Predictive macroscopic modeling of cell growth, metabolism and monoclonal antibody production: Case study of a CHO fed-batch production.

We describe a systematic approach to establish predictive models of CHO cell growth, cell metabolism and monoclonal antibody (mAb) formation during biopharmaceutical production. The prediction is based on a combination of an empirical metabolic model connecting extracellular metabolic fluxes with cellular growth and product formation with mixed Monod-inhibition type kinetics that we generalized to every possible external metabolite. We describe the maximum specific growth rate as a function of the integral viable cell density (IVCD). Moreover, we also take into account the accumulation of metabolites in intracellular pools that can influence cell growth. This is possible even without identification and quantification of these metabolites as illustrated with fed-batch cultures of Chinese Hamster Ovary (CHO) cells producing a mAb. The impact of cysteine and tryptophan on cell growth and cell productivity was assessed, and the resulting macroscopic model was successfully used to predict the impact of new, untested feeding strategies on cell growth and mAb production. This model combining piecewise linear relationships between metabolic rates, growth rate and production rate together with Monod-inhibition type models for cell growth did well in predicting cell culture performance in fed-batch cultures even outside the range of experimental data used for establishing the model. It could therefore also successfully be applied for in silico prediction of optimal operating conditions.

In-depth characterization of genome-scale network reconstructions for the in vitro synthesis in cell-free systems.

Cell-free systems containing multiple enzymes are becoming an increasingly interesting tool for one-pot syntheses of biochemical compounds. To extensively explore the enormous wealth of enzymes in the biological space, we present methods for assembling and curing data from databases to apply them for the prediction of pathway candidates for directed enzymatic synthesis. We use Kyoto Encyclopedia of Genes and Genomes to establish single organism models and a pan-organism model that is combining the available data from all organisms listed there. We introduce a filtering scheme to remove data that are not suitable, for example, generic metabolites and general reactions. In addition, a valid stoichiometry of reactions is required for acceptance. The networks created are analyzed by graph theoretical methods to identify a set of metabolites that are potentially reachable from a defined set of starting metabolites. Thus, metabolites not connected to such starting metabolites cannot be produced unless new starting metabolites or reactions are introduced. The network models also comprise stoichiometric and thermodynamic data that allow the definition of constraints to identify potential pathways. The resulting data can be directly applied using existing or future pathway finding tools.

Metabolic switches from quiescence to growth in synchronized Saccharomyces cerevisiae.

INTRODUCTION: The switch from quiescence (G0) into G1 and cell cycle progression critically depends on specific nutrients and metabolic capabilities. Conversely, metabolic networks are regulated by enzyme-metabolite interaction and transcriptional regulation that lead to flux modifications to support cell growth. How cells process and integrate environmental information into coordinated responses is challenging to analyse and not yet described quantitatively. OBJECTIVES: To quantitatively monitor the central carbon metabolism during G0 exit and the first 2 h after reentering the cell cycle from synchronized Saccharomyces cerevisiae. METHODS: Dynamic tailored 13C metabolic flux analysis was used to observe the intracellular metabolite flux changes, and the metabolome and proteome were observed to identify regulatory mechanisms. RESULTS: G0 cells responded immediately to an extracellular increase of glucose. The intracellular metabolic flux changed in time and specific events were observed. High fluxes into trehalose and glycogen synthesis were observed during the G0 exit. Both fluxes then decreased, reaching a minimum at t = 65 min. Here, storage degradation contributed significantly (i.e. 21%) to the glycolytic flux. In contrast to these changes, the glucose uptake rate remained constant after the G0 exit. The flux into the oxidative pentose phosphate pathway was highest (29-fold increase, 36.4% of the glucose uptake) at t = 65 min, while it was very low at other time points. The maximum flux seems to correlate with a late G1 state preparing for the S phase transition. In the G1/S phase (t = 87 min), anaplerotic reactions such as glyoxylate shunt increased. Protein results show that during this transition, proteins belonging to clusters related with ribosome biogenesis and assembly, and initiation transcription factors clusters were continuously synthetised. CONCLUSION: The intracellular flux distribution changes dynamically and these major rearrangements highlight the coordinate reorganization of metabolic flux to meet requirements for growth during different cell state.

Hepatocytes of Wistar and Sprague Dawley rats differ significantly in their central metabolism.

