Compartment-specific metabolome analysis reveals the tight link between IgG1 formation and necessarily high mitochondrial shuttle activities in Chinese Hamster Ovary cells

Chinese hamster ovary (CHO) cells are the dominating host for the production of pharmaceutical proteins, in particular monoclonal antibodies (mABs). Although production titers improved more than 100 fold during the last 2 decades, similar enhancements of cell specific productivities are less pronounced. They demand for detailed subcellular studies to identify promising metabolic engineering targets. In this context, our study focused on compartment specific metabolome analysis to measure metabolic patterns in the cytosol and in the mitochondrion during cell cultivation. Thereof, in vivo shuttle activities were calculated and correlated with cell specific IgG1 formation rates. The compartment-specific metabolome and labelling analysis (13C) distinguishes between cytosol and mitochondrion. Metabolomics and instationary 13C metabolic flux analysis build on preliminary own studies of 13C analytics (Teleki et al., Anal Biochem 2015; Teleki et al. Metab Eng 2017) and compartment-specific metabolomics (Matuszczyk et al., Biotechnol J 2015; Pfitzenmaier et al., Biotechnol J 2016). Further development and optimization has been performed finally reaching the current status that allows monitoring compartment-specific flux distributions and shuttle activities during the course of cell cultivation. Studying multiple periods of an IgG1 production process the crucial role of the mitochondrion not only as a provider of ATP but also as an essential part of metabolism was unraveled. 13C flux analysis disclosed the time-variant activities of the mitochondrial shuttles that are tightly linked to mitochondrial and cytosolic metabolism. Clear evidence was found that mAB production strongly depends on sufficient NADPH supply provided by cytosolic malic enzyme activity and malate export from the mitochondrio

The Less the Better: How Suppressed Base Addition Boosts Production of Monoclonal Antibodies With Chinese Hamster Ovary Cells

Biopharmaceutical production processes strive for the optimization of economic efficiency. Among others, the maximization of volumetric productivity is a key criterion. Typical parameters such as partial pressure of CO2 (pCO2) and pH are known to influence the performance although reasons are not yet fully elucidated. In this study the effects of pCO2 and pH shifts on the phenotypic performance were linked to metabolic and energetic changes. Short peak performance of qmAb (23 pg/cell/day) was achieved by early pCO2 shifts up to 200 mbar but followed by declining intracellular ATP levels to 2.5 fmol/cell and 80% increase of qLac. On the contrary, steadily rising qmAb could be installed by slight pH down-shifts ensuring constant cell specific ATP production (qATP) of 27 pmol/cell/day and high intracellular ATP levels of about 4 fmol/cell. As a result, maximum productivity was achieved combining highest qmAb (20 pg/cell/day) with maximum cell density and no lactate formation. Our results indicate that the energy availability in form of intracellular ATP is crucial for maintaining antibody synthesis and reacts sensitive to pCO2 and pH-process parameters typically responsible for inhomogeneities after scaling up

Correlations of differentially expressed gap junction connexins cx26, cx30, cx32, cx43 and cx46 with breast cancer progression and prognosis.

BACKGROUND AND AIMS: Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers. MATERIALS AND METHODS: Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models. RESULTS: The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other's prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively. CONCLUSION: Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers

Az ingatlanpiac helyzete és értékelési módszerei

Dolgozatomban azokra a kérdésekre kerestem a választ, hogy milyen sajátosságokkal rendelkezik a magyarországi ingatlanpiac és azon belül is a lakáspiac, milyen tényezők határozzák meg a lakások árát és milyen módszerekkel lehet meghatározni az ingatlanok értékét. Dolgozatomban bemutatok egy - a Fészek lakóparkban található - lakás értékét meghatározó tényezőket. Célkitűzéseim: - Az ingatlan műszaki leírásának bemutatása. - Az ingatlan értékének meghatározás. - Ismertetem az ingatlan értékének várható alakulását.M

Systems biology studies for the optimization of sulfur-containing amino acid producing microorganisms

