Modified monoclonal antibody production kinetics kappa/gamma mRNA levels, and metabolic activities in a murine hybridoma selected by continuous Culture

During long-term continuous culture of the hybridoma cell line 11317, a better-producing subclone (I1317-SF11), giving improved productivity, has been selected. The comparison of the original cell line (I1317-DC) with this subclone revealed that although the growth patterns of both clones were similar, both in continuous and in batch cultures, considerable differences could be seen between the clones with respect to monoclonal antibody (MAB) accumulation, MAB production rate, the levels of mRNA coding for heavy and light chains of IgG, and some metabolic activities. In continuous culture as well as in batch culture, I1317-SF11 showed increased levels of mRNA coding for kappa and gamma chains compared with I1317-DC and/or a modified ratio of the mRNA species when compared to that in I1317-DC. Using pulse experiments, it could be established that the biosynthesis of both chains was augmented in I1317-SF11. Although the kappa and gamma mRNA levels were modified or inversed for I1317-SF11, the cells always synthesized more kappa than gamma chains. The overall increase in the synthetic activity of I1317-SF11 is suggested as one reason for the considerable increase of IgG productivity and product accumulation in continuous culture as well as in repeated batch cultures. Tests concerning metabolic activity revealed that I1317-SF11 had a predominantly glycolytic metabolism independent of growth requirements, whereas for I1317-DC the metabolism became increasingly glycolytic with increased growth. The antibody yield coefficient of I1317-SF11 on glutamine was significantly higher than that of I1317-DC for the continuous culture, whereas the antibody coefficients on glucose were almost similar for both clones under the different culture conditions used. Both antibody coefficients were considerablly influenced by the specific growth rate.All these facts together lead to the conclusion that subclone I1317-SF11 uses more of the energy available, or it was the energy and/or precursors available for the synthesis and production of MAB more efficiently than the thesis and production of MAB more efficiently than the original cell line. Although the levels of mRNA coding for heavy and light chains of IgG were modified, it could be confirmed that the overall regulation of MAB-synthesis and -production occurs post-translationally and that at higher growth rates, more biosynthetic activity is diverted to biomass production. (c) 1994 John Wiley & Sons, Inc.     

Determinants and rate laws of growth and death of hybridoma cells in continuous culture

Experimental data from six hybridoma cell lines grown under diverse experimental conditions in both normal continuous and perfusion cultures are analyzed with respect to the significance of nutrients and products in determining the growth and death rates of cells and with respect to their mathematical modeling. It is shown that neither nutrients (glucose and glutamine) nor the common products lactic acid, ammonia, and monoclonal antibody can be generally assumed to be the clear-limiting or inhibiting factors for most of the cultures. Correspondingly, none of the unstructured models existing in the literature can be generally applied to describe the experimental data obtained over a relatively wide range of cultivation conditions as considered in this work. Surprisingly, for all cultures the specific growth rate (mu) almost linearly correlates with the ratio of the viable cell concentration (NV) to the dilution (perfusion) rate (D). Similarly, the specific death rate (kd) is a function of the ratio of the total cell concentration (Nt) to the dilution (perfusion) rate. These results strongly suggest the formation of not yet identified critical factors or autoinhibitors that determine both the growth and death rates of hybridoma cells. Based on these observations, simple kinetic models are developed for mu and kd which describe the experimental data satisfactorily. Analysis of the experimental data with the kinetic models reveals that under the current cultivation conditions the formation rate of the autoinhibitor(s) or the sensitivity of cell growth and death to the autoinhibitor(s) is mainly affected by the medium composition. Irrespective of the cell lines, cells grown on serum-containing media have almost the same model parameters, which are distinctively different from those of cells grown on serum-free media. Furthermore, in contrast to the prevailing view, kd is shown to positively correlate with mu if the effects of cell concentration and dilution (perfusion) rate are considered. Several important implications of these findings are discussed for the optimization and control of animal cell culture.     