Wistar and Sprague-Dawley (SD) rats are most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and SD rat hepatocytes from middle (6 months) and old (23 months) age groups. Principle component analysis (PCA) on the specific uptake and production rates of amino acids, glucose, lactate and urea indicated clear differences between Wistar and SD rat hepatocytes. SD rat hepatocytes showed higher uptake rates of various essential and non-essential amino acids, particularly in early culture phases (0-12 h) compared to later phases (12-24 h). SD hepatocytes seem to be more sensitive to isolation procedure and in vitro culture requiring more amino acids for cellular maintenance and repair. Major differences between Wistar and SD rat hepatocytes were observed for glucose and branched chain amino acid metabolism. We conclude that the observed differences in the central carbon metabolism of isolated hepatocytes from these two rats should be considered when using one or the other rat type in studies on metabolic effects or diseases such as diabetes or obesity.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.

Network design and analysis for multi-enzyme biocatalysis.

BACKGROUND: As more and more biological reaction data become available, the full exploration of the enzymatic potential for the synthesis of valuable products opens up exciting new opportunities but is becoming increasingly complex. The manual design of multi-step biosynthesis routes involving enzymes from different organisms is very challenging. To harness the full enzymatic potential, we developed a computational tool for the directed design of biosynthetic production pathways for multi-step catalysis with in vitro enzyme cascades, cell hydrolysates and permeabilized cells. RESULTS: We present a method which encompasses the reconstruction of a genome-scale pan-organism metabolic network, path-finding and the ranking of the resulting pathway candidates for proposing suitable synthesis pathways. The network is based on reaction and reaction pair data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the thermodynamics calculator eQuilibrator. The pan-organism network is especially useful for finding the most suitable pathway to a target metabolite from a thermodynamic or economic standpoint. However, our method can be used with any network reconstruction, e.g. for a specific organism. We implemented a path-finding algorithm based on a mixed-integer linear program (MILP) which takes into account both topology and stoichiometry of the underlying network. Unlike other methods we do not specify a single starting metabolite, but our algorithm searches for pathways starting from arbitrary start metabolites to a target product of interest. Using a set of biochemical ranking criteria including pathway length, thermodynamics and other biological characteristics such as number of heterologous enzymes or cofactor requirement, it is possible to obtain well-designed meaningful pathway alternatives. In addition, a thermodynamic profile, the overall reactant balance and potential side reactions as well as an SBML file for visualization are generated for each pathway alternative. CONCLUSION: We present an in silico tool for the design of multi-enzyme biosynthetic production pathways starting from a pan-organism network. The method is highly customizable and each module can be adapted to the focus of the project at hand. This method is directly applicable for (i) in vitro enzyme cascades, (ii) cell hydrolysates and (iii) permeabilized cells.

Segmented linear modeling of CHO fed-batch culture and its application to large scale production.

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed-batch cultures. Using the model structure and parameter values from a small-scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed-batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785-797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Synthesis of natural variants and synthetic derivatives of the cyclic nonribosomal peptide luminmide in permeabilized E. coli Nissle and product formation kinetics.

We used a recombinant, permeabilized E. coli Nissle strain harbouring the plu3263 gene cluster from Photorhabdus luminescens for the synthesis of luminmide type cyclic pentapeptides belonging to the class of nonribosomally biosynthesized peptides (NRP). Cells could be fully permeabilized using 1 % v/v toluene. Synthesis of luminmides was increased fivefold when 0.3 mM EDTA was added to the substrate mixture acting as an inhibitor of metal proteases. Luminmide formation was studied applying different amino acid concentrations. Apparent kinetic parameters for the synthesis of the main product luminmide A from leucine, phenylalanine and valine were calculated from the collected data. K sapp values ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and r maxapp was about 3 × 10-8 mmol min-1(g CDW)-1). By removing phenylalanine from the substrate mixture, the formation of luminmide A was reduced tenfold while luminmide B was increased from 50 to 500 µg/l becoming the main product. Two new luminmides were synthesized in this study. Luminmide H incorporates tryptophan replacing phenylalanine in luminmide A. In luminmide I, leucine was replaced with 4,5-dehydro-leucine, a non-proteinogenic amino acid fed to the incubation mixture. Our study shows new opportunities for increasing the spectrum of luminmide variants produced, for improving production selectivity and for kinetic in vitro studies of the megasynthetases.

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized Schizosaccharomyces pombe expressing human UDP-glucose 6-dehydrogenase.

