Rapid media transition: An experimental approach for steady state analysis of metabolic pathways

Commonly steady state analysis of microbial metabolism is performed under well defined physiological conditions in continuous cultures with fixed external rates. However, most industrial bioprocesses are operated in fed‐batch mode under non‐stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup—rapid media transition—enables steady state perturbation of metabolism on a time scale of several minutes in parallel to operating bioprocesses. For this purpose, cells are separated from the production process and transferred into a lab‐scale stirred‐tank reactor with modified environmental conditions. This new approach was evaluated experimentally in four rapid media transition experiments with Escherichia coli from a fed‐batch process. We tested the reaction to different carbon sources entering at various points of central metabolism. In all cases, the applied substrates (glucose, succinate, acetate, and pyruvate) were immediately utilized by the cells. Extracellular rates and metabolome data indicate a metabolic steady state during the short‐term cultivation. Stoichiometric analysis revealed distribution of intracellular fluxes, which differs drastically subject to the applied carbon source. For some reactions, the variation of flux could be correlated to changes of metabolite concentrations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Membrane Inlet Mass Spectrometry for the on‐line Measurement of Metabolic Fluxes: Case Lysine‐Production by Corynebacterium glutamicum

A miniaturized reactor system with on‐line measurement of respiration rates by membrane inlet mass spectrometry was applied for the on‐line metabolic flux analysis at different phases of a 1.2 L batch culture of lysine‐producing Corynebacterium glutamicum. For this purpose, cells taken from the batch culture were transferred into the 12 mL mini reactor, and incubated for 15 min with [1‐¹⁸O]glucose. Quantification of oxygen uptake rate and CO₂ mass isotopomer production rates in combination with a simple metabolic model allowed the estimation of the flux partitioning ratio between the pentose phosphate pathway and glycolysis during the process. The relative flux into the pentose pathway increased during growth, and reached maxima at 11 and 17 h cultivation time coinciding with maxima of the differential lysine yield. The developed system is a promising tool for determination of metabolic flux dynamics in industrially relevant batch and fed‐batch cultures.     

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.     

Metabolic analysis of antibody producing CHO cells in fed‐batch production

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed‐batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed‐batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth. Biotechnol. Bioeng. 2013; 110: 1735–1747. © 2013 Wiley Periodicals, Inc.     

Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed‐batch CHO cell culture

Cell metabolism can vary considerably over the course of a typical fed‐batch antibody production process. However, the intracellular pathway alterations associated with various phases of growth and antibody production have yet to be fully elucidated using industrially relevant production hosts. Therefore, we performed ¹³C labeling experiments and metabolic flux analysis (MFA) to characterize CHO cell metabolism during four separate phases of a fed‐batch culture designed to closely represent industrial process conditions. First, we found that peak specific growth rate was associated with high lactate production and minimal TCA cycling. Conversely, we found that lactate metabolism switched from net production to net consumption as the culture transitioned from peak growth to peak antibody production. During the peak antibody production phase, energy was primarily generated through oxidative phosphorylation, which was also associated with elevated oxidative pentose phosphate pathway (oxPPP) activity. Interestingly, as TCA cycling and antibody production reached their peaks, specific growth rate continued to diminish as the culture entered stationary phase. However, TCA cycling and oxPPP activity remained high even as viable cell density began to decline. Overall, we found that a highly oxidative state of metabolism corresponded with peak antibody production, whereas peak cell growth was characterized by a highly glycolytic metabolic state. Biotechnol. Bioeng. 2013; 110: 2013–2024. © 2013 Wiley Periodicals, Inc.     

Optimizing performance of semi‐continuous cell culture in an ambr15™ microbioreactor using dynamic flux balance modeling

The ambr bioreactors are single‐use microbioreactors for cell line development and process optimization. With operating conditions for large‐scale biopharmaceutical production properly scaled down, microbioreactors such as the ambr15? can potentially be used to predict the effect of process changes such as modified media or different cell lines. While there have been some recent studies evaluating the ambr15? technology as a scale‐down model for fed‐batch operations, little has been reported for semi‐continuous or continuous operation. Gassing rates and dilution rates in the ambr15? were varied in this study to attempt to replicate performance of a perfusion process at the 5 L scale. At both scales, changes to metabolite production and consumption, and cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15? bioreactor that produced metabolic shifts and specific metabolic and protein production rates that are characteristic of the corresponding 5 L perfusion process. A dynamic flux balance (DFB) model was employed to understand and predict the metabolic changes observed. The DFB model predicted trends observed experimentally, including lower specific glucose consumption and a switch from lactate production to consumption when dissolved CO₂ was maintained at higher levels in the broth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:420–431, 2018     

Acetate formation during recombinant protein production in Escherichia coli K‐12 with an elevated NAD(H) pool