A kinetic model for product formation of microbial and mammalian cells

Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. (c) 1995 John Wiley & Sons, Inc.     

Continuous microalgal cultivation in a laboratory-scale photobioreactor under seasonal day–night irradiation: experiments and simulation

In this work, the production of Scenedesmus obliquus in a continuous flat-plate laboratory-scale photobioreactor (PBR) under alternated day–night cycles was tested both experimentally and theoretically. Variation of light intensity according to the four seasons of the year were simulated experimentally by a tunable LED lamp, and effects on microalgal growth and productivity were measured to evaluate the conversion efficiency of light energy into biomass during the different seasons. These results were used to validate a mathematical model for algae growth that can be applied to simulate a large-scale production unit, carried out in a flat-plate PBR of similar geometry. The cellular concentration in the PBR was calculated in both steady-state and transient conditions, and the value of the maintenance kinetic term was correlated to experimental profiles. The relevance of this parameter was finally outlined.     

An energetic model for oxygen-limited metabolism

Microbial production of 2,3-butanediol by Klebsiella oxytoca occurs under conditions of an oxygen limitation. The extent to which substrate is oxidized to 2,3-butanediol and its coproducts, (acetic acid, acetoin, and ethanol) and the relative flow rates of substrate to energetic and biosynthetic pathways are controlled by the degree of oxygen limitation. Two energetic relationships which describe the response to an oxygen limitation have been derived. The first relationship describes the coupling between growth and energy production observed under oxygen-limited conditions. This allows calculation of energetic parameters and modeling of the cell mass and substrate profiles in terms of the degree of oxygen limitation only. The second relationship describes the average degree of oxidation and the rate of the end-product flow. The model has been tested with both batch and continuous culture. During these kinetic studies, two phases of growth have been observed: energy-coupled growth, which was described above; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal culture performance with respect to 2,3-butanediol productivity occurs during energy-coupled growth. (c) 1993 John Wiley & Sons, Inc.     

Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum

Stoichiometric analysis is applied to continuous glycerol fermentation by Clostridium butyricum to calculate theoretical maximum yields and to predict preferred pathways under different conditions. The upper limits of product concentration and productivity as a function of dilution rate in continuous culture is also predicted from product inhibition kinetic. The theoretical maximum propanediol yield (0.72 mol/mol glycerol) which is calculated for a culture without hydrogen and butyric acid formation agrees well with the experimental maximum value (around 0.71 mol/mol). Comparisons of experimental results (product concentration and productivity) with theoretical calculations and those of the glycerol fermentation by Klebsiella pneumoniae reveal that the production of 1,3-propanediol by C. butyricum is far below the optimum performance available with the present strain. One of the reasons is the relatively high formation of butyric acid under the culture conditions so far applied. The distribution of reducing equivalents to propanediol and hydrogen is also suboptimal. The utilization of the reducing power from pyruvate oxidation for propanediol production is about 60–70% of the theoretical maximum under the present experimental conditions.     

Analysis of cell growth kinetics and substrate diffusion in a polymer scaffold.

The cultivation of cartilage cells (chondrocytes) in polymer scaffolds leads to implants that may potentially be used to repair damaged joint cartilage or for reconstructive surgery. For this technique to be medically applicable, the physical parameters that govern cell growth in a polymer scaffold must be understood. This understanding of cell behavior under in vitro conditions, where diffusion is the primary mode of transport of nutrients, may aid in the scale-up of the cartilage generation process. A mathematical model of chondrocyte generation and nutrient consumption is developed here to analyze the behavior of cell growth in a biodegradable polymer matrix for a series of different thickness polymers. Recent literature has implied that the diffusion of nutrients is a major factor that limits cell growth (Freed et al., 1994). In the present paper, a mathematical model is developed to directly relate the effects of increasing cell mass in the polymer matrix on the transport of nutrients. Reaction and diffusion of nutrients in the cell-polymer system are described using the fundamental species continuity equations and the volume averaging method. The volume averaging method is utilized to derive a single averaged nutrient continuity equation that includes the effective transport properties. This approach allows for the derivation of effective diffusion and rate coefficients as functions of the cell volume fraction. The cell volume fraction as a function of time is determined by solution of a material balance on cell mass. Growth functions including the Moser, a modified Contois, and an nth-order heterogeneous growth kinetic model are evaluated through a parameter analysis, and the results are compared to experimental data found in the literature. The results indicate that cellular functions in conjunction with mass transfer processes can account partially for the general trends in the cell growth behavior for various thickness polymers. The Contois growth function appeared to describe the data more accurately in terms of the lag period at early times and the long time limits. However, all kinetic growth functions required variations in the kinetic parameters to fully describe the effects of polymer thickness. This result implies that restricted diffusion of nutrients is not the sole factor limiting cell growth when the thickness of the polymer is changed. Therefore, further experimental data and model improvements are needed to accurately describe the cell growth process.     