OBJECTIVES: To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose. RESULTS: In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD(+). Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h. CONCLUSIONS: Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

Mass isotopomer analysis of nucleosides isolated from RNA and DNA using GC/MS.

Nucleosides are biosynthesized from metabolites that are at key nodes of intermediary metabolism. Therefore, (13)C labeling patterns in nucleosides from ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in suitably designed isotopic tracer studies provide information on metabolic flux distributions of proliferating cells. Here, we present a gas chromatography (GC)-mass spectrometry (MS)-based approach that permits one to exploit that potential. In order to elucidate positional isotopomers of nucleosides from RNA and DNA, we screened the fragmentation spectra of their trimethylsilyl derivatives. We identified the molecular ion moieties retained in the respective fragment ions, focusing particularly on the carbon backbone. Nucleosides fragmented at the N-glycosidic bond provide nucleobase and/or ribose or 2'-deoxyribose fragment ions and fragments thereof. Nucleoside fragments composed of the nucleobase plus some carbons of the ribose ring were also observed. In total, we unequivocally assigned 31 fragments. The mass-isotopic distribution of the assigned fragments provides valuable information for later (13)C metabolic flux analysis as indicated by a labeling experiment applying [1-(13)C]glucose in a yeast culture.

Identification of active elementary flux modes in mitochondria using selectively permeabilized CHO cells.

Metabolic compartmentation is a key feature of mammalian cells. Mitochondria are the powerhouse of eukaryotic cells, responsible for respiration and the TCA cycle. We accessed the mitochondrial metabolism of the economically important Chinese hamster ovary (CHO) cells using selective permeabilization. We tested key substrates without and with addition of ADP. Based on quantified uptake and production rates, we could determine the contribution of different elementary flux modes to the metabolism of a substrate or substrate combination. ADP stimulated the uptake of most metabolites, directly by serving as substrate for the respiratory chain, thus removing the inhibitory effect of NADH, or as allosteric effector. Addition of ADP favored substrate metabolization to CO2 and did not enhance the production of other metabolites. The controlling effect of ADP was more pronounced when we supplied metabolites to the first part of the TCA cycle: pyruvate, citrate, α-ketoglutarate and glutamine. In the second part of the TCA cycle, the rates were primarily controlled by the concentrations of C4-dicarboxylates. Without ADP addition, the activity of the pyruvate carboxylase-malate dehydrogenase-malic enzyme cycle consumed the ATP produced by oxidative phosphorylation, preventing its accumulation and maintaining metabolic steady state conditions. Aspartate was taken up only in combination with pyruvate, whose uptake also increased, a fact explained by complex regulatory effects. Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase were identified as the key regulators of the TCA cycle, confirming existent knowledge from other cells. We have shown that selectively permeabilized cells combined with elementary mode analysis allow in-depth studying of the mitochondrial metabolism and regulation.

Macroscopic modeling of mammalian cell growth and metabolism.

We review major modeling strategies and methods to understand and simulate the macroscopic behavior of mammalian cells. These strategies comprise two important steps: the first step is to identify stoichiometric relationships for the cultured cells connecting the extracellular inputs and outputs. In a second step, macroscopic kinetic models are introduced. These relationships together with bioreactor and metabolite balances provide a complete description of a system in the form of a set of differential equations. These can be used for the simulation of cell culture performance and further for optimization of production.

Engineering the supply chain for protein production/secretion in yeasts and mammalian cells.

Metabolic bottlenecks play an increasing role in yeasts and mammalian cells applied for high-performance production of proteins, particularly of pharmaceutical ones that require complex posttranslational modifications. We review the present status and developments focusing on the rational metabolic engineering of such cells to optimize the supply chain for building blocks and energy. Methods comprise selection of beneficial genetic modifications, rational design of media and feeding strategies. Design of better producer cells based on whole genome-wide metabolic network analysis becomes increasingly possible. High-resolution methods of metabolic flux analysis for the complex networks in these compartmented cells are increasingly available. We discuss phenomena that are common to both types of organisms but also those that are different with respect to the supply chain for the production and secretion of pharmaceutical proteins.

Metabolic control at the cytosol-mitochondria interface in different growth phases of CHO cells.