Acetate formation is a disadvantage in the use of Escherichia coli for recombinant protein production, and many studies have focused on optimizing fermentation processes or altering metabolism to eliminate acetate accumulation. In this study, E. coli MEC697 (MG1655 nadR nudC mazG) maintained a larger pool of NAD(H) compared to the wild‐type control, and also accumulated lower concentrations of acetate when grown in batch culture on glucose. In steady‐state cultures, the elevated total NAD(H) found in MEC697 delayed the threshold dilution rate for acetate formation to a growth rate of 0.27 h^?1. Batch and fed‐batch processes using MEC697 were examined for the production of β‐galactosidase as a model recombinant protein. Fed‐batch culture of MEC697/pTrc99A‐lacZ compared to MG1655/pTrc99A‐lacZ at a growth rate of 0.22 h^?1 showed only a modest increase of protein formation. However, 1 L batch growth of MEC697/pTrc99A‐lacZ resulted in 50% lower acetate formation compared to MG1655/pTrc99A‐lacZ and a two‐fold increase in recombinant protein production.     

Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported K(s) vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NH(x)/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures. (c) 1994 John Wiley & Sons, Inc.     

Investigation of metabolic variability observed in extended fed batch cell culture

A 13‐day fed‐batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell‐specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1519–1527, 2013     

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.Institut National Polytechnique de Lorraine, ENSAIA BP 172, 2 avenue de la forêt de Haye, 54505, Vandoeuvre Cedex France     

A structured kinetic modeling framework for the dynamics of hybridoma growth and monoclonal antibody production in continuous suspension cultures

A structured kinetic model is developed to describe the dynamics of hybridoma growth and the production of monoclonal antibodies and metabolic waste products in suspension culture. The crucial details of known metabolic processes in hybridoma cells are incorporated by dividing the cell mass into four intracellular metabolic pools. The model framework and structure allow the dynamic calculation of the instantaneous specific growth rate of a hybridoma culture. The steady state and dynamic simulations of the model equations exhibit excellent agreement with experimentally observed trends in substrate utilization and product formation. The model represents the first to include any degree of metabolic detail and structure in describing a hybridoma culture. In so doing, it provides the basic modeling framework for incorporating further details of metabolism and can be a useful tool to study various strategies for enhancing hybridoma growth as well as viability and the production of monoclonal antibodies in suspension cultures.     

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.     

Metabolic flux analysis for calcium dependent antibiotic (CDA) production in Streptomyces coelicolor

The calcium dependent antibiotic (CDA) is a nonribosomal lipopeptide produced by Streptomyces coelicolor. We constructed a metabolic network of more than 400 reactions for the primary and secondary metabolism of S. coelicolor and used computational metabolic flux balancing to investigate some of the factors affecting growth and production of CDA. Computational results indicated that the CDA production was concomitant with growth. Computational specific growth rates were twice as high as the experimental specific growth rates. Metabolic flux distributions and sensitivity analyses computed for various phases of the batch culture indicated that the specific CDA production rate was affected by nitrogen assimilation, pentose phosphate pathway, shikimate biosynthesis, and oxoglutarate fluxes. Consequently, these metabolic targets were tested using genetic deletions in the model which increased the in silico specific CDA production rate.     

Elucidation of metabolism in hybridoma cells grown in fed‐batch culture by genome‐scale modeling

Genome‐scale modeling of mouse hybridoma cells producing monoclonal antibodies (mAb) was performed to elucidate their physiological and metabolic states during fed‐batch cell culture. Initially, feed media nutrients were monitored to identify key components among carbon sources and amino acids with significant impact on the desired outcome, for example, cell growth and antibody production. The monitored profiles indicated rapid assimilation of glucose and glutamine during the exponential growth phase. Significant increase in mAb concentration was also observed when glutamine concentration was controlled at 0.5 mM as a feeding strategy. Based on the reconstructed genome‐scale metabolic network of mouse hybridoma cells and fed‐batch profiles, flux analysis was then implemented to investigate the cellular behavior and changes in internal fluxes during the cell culture. The simulated profile of the cell growth was consistent with experimentally measured specific growth rate. The in silico simulation results indicated (i) predominant utilization of glycolytic pathway for ATP production, (ii) importance of pyruvate node in metabolic shifting, and (iii) characteristic pattern in lactate to glucose ratio during the exponential phase. In future, experimental and in silico analyses can serve as a promising approach to identifying optimal feeding strategies and potential cell engineering targets as well as facilitate media optimization for the enhanced production of mAb or recombinant proteins in mammalian cells. Biotechnol. Bioeng. 2009;102: 1494–1504. © 2008 Wiley Periodicals, Inc.     