Explicit Kinetic Heterogeneity: Mathematical Models for Interpretation of Deuterium Labeling of Heterogeneous Cell Populations

Estimation of division and death rates of lymphocytes in different conditions is vital for quantitative understanding of the immune system. Deuterium, in the form of deuterated glucose or heavy water, can be used to measure rates of proliferation and death of lymphocytes in vivo. Inferring these rates from labeling and delabeling curves has been subject to considerable debate with different groups suggesting different mathematical models for that purpose. We show that the three most common models, which are based on quite different biological assumptions, actually predict mathematically identical labeling curves with one parameter for the exponential up and down slope, and one parameter defining the maximum labeling level. By extending these previous models, we here propose a novel approach for the analysis of data from deuterium labeling experiments. We construct a model of “kinetic heterogeneity” in which the total cell population consists of many sub-populations with different rates of cell turnover. In this model, for a given distribution of the rates of turnover, the predicted fraction of labeled DNA accumulated and lost can be calculated. Our model reproduces several previously made experimental observations, such as a negative correlation between the length of the labeling period and the rate at which labeled DNA is lost after label cessation. We demonstrate the reliability of the new explicit kinetic heterogeneity model by applying it to artificially generated datasets, and illustrate its usefulness by fitting experimental data. In contrast to previous models, the explicit kinetic heterogeneity model 1) provides a novel way of interpreting labeling data; 2) allows for a non-exponential loss of labeled cells during delabeling, and 3) can be used to describe data with variable labeling length.     

Mathematical modeling and analysis of glucose and glutamine utilization and regulation in cultures of continuous mammalian cells

A number of factors have been shown to affect the metabolism of glucose and glutamine in mammalian cells and their mechanisms have been partially elucidated. Despite these efforts, a quantitative knowledge of the significance of these factors, the regulation of glucose and glutamine utilization, and particularly the interactions of these two nutrients is still lacking. Controversies exist in the literature. To clarify some of these controversies, mathematical models are proposed in this work which enable to separate and identify the effects of individual factors. Experimental data from five cell lines obtained in batch, fed-batch, and continuous cultures, both under steady-state and transient conditions, were used to verify the model formulations. The resulting kinetic models successfully describe all these cultures. According to the models, the specific consumption rate of glucose (Q(Glc)) of continuous animal cells under normal culture conditions can be expressed as a sum of three parts: a part owing to cell growth; a part owing to glucose excess; and a part owing to glutamine regulation. The specific consumption rate of glutamine (q(Glc)7) can be expressed as a sum of only two parts: a part owing to cell growth; and a part owing to glutamine excess. Using the kinetic models the interaction and regulation of glucose and glutamine utilizations are quantitatively analyzed. The results indicate that, whereas q(Glc) is affected by glutamine, q(Gln) appears to be not or less significantly affected by glucose. It is also shown that the relative utilizations of glucose and glutamine by anabolism and catabolism are mainly affected by the residual concentrations of the respective compounds and are less sensitive to growth rate and the nature of growth limitation.(c) 1995 John Wiley & Sons, Inc.     

