Novel stable isotope methods to identify flux bottlenecks in photosynthetic hosts

Engineering host cell metabolism to promote high yield and specific productivity is a major goal of the biotech industry. 13C metabolic flux analysis (MFA) provides a rigorous approach to quantify host metabolic phenotypes by applying isotope tracers to map the flow of carbon through intracellular biochemical pathways. In particular, transient measurements of isotope incorporation following a step change from unlabeled to labeled CO2 can be used to estimate photosynthetic carbon fluxes by applying isotopically nonstationary MFA (INST-MFA) [1]. We have previously developed a package of MATLAB routines called INCA [2] that automates the computational workflow of INST-MFA. INCA is the first publicly available software package that can perform INST-MFA on metabolic networks of arbitrary size and complexity. We have recently applied INCA to model the photoautotrophic metabolism of cyanobacteria that have been engineered to produce isobutyraldehyde (IBA) [3]. The flux analysis identified an alternative three-step route from PEP to pyruvate, which supplied the majority of carbon for IBA synthesis. Based on these results, we overexpressed each single enzyme involved in this pathway and identified strains with significant improvements in IBA production. We next adapted our INST-MFA modeling approach to a terrestrial plant system[4]. We performed in vivo isotopic labeling of Arabidopsis thaliana leaves with 13CO2, measured the transient labeling of 37 metabolite fragment ions using mass spectrometry, and estimated fluxes throughout leaf photosynthetic metabolism using INCA. Leaves were acclimated to either 200 (LL) or 500 (HL) μmol/m2/s light intensity. Approximately 1,400 independent mass isotopomer measurements were regressed to estimate 136 fluxes under each condition. Despite a doubling in the carboxylation rate, the photorespiratory flux increased from 17% to 28% of net CO2 assimilation in HL acclimated plants. Photorespiration is considered a wasteful metabolic process that results in losses of energy and fixed carbon, and the ability to precisely quantify photorespiratory flux with INST-MFA is now being leveraged to guide metabolic engineering efforts to improve photosynthetic efficiency in plants. These studies have established 13C INST-MFA and the INCA software package as a comprehensive platform to map carbon fluxes in cyanobacteria, plants, and other photosynthetic host organisms. 1. Young, J.D., et al., Mapping photoautotrophic metabolism with isotopically nonstationary 13C flux analysis. Metab Eng, 2011. 13(6): p. 656-65. 2. Young, J.D., INCA: a computational platform for isotopically non-stationary metabolic flux analysis. Bioinformatics, 2014. 30(9): p. 1333-5. 3. Jazmin, L.J., et al., Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production. Metab Eng, 2017. revision submitted. 4. Ma, F., et al., Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation. Proc Natl Acad Sci U S A, 2014. 111(47): p. 16967-72

13C flux analysis in industrial CHO cell culture applications

Industrial bioprocesses place extraordinary demands on the intermediary metabolism of host cells to meet the biosynthetic requirements for maximal growth and protein expression. 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 impacts of IgG expression and other physiological stresses on CHO cell metabolism. First, we performed 13C MFA to characterize the metabolism of a IgG-expressing DHFR-deficient CHO host during four separate phases of a fed-batch culture. We found that peak specific growth rate during early exponential phase was associated with high lactate production and minimal citric acid cycle (CAC) flux. Conversely, we found that lactate metabolism switched from net production to net consumption as the culture transitioned from peak growth to peak IgG production. During stationary phase when IgG production peaked, energy was primarily generated through CAC and oxidative phosphorylation. Second, we examined nine CHOK1SV (Lonza) clones cultured in 3-liter fed-batch bioreactors, to assess their metabolism during stationary phase. Three of the clones did not express IgG. Six of the clones used the GS SystemTM to express one of three different IgGs. Four of the clones were genetically manipulated to be apoptosis-resistant by expressing Bcl-2Δ. Hierarchical clustering was performed to assess correlations amongst flux phenotypes of the nine clones. The six IgG-producing clones clustered together and were separated by host background (Bcl-2Δ or CHOK1SV). The lactate dehydrogenase (LDH) flux was most closely associated with specific IgG productivity: as IgG productivity increased, lactate production decreased. Additionally, elevated CAC fluxes corresponded strongly with increased specific productivity. This study provided further evidence of enhanced oxidative metabolism in high-producing CHO cell lines. Finally, 13C MFA was used to characterize the metabolic response of CHO cells to a novel medium variant designed to reduce ammonia production. Ammonia production was reduced by manipulating the amino acid composition of the culture medium; specifically, glutamine, glutamate, asparagine, aspartate, and serine levels were adjusted. Parallel 13C flux analysis experiments determined that, while ammonia production decreased by roughly 40%, CHO cell metabolic phenotype, growth, viability, and monoclonal antibody (mAb) titer were not significantly altered by the changes in media composition. This study illustrates how 13C flux analysis can be applied to assess the metabolic effects of media manipulations on mammalian cell cultures. The analysis revealed that adjusting the amino acid composition of CHO cell culture media can effectively reduce ammonia production while preserving fluxes throughout central carbon metabolism. Taken together, these studies provide several useful examples of how 13C MFA can be applied to assess metabolic responses of CHO cell cultures to high-yield IgG production and changing bioprocess conditions. This presentation will describe the methodology and its application to develop engineering strategies to enhance IgG productivity and titer of industrial CHO hosts