Trichloroethene and cis‐1,2‐dichloroethene concentration‐dependent toxicity model simulates anaerobic dechlorination at high concentrations: I. batch‐fed reactors

A model was developed to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures under batch‐growth conditions. A reductively dechlorinating anaerobic Evanite subculture (EV‐cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis‐1,2‐dichloroethene (cDCE) in batch‐fed reactors. A second Point Mugu (PM) culture was also studied in the cDCE accumulating batch‐fed experiment, as well as in a time‐ and concentration‐dependent cDCE exposure experiment. Both cultures accumulated cDCE to concentrations ranging from 9,000 to 12,000 µM before cDCE production from TCE ceased. Exposure to approximately 3,000 and 6,000 µM cDCE concentrations for 5 days during continuous TCE dechlorination exhibited greater loss in activity proportional to both time and concentration of exposure than simple endogenous decay. Various inhibition models were analyzed for the two cultures, including the previously proposed Haldane inhibition model and a maximum threshold inhibition model, but neither adequately fit all experimental observations. A concentration‐dependent toxicity model is proposed, which simulated all the experimental observations well. The toxicity model incorporates CAH toxicity terms that directly increase the cell decay coefficient in proportion with CAH concentrations. We also consider previously proposed models relating toxicity to partitioning in the cell wall (K_M/B), proportional to octanol–water partitioning (K_OW) coefficients. A reanalysis of previously reported modeling of batch tests using the Haldane model of Yu and Semprini, could be fit equally well using the toxicity model presented here, combined with toxicity proportioned to cell wall partitioning. A companion paper extends the experimental analysis and our modeling approach to a completely mixed reactor and a fixed film reactor. Biotechnol. Bioeng. 2010;107: 529–539. © 2010 Wiley Periodicals, Inc.     

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q_p) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q_p(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed‐batch experiments, leads to errors in the prediction, because q_p is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate‐limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin‐N synthase (IPNS) was the main rate‐limiting enzyme for penicillin‐G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady‐state as well as the dynamics during chemostat start‐up and fed‐batch cultivation. Biotechnol. Bioeng. 2010;106: 608–618. © 2010 Wiley Periodicals, Inc.     

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO₂ concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO₂ in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO₂ values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO₂. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO₂ concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO₂ accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO₂ might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.     

Homeostatic adjustment and metabolic remodeling in glucose-limited yeast cultures

We studied the physiological response to glucose limitation in batch and steady-state (chemostat) cultures of Saccharomyces cerevisiae by following global patterns of gene expression. Glucose-limited batch cultures of yeast go through two sequential exponential growth phases, beginning with a largely fermentative phase, followed by an essentially completely aerobic use of residual glucose and evolved ethanol. Judging from the patterns of gene expression, the state of the cells growing at steady state in glucose-limited chemostats corresponds most closely with the state of cells in batch cultures just before they undergo this "diauxic shift." Essentially the same pattern was found between chemostats having a fivefold difference in steady-state growth rate (the lower rate approximating that of the second phase respiratory growth rate in batch cultures). Although in both cases the cells in the chemostat consumed most of the glucose, in neither case did they seem to be metabolizing it primarily through respiration. Although there was some indication of a modest oxidative stress response, the chemostat cultures did not exhibit the massive environmental stress response associated with starvation that also is observed, at least in part, during the diauxic shift in batch cultures. We conclude that despite the theoretical possibility of a switch to fully aerobic metabolism of glucose in the chemostat under conditions of glucose scarcity, homeostatic mechanisms are able to carry out metabolic adjustment as if fermentation of the glucose is the preferred option until the glucose is entirely depleted. These results suggest that some aspect of actual starvation, possibly a component of the stress response, may be required for triggering the metabolic remodeling associated with the diauxic shift.     

13C‐metabolic flux analysis for batch culture of Escherichia coli and its pyk and pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites

Since most bio‐production processes are conducted in a batch or fed‐batch manner, the evaluation of metabolism with respect to time is highly desirable. Toward this aim, we applied ¹³C‐metabolic flux analysis to nonstationary conditions by measuring the mass isotopomer distribution of intracellular metabolites. We performed our analysis on batch cultures of wild‐type Escherichia coli, as well as on Pyk and Pgi mutants, obtained the fluxes and metabolite concentrations as a function of time. Our results for the wild‐type indicated that the TCA cycle flux tended to increase during growth on glucose. Following glucose exhaustion, cells controlled the branch ratio between the glyoxylate pathway and the TCA cycle, depending on the availability of acetate. In the Pyk mutant, the concentrations of glycolytic intermediates changed drastically over time due to the dumping and feedback inhibition caused by PEP accumulation. Nevertheless, the flux distribution and free amino acid concentrations changed little. The growth rate and the fluxes remained constant in the Pgi mutant and the glucose‐6‐phosphate dehydrogenase reaction was the rate‐limiting step. The measured fluxes were compared with those predicted by flux balance analysis using maximization of biomass yield or ATP production. Our findings indicate that the objective function of biosynthesis became less important as time proceeds on glucose in the wild‐type, while it remained highly important in the Pyk mutant. Furthermore, ATP production was the primary objective function in the Pgi mutant. This study demonstrates how cells adjust their metabolism in response to environmental changes and/or genetic perturbations in the batch cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010