Die schwefelhaltige und essentielle Aminosäure L-Methionin besitzt vor allem aufgrund ihres Einsatzes zur Supplementierung von pflanzlichem Tierfutter eine enorme wirtschaftliche Bedeutung. Der globale Jahresbedarf wird auf ca. 1.000.000 Tonnen geschätzt und weist angesichts eines ansteigenden Fleischbedarfs, insbesondere in Schwellenländern, stabile jährliche Wachstumsraten von ca. 5-6% auf. Methionin wird derzeit nahezu ausschließlich durch chemische Synthese (Evonik-Verfahren) als DL­-Razemat hergestellt. Ein moderner biotechnologischer Prozess würde eine nachhaltige Alternative zu bisher petrochemisch-basierten Verfahren darstellen und bietet das Potential einer erheblichen Kostensenkung, höherer Substratflexibilität sowie der Herstellung von enantiomer-reinem L-Methionin. Eine Überproduktion von L-Methionin stellt allerdings aufgrund der Komplexität zugrundeliegender Stoffwechselwege, einer strikten zellulären Regulation und eines vergleichsweise hohen energetischen Syntheseaufwands innerhalb gängiger mikrobieller Plattformen eine große Herausforderung dar. In der vorliegenden Arbeit wurde mittels systembiologischer Ansätze ein durch die Evonik Industries AG bereitgestellter Modellproduzent (E. coli SPOT01) auf Potentiale zu einer gezielten Methionin-Überproduktion untersucht. Rationale Stammoptimierungen basieren auf systematische genetische Modifikationen und setzen ein dynamisch-quantitatives Verständnis zugrundeliegender intrazellulärer Stoffwechselnetzwerke voraus (Systems Metabolic Engineering). Innerhalb zielgerichteter metabolischer Perturbationsanalysen können Kontrolleinflüsse beteiligter Enzyme auf die zu optimierenden Zielflüsse auf systemischer Ebene quantifiziert werden (Metabolic Control Analysis). Derartige Ansätze erfordern eine abgestimmte Anwendung intrazellulärer Metabolomanalysen, adäquater Perturbations- und Probennahmestrategien sowie mathematischer Modellierung. Hierzu wurde eine universelle LC-ESI-MS/MS-Methode entwickelt, die eine sensitive und selektive Quantifizierung eines breiten Spektrums niedermolekularer Metabolite des zellulären Stoffwechsels ermöglicht (Metabolic Profiling). In umfangreichen Transportkinetik-Studien konnte anschließend mit L-Serin ein alternatives und netzwerk-inhärentes Stimulussubstrat identifiziert werden, das eine direkte und zielgerichtete Perturbation des L-Methionin-Synthesenetzwerks ermöglicht. Unter Anwendung adaptierter Stimulusszenarien sowie semi-automatisierter Beprobungsstrategien konnten innerhalb produktionsrelevanter Kultivierungsbedingungen von E. coli SPOT01 aussagekräftige Konzentrationsdynamiken fokussierter Metabolitpools erzielt werden. Für die quantitative Auswertung resultierender Perturbationsprofile wurde ein modellbasierter Ansatz unter Verwendung einer nicht-mechanistischen LinLog-Kinetik sowie ein rein datengetriebener Ansatz unter Verwendung des PEC-Kriteriums (Pool Efflux Capacity) verfolgt. Insbesondere die PEC-Analyse stellte sich hierbei als zielführender Ansatz heraus, der aufgrund seiner hohen Robustheit auch bei messtechnischen Unsicherheiten valide Aussagen im Rahmen der Kontrollanalyse erlaubt. Innerhalb unterschiedlicher Perturbationsstudien konnten hierbei übereinstimmend die Cystathionin-β-Lyase (MetC), die Methioninsynthase (MetH/E) und das Methionin-Exportsystem (YjeH) als Enzyme mit hoher Flusskontrolle identifiziert werden. Aufgrund einer ausgeprägten Akkumulation des Vorläufermetabolits L-Homocystein und eines zunehmenden Übergewichts gegenüber L-Cystein, erfolgt die unspezifische Umsetzung des cytotoxischen Intermediats zum Akkumulationsprodukt Homolanthionin. Der unspezifische Abbau von Homolanthionin führt zur erneuten Bildung von L-Homocystein und stellt den Ausgangspunkt für eine alternative Syntheseroute des Haupt-Nebenprodukts L-Isoleucin. Dieses für die L-Methioninsynthese höchst unvorteilhafte Akkumulationsszenario (Kohlenstoff- und Energieverlust) konnte letztlich auf ein Ungleichgewicht in der Bereitstellung von L-Aspartat (TCA-Syntheseweg) und L-Serin (EMP-Syntheseweg) zurückgeführt werden. Unter Einsatz des vollmarkierten Stimulussubstrats [U13C]-L-Serin konnte der Informationsgehalt resultierender instationärer Datensätze weiter gesteigert werden. Die hohe Flusskontrolle durch die Methioninsynthase (MetH/E) konnte hierbei eindeutig auf eine limitierte Transmethylierungskapazität durch den C1-Stoffwechsel zurückgeführt werden. Ein Schlüsselenzym in diesem Zusammenhang stellt die Hydroxymethyltransferase (GlyA) dar, welche unter Umsetzung von L-Serin Methylgruppen für die finale