Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

Mapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.Institute of Cell Culture Technology (University Bielefeld, Germany) ; the BMBF (German Federal Ministry of Education and Research

Probing CHO cells metabolism using metabolomics and fluxomics tools

Chinese hamster ovary (CHO) cells are preferred mammalian hosts for industrial production of therapeutic glycoproteins such as monoclonal antibodies (mAbs), used to treat cancer and immunological disorders. In these cells, the glutamine synthetase (GS) expression system has been widely used for efficient selection of high-yielding clones. However, fundamental knowledge on the metabolic behavior of GS-CHO cells in culture and its impact on product yields have not been yet systematized. The overall objective of this thesis was to comprehensively analyse the metabolome and fluxome of CHO cells exploring how the metabolism is affected by clonal variability and culture conditions. This information is then used to generate hypotheses on how metabolic wiring impacts mAb production.(...)PTDC/BBB-BSS/0518/201

Predictive macroscopic modeling of Chinese hamster ovary cells in fed-batch processes

This thesis focuses on developing a systematic modeling method that can capture the essential features for prediction of cell metabolism, growth and monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells. In a first step all specific consumption rates are calculated based on time courses of extracellular metabolites, viable cell density and mAb. Then the metabolic phases within which the metabolic pseudo-steady state approximation is verified are identified. In a third step, all metabolic rates are expressed as a function of the specific growth rate within each metabolic phase. We have applied this method to a set of small bioreactor data and have shown that the model obtained can predict specific conversion rates both small and also at large scale. In the second part of this thesis, a kinetic model of the cell growth has been developed. Together with previously described methodology, this kinetic model results in a predictive metabolic model for each experimental cell growth data are not required. The kinetic model is based on Monod kinetics with a few modifications such as a varying the maximum specific growth rate as a function of the integral viable cell density. The full kinetic model can be used off line to design optimal feeding profiles. The results of this thesis demonstrate that rich knowledge can be derived from macroscopic data that can then be used to predict new production conditions in an industrial environment at small and large scale.Der Schwerpunkt dieser Dissertation liegt auf der systematischen Entwicklung Modellen für die Vorhersage des zellulären Stoffwechsels, des Wachstums und der Produktion von monoklonalen Antikörpern (mAb) in Kulturen von Chinesischen Hamster-Ovarzellen (CHO). Zunächst wurden mit segmentierter linearer Regression metabolischer Phasen identifiziert. Diese Identifizierung beruht auf der Annahme eines pseudo-stationären Zustands und somit, dass in einer Phase alle Raten linear miteinander korreliert waren. Die spezifischen Raten wurden aus den Zeitverläufen der Konzentrationen der Metabolite und des mAb sowie der Lebendzellzahl bestimmt. Durch die Korrelation konnten alle Raten über die Wachstumsrate im 2 L und im 2000 L Maßstab berechnet werden. Danach wurde ein kinetisches Modell des Wachstums der Zellen etabliert, was die Vorhersage aller Raten auch in fed-batch Kulturen erlaubt. Die Kinetik basiert auf der Monod-Kinetik modifiziert mit einer variablen maximalen spezifischen Wachstumsrate. Das kinetische Modell erlaubt eine rechnerische Optimierung der Substratzuführung für eine maximale Produktion. Damit wurde gezeigt, dass aus makroskopischen Daten, d.h. ohne intrazelluläre Messungen, wesentliche Informationen erhalten werden können, mit denen neue Experimente in einem industriellen Umfeld vorhergesagt werden können. Diese innovative und systematische Vorgangsweise eröffnet neue Perspektiven für die Reduzierung von Kosten und für eine Beschleunigung der Prozessentwicklung

Réduction de l’effet Warburg chez une lignée de cellules de mammifères productrices d’anticorps

