total of 232 net fluxes of central and peripheral metabolism could be resolved in the <i>E. coli</i> network. The increase in scope was shown to cover the full biosynthetic route to an expanded set of bioproduction pathways, which should facilitate applications such as the design of more complex bioprocessing strains and aid in identifying new antimicrobials. Importantly, it was found that there was no loss in precision of core fluxes when compared to a traditional core model, and additionally there was an overall increase in precision when considering all observable reactions

Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat

The liver—a central metabolic organ that integrates whole-body metabolism to maintain glucose and fatty-acid regulation, and detoxify ammonia—is susceptible to injuries induced by drugs and toxic substances. Although plasma metabolite profiles are increasingly investigated for their potential to detect liver injury earlier than current clinical markers, their utility may be compromised because such profiles are affected by the nutritional state and the physiological state of the animal, and by contributions from extrahepatic sources. To tease apart the contributions of liver and non-liver sources to alterations in plasma metabolite profiles, here we sought to computationally isolate the plasma metabolite changes originating in the liver during short-term fasting. We used a constraint-based metabolic modeling approach to integrate central carbon fluxes measured in our study, and physiological flux boundary conditions gathered from the literature, into a genome-scale model of rat liver metabolism. We then measured plasma metabolite profiles in rats fasted for 5–7 or 10–13 h to test our model predictions. Our computational model accounted for two-thirds of the observed directions of change (an increase or decrease) in plasma metabolites, indicating their origin in the liver. Specifically, our work suggests that changes in plasma lipid metabolites, which are reliably predicted by our liver metabolism model, are key features of short-term fasting. Our approach provides a mechanistic model for identifying plasma metabolite changes originating in the liver

Dysfunctional BMPR2 signaling drives an abnormal endothelial requirement for glutamine in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is increasingly recognized as a systemic disease driven by alteration in the normal functioning of multiple metabolic pathways affecting all of the major carbon substrates, including amino acids. We found that human pulmonary hypertension patients (WHO Group I, PAH) exhibit systemic and pulmonary-specific alterations in glutamine metabolism, with the diseased pulmonary vasculature taking up significantly more glutamine than that of controls. Using cell culture models and transgenic mice expressing PAH-causing BMPR2 mutations, we found that the pulmonary endothelium in PAH shunts significantly more glutamine carbon into the tricarboxylic acid (TCA) cycle than wild-type endothelium. Increased glutamine metabolism through the TCA cycle is required by the endothelium in PAH to survive, to sustain normal energetics, and to manifest the hyperproliferative phenotype characteristic of disease. The strict requirement for glutamine is driven by loss of sirtuin-3 (SIRT3) activity through covalent modification by reactive products of lipid peroxidation. Using 2-hydroxybenzylamine, a scavenger of reactive lipid peroxidation products, we were able to preserve SIRT3 function, to normalize glutamine metabolism, and to prevent the development of PAH in BMPR2 mutant mice. In PAH, targeting glutamine metabolism and the mechanisms that underlie glutamine-driven metabolic reprogramming represent a viable novel avenue for the development of potentially disease-modifying therapeutics that could be rapidly translated to human studies

ETA: Robust software for determination of cell specific rates from extracellular time courses †

ABSTRACT Accurate quantification of cell specific rates and their uncertainties is of critical importance for assessing metabolic phenotypes of cultured cells. We applied two different methods of regression and error analysis to estimate specific metabolic rates from time-course measurements obtained in exponentially growing cell cultures. Using simulated data sets to compute specific rates of growth, glucose uptake, and lactate excretion, we found that Gaussian error propagation from prime variables to the final calculated rates was the most accurate method for estimating parameter uncertainty. We incorporated this method into a MATLAB-based software package called Extracellular Time-Course Analysis (ETA), which automates the analysis workflow required to (i) compute cell specific metabolic rates and their uncertainties, (ii) test the goodness-of-fit of the experimental data to the regression model, and (iii) rapidly compare the results across multiple experiments. ETA was used to estimate the uptake or excretion rate of glucose, lactate, and 18 different amino acids in a B-cell model of c-Myc-driven cancer. We found that P493-6 cells with High Myc expression increased their specific uptake of glutamine, arginine, serine, lysine, and branched-chain amino acids by 2-to 3-fold in comparison to Low Myc cells, but exhibited only modest increases in glucose uptake and lactate excretion. By making the ETA software package freely available to the scientific community, we expect that it will become an important tool for rigorous estimation of specific rates required for metabolic flux analysis and other quantitative metabolic studies

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fluxomers: A new approach for 13 C metabolic flux analysis

PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Fluxomers: A new approach for 13C metabolic flux analysi