Transmethylierung bereitstellt. Durch eine gezielte Erhöhung der enzymatischen Affinität von GlyA zu seinem Substrat L-Serin kann eine Kompensation des TCA/EMP-Ungleichgewicht unter einer gleichzeitigen Aufhebung des Akkumulationszyklus erfolgen.The sulfurous and essential amino acid L-methionine is of enormous industrial importance, mainly due to its addition to plant-based animal feed products. The annual global demand is estimated at about 1,000,000 tons and the annual growth rates are stable at 5-6%, caused by an increasing need for meat, particularly in developing countries. Currently, methionine is produced exclusively by chemical synthesis as DL-racemate (Evonik procedure). Modern biotechnological processes represent a sustainable alternative to the hitherto existing petrochemical based syntheses and offer the potential of significant cost reduction, higher substrate flexibility and production of enantiopure L-methionine. However, an overproduction of L-methionine in common microbial producers is challenging due to the complexity of underlying metabolic pathways, a strict cellular regulation and comparably high energetic efforts for the synthesis. In the present study a promising L-methionine producer provided by the Evonik Industries AG (E. coli SPOT01) was investigated by systems biology tools triggering product synthesis. Rational strain developments are based on targeted genetic modifications and require an extensive quantitative understanding of corresponding intracellular metabolic networks (Systems Metabolic Engineering). In this context, metabolic pulse experiments enable the quantification of control properties of involved enzymes on intracellular target fluxes at a systemic level (Metabolic Control Analysis). Such experimental concepts require a concerted application of intracellular metabolome analysis, appropriate pulse and sampling strategies, as well as mathematical modeling. For this purpose, a comprehensive LC-ESI-MS/MS method has been developed for a quantitative analysis of a broad range of common metabolites of the cellular metabolism with high selectivity and sensitivity (Metabolic Profiling). Through extensive transport kinetic studies, L-serine has subsequently been identified as an alternative and network-inherent stimulus substrate, allowing a direct and targeted perturbation of the L-methionine synthesis network. Adapted stimulus scenarios and rapid semi-automatized sampling strategies were applied to monitor metabolic dynamics as a function of external stimuli in connection to production-related cultivation conditions of E. coli SPOT01. Transient metabolic data were analyzed quantitatively by a non-mechanistic linlog kinetic model and purely data-driven approach using the PEC criterion (Pool Efflux Capacity). Especially the PEC analysis emerges as a straight-forward approach, which enables valid results even in case of metrological uncertainties due to its inherent robustness. As the result of various perturbation studies, the cystathionine-β-lyase (MetC), the methionine synthase (MetH/E) and the methionine exporter system (YjeH) were identified concurringly as enzymes with high flux control. In this regard, a distinct accumulation of the precursor L-homocysteine and an increasing overweight towards to L-cysteine, results in an unspecific metabolization of the cytotoxic intermediate to the accumulation product homolanthionine. The unspecific degradation of homolanthionine leads to a renewed formation of L-homocysteine and represents the entry point for an alternative synthesis route of the main byproduct L-isoleucine. This highly unfavorable accumulation scenario concerning the methionine synthesis (loss of carbon and energy) is ultimately based on an imbalance in the supply of L-aspartate (TCA synthesis route) and L-serine (EMP synthesis route). In order to enhance the information content of resulting transient metabolic data, the experimental design was additional adapted by utilization of full labeled [U13C]-L-serine as stimulus substrate. The high flux control by the methionine synthase (MetH/E) is certain to being attributed to a limited transmethylation capacity by the C1 metabolism. In this connection the hydroxymethyltransferase (GlyA) represents a key enzyme which provides the final transmethylation of L-homocysteine with methyl groups by degradation of L-serine. A targeted enhancement of the enzymatic affinity of GlyA to its substrate L-serine could lead to a compensation of the TCA/EMP imbalance and a simultaneous elimination of the accumulation cycle