RÉSUMÉ L’effet Warburg, ou glycolyse aérobie, est l’une des signatures métaboliques du cancer. Il est aussi largement observé chez les cellules d’ovaires de hamster chinois (CHO), plateforme privilégiée par l’industrie pharmaceutique pour la production de médicaments. L’utilisation de la glycolyse aérobie, plutôt que de la respiration mitochondriale, comme voie catabolique principale est à l’origine d’un manque d’efficacité énergétique et d’une production accrue de lactate pouvant inhiber la croissance et la production. C’est pourquoi de nombreuses stratégies existent pour réduire l’effet Warburg durant les bioprocédés de cultures de cellules CHO. La plupart du temps, elles orientent le métabolisme vers la respiration en faisant appel à des modifications génétiques, activant ou inhibant des enzymes du métabolisme, ou à l’optimisation des conditions de culture, par le contrôle précis des échanges avec le milieu extracellulaire. Nous proposons dans ce mémoire une troisième approche encore peu explorée. En s’inspirant des thérapies métaboliques appliquées pour diminuer l’effet Warburg dans le cadre du cancer, nous avons ajouté au milieu de culture des molécules interagissant avec certaines enzymes clés du métabolisme énergétique. Deux candidats ont été sélectionnés pour notre étude : l’acide lipoïque, connu pour activer l’enzyme responsable de l’entrée du flux glycolytique dans la mitochondrie, et le bleu de méthylène, connu pour faciliter les échanges d’électrons à la membrane mitochondriale. Après avoir déterminé la dose maximale n’altérant pas la croissance pour chacune des drogues sur une lignée test de notre laboratoire capable de produire un anticorps monoclonal, nous avons mesuré leur effet sur la croissance, la viabilité, la consommation et la production de plusieurs métabolites importants, ainsi que sur des marqueurs directs de l’activité mitochondriale. Ce faisant, nous sommes parvenus à caractériser quelles voies métaboliques étaient activées et inhibées par la supplémentation de chacune des drogues et de leur combinaison. L’acide lipoïque semble encourager l’approvisionnement de la mitochondrie en intermédiaires réactionnels, tandis que le bleu de méthylène favorise les réactions d’oxydoréduction à la membrane mitochondriale et diminue le stress oxydatif qui en résulte. Ce dernier paramètre a un impact positif sur la productivité en anticorps, avec un titre final augmenté de 24 % par rapport au contrôle non traité. Ces résultats encourageants donnent un meilleur aperçu de la régulation métabolique des CHOs et présentent la supplémentation de drogues métaboliques comme une stratégie prometteuse pour augmenter la productivité des bioprocédés pharmaceutiques.----------ABSTRACT The Warburg effect, or aerobic glycolysis, is one of the metabolic signatures of cancer. It is also widely observed in Chinese hamster ovary (CHO) cells, the pharmaceutical industry's preferred platform for the production of drugs. The use of aerobic glycolysis, rather than mitochondrial respiration, as the main catabolic pathway impairs energy efficiency and increases lactate production, undermining cellular growth and production. This is why many existing strategies aim to reduce the Warburg effect during CHO-based bioprocesses. Most of the time, they direct the metabolism towards respiration by using genetic modifications, through the activation or inhibition of metabolic enzymes, or by optimizing culture conditions, through the precise control of exchanges with the extracellular environment. In this work, we propose a third approach still scarcely investigated. Based on the example of metabolic therapies used in cancer treatments, we added effectors of certain energetic metabolism enzymes in the culture medium. Two candidates have been selected for our study: lipoic acid, known to activate the enzyme responsible for directing the glycolytic flux in the mitochondria, and methylene blue, known to facilitate electrons exchange at the mitochondrial membrane. After determining the maximal dose without growth effects for each drug on an antibody-producing cell line available in our laboratory, we measured their effect on growth, viability, consumption and production of key metabolites, as well as direct markers of mitochondrial activity. Therefore, we managed to characterize which metabolic pathways were activated and inhibited by the supplementation of each drug and their combination. Lipoic acid appears to promote the supply of mitochondrial reaction intermediates, while methylene blue promotes redox reactions at the mitochondrial membrane and decreases the resulting oxidative stress. This last parameter has a positive impact on antibody productivity, with a final titer increased by 24%, compared to untreated control. These encouraging results provide a better insight into the metabolic regulation of CHOs and present metabolic drug supplementation as a promising strategy for increasing the productivity of pharmaceutical bioprocesses

Investigating Cyanobacteria Metabolism and Channeling-based Regulations via Isotopic Nonstationary Labeling and Metabolomic Analyses