Application of 13C flux analysis to identify high-productivity CHO metabolic phenotypes

Industrial bioprocesses place high demands on the intermediary metabolism of host cells to meet the biosynthetic requirements for maximal growth and protein expression. 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 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 recombinant IgG expression using two common CHO expression systems, the glutamine synthetase (GS) and dihydrofolate reductase (DHFR) systems. First, we performed 13C MFA to characterize the metabolism of a IgG-expressing DHFR host (Amgen) during four separate phases of a fed-batch culture. We found that peak specific growth rate during early exponential phase was associated with high lactate production and minimal citric acid cycle (CAC) flux. Conversely, we found that lactate metabolism switched from net production to net consumption as the culture transitioned from peak growth to peak IgG production. During stationary phase when IgG production peaked, energy was primarily generated through CAC and oxidative phosphorylation. Second, we examined nine CHOK1SV (Lonza) clones cultured in 3-liter fed-batch bioreactors, to assess their metabolism during stationary phase. Three of the clones did not express IgG. Six of the clones used the GS SystemTM to express one of three different IgGs. Four of the clones were genetically manipulated to be apoptosis-resistant by expressing Bcl-2Δ. Hierarchical clustering was performed to assess correlations amongst flux phenotypes of the nine clones. The six antibody-producing clones clustered together and were separated by host background (Bcl-2Δ or CHOK1SV). The lactate dehydrogenase (LDH) flux was most closely associated with specific IgG productivity: as IgG productivity increased, lactate production decreased. Additionally, elevated CAC fluxes corresponded strongly with increased specific productivity. Taken together, these studies indicate that oxidative metabolism is enhanced in high-producing CHO cell lines. This presentation will discuss the central metabolic trends observed among both GS and DHFR expression systems as a means to provide potential metabolic engineering strategies to further enhance IgG productivity and titer of industrial CHO hosts

13C flux analysis in industrial CHO cell culture applications

Industrial bioprocesses place extraordinary demands on the intermediary metabolism of host cells to meet the biosynthetic requirements for maximal growth and protein expression. 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 multiple stressors on CHO cell metabolism. First, we analyzed the effects of various media compositions and supplementation regimens on CHO cell metabolism. The basal media developed in-house by an industrial collaborator was chemically altered to cause cells to produce less ammonia byproduct. This was tested against the basal media and the basal media supplemented with experimental levels of ammonia. From the comparison of the 13C flux analysis of CHO cells grown identically in the three media types, we have found that neither the chemical composition of the media nor the mere presence of ammonia in the cultures significantly altered cell metabolism. This suggests that the collaborator can use their new medium formulation without altering the metabolic phenotype of their IgG producing CHO cell lines. We are also implementing 13C MFA studies in several IgG producing cell lines to elucidate metabolic phenotypes associated with high-yield recombinant protein expression. From previous studies, it has been established that there exists a high-productivity metabolic phenotype largely identifiable by an increase in oxidative metabolism. We are engineering these proprietary IgG-producing CHO cells to up-regulate their citric acid cycle (CAC) metabolism to potentially increase IgG productivities. Through 13C stable isotope tracing, we can verify increased flux through the CAC and confirm the rational engineering of a high-productivity phenotype. These studies prove the value of 13C MFA in assessing the metabolic response to changing medium formulations or rational engineering of the host cell genome. This poster will outline the methodology used to elucidate CHO cell metabolic phenotypes in these studies as well as the potential use for this method in future studies to further increase IgG productivity and titer of industrial host lines

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

Beyond exponential phase: Metabolic phenotypes in the stationary phase of CHO cell cultures

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

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Scalable iPSC-based platform to produce tissue-specific Extracellular Vesicles

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Mapping cancer cell metabolism with 13 C flux analysis: Recent progress and future challenges

The reprogramming of energy metabolism is emerging as an important molecular hallmark of cancer cells. Recent discoveries linking specific metabolic alterations to cancer development have strengthened the idea that altered metabolism is more than a side effect of malignant transformation, but may in fact be a functional driver of tumor growth and progression in some cancers. As a result, dysregulated metabolic pathways have become attractive targets for cancer therapeutics. This review highlights the application of 13 C metabolic flux analysis (MFA) to map the flow of carbon through intracellular biochemical pathways of cancer cells. We summarize several recent applications of MFA that have identified novel biosynthetic pathways involved in cancer cell proliferation and shed light on the role of specific oncogenes in regulating these pathways. Through such studies, it has become apparent that the metabolic phenotypes of cancer cells are not as homogeneous as once thought, but instead depend strongly on the molecular alterations and environmental factors at play in each case