Scale-up effects on kinetic parameters and on predictions of a yeast recycle continuous ethanol fermentation model incorporating loss of cell viability

The scale-up effects on kinetic parameters and on predictions of a yeast recycle continuous ethanol fermentation model incorporating loss of cell viability were evaluated. The average level of cell viability estimated for large scale was similar to that estimated for small scale, although with a major standard deviation. The values of specific rate of cell viability loss were equal for the two scales. These results were due to the utilization of the same aeration rate for both scales, one of the main factors for cell-viability maintenance. The kinetic parameters were not significantly affected by the scale-up of the fermentation process. Major differences were observed for the maximum specific growth rate and for maximum ethanol concentrations for which, growth and ethanol production are totally inhibited. The scale-up did not result in lack of fit of the mathematical model to the experimental data.     

Synchronized mammalian cell culture: Part II—population ensemble modeling and analysis for development of reproducible processes

The consideration of inherent population inhomogeneities of mammalian cell cultures becomes increasingly important for systems biology study and for developing more stable and efficient processes. However, variations of cellular properties belonging to different sub‐populations and their potential effects on cellular physiology and kinetics of culture productivity under bioproduction conditions have not yet been much in the focus of research. Culture heterogeneity is strongly determined by the advance of the cell cycle. The assignment of cell‐cycle specific cellular variations to large‐scale process conditions can be optimally determined based on the combination of (partially) synchronized cultivation under otherwise physiological conditions and subsequent population‐resolved model adaptation. The first step has been achieved using the physical selection method of countercurrent flow centrifugal elutriation, recently established in our group for different mammalian cell lines which is presented in Part I of this paper series. In this second part, we demonstrate the successful adaptation and application of a cell‐cycle dependent population balance ensemble model to describe and understand synchronized bioreactor cultivations performed with two model mammalian cell lines, AGE1.HN_AAT and CHO‐K1. Numerical adaptation of the model to experimental data allows for detection of phase‐specific parameters and for determination of significant variations between different phases and different cell lines. It shows that special care must be taken with regard to the sampling frequency in such oscillation cultures to minimize phase shift (jitter) artifacts. Based on predictions of long‐term oscillation behavior of a culture depending on its start conditions, optimal elutriation setup trade‐offs between high cell yields and high synchronization efficiency are proposed. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:175–185, 2015     

Kinetic study of instability of recombinant plasmid pPLc23trpAl in E. coli using two-stage continuous culture system

Derepression of the phage lambda p(L) promoter on recombinant plasmid pPLc 23-trpAl caused a rapid increase of plasmid free segregants in the population. In continuous culture, increased production of trpA protein follwing derepression was accompanied by a continuous deceleration of specific growth rate. In the repressed condition, plasmid loss per generation in continuous culture decreased as dilution rate increased from 0.06 to 1.08 h(-1). Over this range, the concentration of plasmid DNA within the cell decreased eightfold corresponding to a decrease in plasmid number from 74 to 32 molecules/cell. The use of a two-stage continuous culture system coupled with a temperature sensitive expression system allows a high trpA productivity from the derepressed plasmid for more than 48 h and also offers a possibility of minimizing the instability problem of high expression recombinants. Such a system also permits the critical study of the effects of fermentation and other regulatory parameters on expression under better controlled conditions than is possible in a batch culture or single-stage continous culture.     

A comparison of different culture methods for hybridoma propagation and monoclonal antibody production

A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr^–1.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

用遗传算法进行重组大肠杆菌发酵动力学的分析

重组大肠杆菌在诱导表达人表皮生长因子的过程促使细菌的生长受到抑制,一部分重组菌丧失了分裂能力,但仍保持着一定的代谢活力,分离成为存活但不能培养的细菌,根据大肠杆菌在表达外源蛋白过程中细胞生理状态的不同将细菌分为三类,提出一个描述诱导表达过程中重组大肠杆菌分离、生长的动力学模型．应用遗传算法对不同底物浓度的细胞生长、分离和产物合成的动力学参数进行了有效地估计,避免了传统算法可能陷于局部最优的问题,模型计算结果与实验结果吻合良好．分离模型在初始糖浓为5-20g/L的范围内可以较好地描述发酵过程中细胞生长、分离和目标产物表达的过程并具有一定的预测能力．     