Cyanobacteria have the potential to be low-cost and sustainable cell factories for bio-products; however, many challenges face cyanobacteria as biorefineries. This dissertation seeks to advance non-model photosynthetic organisms for biotechnology applications by characterizing central carbon metabolism and its regulations. Cyanobacteria phenotypes for bio-production are examined and their intracellular metabolism is quantified. Using isotopic labeling experiments, phenotypic relationships between biomass composition, central carbon fluxes, and metabolite pool sizes are investigated. Metabolic analyses of cyanobacteria led to new investigations of flux regulation mechanisms via protein spatial organizations or metabolite channeling. Metabolite channeling is further explored as a hypothesis to explain enigmatic labeling patterns and as a method to organize and regulate enzymes for robust central metabolisms. The insights reveal strategies for redirecting central metabolic fluxes for value-added chemicals as well as broad impacts for intracellular modeling approaches. First, Synechococcus UTEX 2973 was probed with isotopic nonstationary metabolic flux analysis under changing growth conditions. Despite similar genetics to Synechococcus 7942, Synechococcus UTEX 2973’s exhibits a fast growth phenotype with greater carbon fixation driven by higher energy charges, optimal ATP/NADPH ratios, low glycogen production during exponential growth, and a central metabolism that reduces CO2 loss. Unusual labeling patterns indicated metabolite channeling as a possible flux regulation mechanism. As cyanobacteria are known to have carboxysomes, a microcompartment that concentrates CO2 for RuBisCO, it was hypothesized that carboxysome mutants may reveal channeling mechanisms. Carboxysome-free mutants (high CO2 requiring phenotypes) were found to accumulate metabolites and reach higher steady state 13C enrichment, indicating more homogenous cytoplasms. Carboxysome-free mutants may provide a method for unlocking cyanobacteria flux constraints, reducing catabolic repression, and providing a way to contain genetically modified cyanobacteria. To ease the constraints of highly regulated and complex metabolic networks, platform or non-model strains can be used to provide a good starting point for small molecules of interest. To take advantage of cyanobacterial native sugar phosphate metabolisms, Synechococcus was engineered for the photoautotrophic production of a high-value polysaccharide, heparosan, which is an unsulfated polysaccharide important for cosmetic and pharmaceutical applications. Via overexpressing two key enzymes, the recombinant strain improves heparosan production by over 50 folds. Synechococcus was also found to naturally synthesize multiple glycosaminoglycans. Lastly, to further explore metabolite channeling as evidenced by isotopic labeling patterns, we developed cell-free glycolysis pathways and compared their performance with in vivo glycolysis functions in E. coli and its PTS mutants. Enzyme assays, dynamic metabolite labeling and flux analysis further confirmed the hypothesized channel of EMP enzymes where the PTS may be an anchor point to initiate enzyme assemblies. In summary, the outcomes of this thesis provide new insights into non-model phototrophic microbial chassis, reveal flux control mechanisms beyond genetic or transcriptional regulations, and offer practical guidelines for sustainable bio-production via synthetic biology approaches

Surexpression de la glucose-6-phosphate déshydrogénase pour l'étude du métabolisme primaire de cellules de mammifères en mode cuvée