Metabolism at the cytosol-mitochondria interface and its regulation is of major importance particularly for efficient production of biopharmaceuticals in Chinese hamster ovary (CHO) cells but also in many diseases. We used a novel systems-oriented approach combining dynamic metabolic flux analysis and determination of compartmental enzyme activities to obtain systems level information with functional, spatial and temporal resolution. Integrating these multiple levels of information, we were able to investigate the interaction of glycolysis and TCA cycle and its metabolic control. We characterized metabolic phases in CHO batch cultivation and assessed metabolic efficiency extending the concept of metabolic ratios. Comparing in situ enzyme activities including their compartmental localization with in vivo metabolic fluxes, we were able to identify limiting steps in glycolysis and TCA cycle. Our data point to a significant contribution of substrate channeling to glycolytic regulation. We show how glycolytic channeling heavily affects the availability of pyruvate for the mitochondria. Finally, we show that the activities of transaminases and anaplerotic enzymes are tailored to permit a balanced supply of pyruvate and oxaloacetate to the TCA cycle in the respective metabolic states. We demonstrate that knowledge about metabolic control can be gained by correlating in vivo metabolic flux dynamics with time and space resolved in situ enzyme activities.

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe--a quantitative approach using 13C-based metabolic flux analysis.

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

Dynamics of growth and metabolism controlled by glutamine availability in Chinese hamster ovary cells.

The physiology of animal cells is characterized by constantly changing environmental conditions and adapting cellular responses. Applied dynamic metabolic flux analysis captures metabolic dynamics and can be applied to industrially relevant cultivation conditions. We investigated the impact of glutamine availability or limitation on the physiology of CHO K1 cells in eight different batch and fed-batch cultivations. Varying glutamine availability resulted in global metabolic changes. We observed dose-dependent effects of glutamine in batch cultivation. Identifying metabolic links from the glutamine metabolism to specific metabolic pathways, we show that glutamine feeding results in its coupling to tricarboxylic acid cycle fluxes and in its decoupling from metabolic waste production. We provide a mechanistic explanation of the cellular responses upon mild or severe glutamine limitation and ammonia stress. The growth rate of CHO K1 decreased with increasing ammonia levels in the supernatant. On the other hand, growth, especially culture longevity, was stimulated at mild glutamine-limiting conditions. Flux rearrangements in the pyruvate and amino acid metabolism compensate glutamine limitation by consumption of alternative carbon sources and facilitating glutamine synthesis and mitigate ammonia stress as result of glutamine abundance.

Metabolism and metabolic burden by α1-antitrypsin production in human AGE1.HN cells.

The metabolic burden on human AGE1.HN cells imposed by the production of recombinant α1-antitrypsin (A1AT) was studied by comparing a selected high-producing clonal cell line with the parental cell line. RNA, lipid, and phosphatidylcholine fractions were higher in the producer cell line causing metabolic changes in the producer, e.g., increased glycine and glutamate production. By simulating the theoretical metabolite demand for production of mature A1AT using a network model, it was found that the differences in metabolic profiles between producer and parental cells match the observed increased C1-unit and nucleotide demand as well as lipid precursor demand in the producer. Additionally, metabolic flux analysis revealed similar energy metabolism in both cell lines. The increased lipid content seems related to activated secretion machinery in the producer cell line. Increased lipid and C1 metabolism seem important targets for further improvement of AGE1.HN and other producing mammalian cells.

A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off-gas.

Chemostat cultivation is a powerful tool for physiological studies of microorganisms. We report the construction and application of a set of eight parallel small-scale bioreactors with a working volume of 10 mL for continuous cultivation. Hungate tubes were used as culture vessels connected to multichannel-peristaltic pumps for feeding fresh media and removal of culture broth and off-gas. Water saturated air is sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allow sufficient mixing and oxygen transfer. Optical sensors are used for non-invasive online measurement of dissolved oxygen, which proved to be a powerful indicator of the physiological state of the cultures, particularly of steady-state conditions. Analysis of culture exhaust-gas by means of mass spectrometry enables balancing of carbon. The capacity of the developed small-scale bioreactor system was validated using the fission yeast Schizosaccharomyces pombe, focusing on the metabolic shift from respiratory to respiro-fermentative metabolism, as well as studies on consumption of different substrates such as glucose, fructose, and gluconate. In all cases, an almost completely closed carbon balance was obtained proving the reliability of the experimental setup.

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate.

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h(-1) where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. (13)C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO2 production and oxygen consumption rates which were in good agreement.