Plasmid stability and kinetics of continuous production of glucoamylase by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in an airlift bioreactor

Production of glucoamylase by recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) in a continuous stirred tank bioreactor was studied at different dilution rates. Plasmid stability was found to be growth (dilution rate) dependent; it increased with the dilution rate. Bioreactor productivity and specific productivity also increased with the dilution rate. A kinetic equation was used to model the plasmid stability kinetics. The growth rate ratio between plasmid-carrying and plasmid-free cells decreased from 1.397 to 1.215, and segregational instability or probability of plasmid loss from each cell division decreased from 0.059 to 0.020 as the dilution rate increased from 0.10 to 0.37 1/h. The specific growth rates increased with dilution rate, while the growth rate difference between plasmid-carrying and plasmid-free cell populations was negligible. This was attributed to the low copy number of the hybrid plasmid pGAC9. Thus, the growth rate had no significant effect on plasmid instability. The proposed kinetics was consistent with experimental results, and the model simulated the experimental data well.     

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.     

Free fatty acid production in Escherichia coli under phosphate-limited conditions

Microbially synthesized fatty acids are an attractive platform for producing renewable alternatives to petrochemically derived transportation fuels and oleochemicals. Free fatty acids (FFA) are a direct precursor to many high-value compounds that can be made via biochemical and ex vivo catalytic pathways. To be competitive with current petrochemicals, flux through these pathways must be optimized to approach theoretical yields. Using a plasmid-free, FFA-producing strain of Escherichia coli, a set of chemostat experiments were conducted to gather data for FFA production under phosphate limitation. A prior study focused on carbon-limited conditions strongly implicated non-carbon limitations as a preferred media formulation for maximizing FFA yield. Here, additional data were collected to expand an established kinetic model of FFA production and identify targets for further metabolic engineering. The updated model was able to successfully predict the strain’s behavior and FFA production in a batch culture. The highest yield observed under phosphate-limiting conditions (0.1 g FFA/g glucose) was obtained at a dilution rate of 0.1 h^?1, and the highest biomass-specific productivity (0.068 g FFA/gDCW/h) was observed at a dilution rate of 0.25 h^?1. Phosphate limitation increased yield (～45 %) and biomass-specific productivity (～300 %) relative to carbon-limited cultivations using the same strain. FFA production under phosphate limitation also led to a cellular maintenance energy ～400 % higher (0.28 g/gDCW/h) than that seen under carbon limitation.     

Kinetic modeling of lipase‐catalyzed esterification reaction between oleic acid and trimethylolpropane: A simplified model for multi‐substrate multi‐product ping–pong mechanisms

Kinetic models are among the tools that can be used for optimization of biocatalytic reactions as well as for facilitating process design and upscaling in order to improve productivity and economy of these processes. Mechanism pathways for multi‐substrate multi‐product enzyme‐catalyzed reactions can become very complex and lead to kinetic models comprising several tens of terms. Hence the models comprise too many parameters, which are in general highly correlated and their estimations are often prone to huge errors. In this study, Novozym^®435 catalyzed esterification reaction between oleic acid (OA) and trimethylolpropane (TMP) with continuous removal of side‐product (water) was carried out as an example for reactions that follow multi‐substrate multi‐product ping‐pong mechanisms. A kinetic model was developed based on a simplified ping‐pong mechanism proposed for the reaction. The model considered both enzymatic and spontaneous reactions involved and also the effect of product removal during the reaction. The kinetic model parameters were estimated using nonlinear curve fitting through unconstrained optimization methodology and the model was verified by using empirical data from different experiments and showed good predictability of the reaction under different conditions. This approach can be applied to similar biocatalytic processes to facilitate their optimization and design. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1422–1429, 2013