Les cellules de mammifères sont l’hôte privilégié pour la production de protéines recombinantes d’intérêt thérapeutique, et parmi elles, les cellules CHO qui dominent le marché et les cellules HEK qui connaissent un regain d’intérêt. Même si elles présentent l’avantage de performer les modifications post-traductionnelles appropriées pour la compatibilité des molécules avec l’être humain, les coûts de production demeurent important devant la demande croissante. De plus, ces cellules présentent un métabolisme lent et n’atteignent pas des densités cellulaires aussi élevées comparé aux microorganismes. De nombreuses améliorations ont été obtenues grâce à l’optimisation des milieux de culture et stratégies de culture, l’amélioration des lignées, et plus récemment l’essor des études sur le métabolisme cellulaire. L’identification des limitations du métabolisme a permis d’orienter les stratégies d’ingénierie des lignées, notamment pour limiter l’effet des sous-produits toxiques de culture ou l’activation des voies apoptotiques. Récemment, des études ont montré un changement de métabolisme entre la phase de croissance et la phase de production. En phase exponentielle, la majorité des flux sont dirigés vers la glycolyse alors que le pic de production correspond à une activité majoritaire des branches oxydatives comme le cycle des acides tricarboxyliques et la voie des pentoses phosphates. C’est dans ce cadre que s’inscrit le présent projet qui consiste en la redirection des flux métaboliques vers la voie des pentoses phosphates. En accord avec les prévisions d’un modèle métabolique disponible dans notre laboratoire, il a été supposé qu’une augmentation de l’activité de la glucose-6-phosphate déshydrogénase, première enzyme de la voie, permettrait de rediriger les flux vers la voie des pentoses phosphates et de soulager la charge métabolique imposée par la production de protéines recombinantes. Cette enzyme a donc été transfectée de manière transitoire dans des cellules HEK293, une lignée parentale et une lignée dérivée exprimant la pyruvate carboxylase, et des cellules CHO afin d’observer son effet sur la production de protéines et la croissance cellulaire en mode cuvée. Les résultats en termes de croissance cellulaire, viabilité, production de protéines et concentrations de métabolites intracellulaires comme extracellulaires ont révélé la robustesse du métabolisme de ces cellules face à la perturbation étudiée. En revanche, les signatures métaboliques des deux lignées cellulaires HEK293 sont très caractéristiques, en raison des changements importants apportés par la pyruvate carboxylase. ---------- Mammalian cells are the prefered host for the production of recombinant proteins of therapeutic interest. Among them, CHO cells are prevailing on the market and HEK are raising interest. Even if they possess the advantage of performing appropriate post-translational modifications for human compatibility of those molecules, production costs are still inhibitory regarding the growing demand. Moreover, mammalian cells possess a slow metabolism and can’t reach high cell densities compared to what is observed in microorganisms. Numerous significant improvements have been made thanks to media and culture strategies optimization, cell lines improvement, and more recently the advances in cell metabolism studies. The identification of metabolism bottlenecks allowed to guide cell line engineering, especially to avoid toxic effects of metabolic by-products or activation of pro-apoptotic signals. Recently, studies have shown a metabolic switch between growth phase and production phase. In exponential phase, most of the flux are routed towards glycolysis. However, pic protein production was associated with an elevated oxidative metabolism in the TCA cycle and the pentose phosphate pathway. Given that, the present work aims at redirecting metabolic fluxes through the pentose phosphate pathway. According to a predictive metabolic model developped in our laboratory, we hypothetized that an increase in glucose-6-phosphate dehydrogenase – the first enzyme in the pathway- activity could reroute the fluxes and aleviate the metabolic burden imposed by recombinant protein secretion. This enzyme was thus transient transfected in two HEK293 cell lines, the parental cells and the derived cell line stably expressing pyruvate carboxylase, and CHO cells in order to assess its effect on protein production and cell growth in batch mode. The results, in terms of growth, viability, recombinant protein production, and the concentrations of intra- and extracellular metabolites demonstrated the robustness of these cells regarding the pertubation studied in this work. However, metabolic signatures of the two HEK293 cell lines is really characteristic of the changes brought by the expression of pyruvate carboxylase in one of the cell lines

Understanding the Warburg Phenotype and the Metabolic Plasticity of Proliferative Mammalian Cells Using \u3csup\u3e13\u3c/sup\u3eC Metabolic Flux Analysis

Proliferative cells, including many types of cancer and pluripotent stem cells, rely primarily on glycolysis and lactate metabolism for energy, regardless of oxygen availability. This metabolic phenotype – referred to as the Warburg effect – results in wasteful lactate accumulation. Although cancer cells and pluripotent stem cells share this central metabolic characteristic, the sensitivities of each of these cell types to lactate stress appear contradictory. While lactate accumulation is thought to adversely impact pluripotent stem cell proliferation and differentiation capacities, cancer cells have been shown to possess bioenergetic plasticity to utilize lactate catabolism for fuel. As a result, lactate buildup within the hypoxic tumor microenvironment has been hypothesized to promote cancer progression and malignancy, in part by selecting for cancer populations capable of catabolizing lactate. Moreover, lactate has been shown to promote stemness gene expression in cancer, suggesting that lactate plays a functional role in regulating pluripotency gene expression. However, an incomplete understanding of the impact of lactate on intracellular metabolism for proliferative cells remains. In this work, 13C-metabolic flux analysis was used to quantify the intracellular metabolic responses of breast cancer cells and induced pluripotent stem cells (iPSCs) to high extracellular lactate; in particular, to determine the role of lactate as a metabolic substrate. In this research, it was demonstrated that both iPSCs and breast cancer cells employ dual consumption of glucose and lactate to support growth. In addition, this is the first study to determine and quantify intracellular contribution of lactate in proliferative iPSC metabolism. These results provide insight into the metabolic flexibility of highly proliferative cells with respect to lactate metabolism and suggest that, much like many types of cancer cells, iPSCs possess the capacity to metabolize lactate to promote exponential growth and maintain pluripotency