Analyzing the dynamics of cell growth and protein production in mammalian cell fed-batch systems using logistic equations

The logistic modeling approach was used to describe experimental viable cell density (X) and product concentration (P) data from two industrial fed-batch mammalian cell culture processes with maximum product concentrations in the 3.0-9.4 g/l range. In both cases, experimental data were well described by the logistic equations and the resulting specific growth rate and protein productivity profiles provided useful insights into the process kinetics. Subsequently, sensitivity equations for both the X and P models were analyzed which helped characterize the influence of model parameters on X and P time courses. This was augmented by conventional sensitivity analyses where five values of each model parameter, 25% apart, were used to generate X and P time courses. Finally, results from sensitivity analysis were used to simulate X and P time courses that were reflective of typical early- and late-stage fed-batch cell culture processes. Different combinations of the logistic model parameters were used to arrive at the same final product concentration demonstrating the ability of the logistic approach to describe the multitude of process paths that result in the same final product concentration. Overall, the capability of the logistic equations to well describe X and P data from fed-batch cultures, coupled with their ability to simulate the multitude of paths leading up to the desired cell density and product concentration profiles, make them a useful tool during mammalian cell fed-batch process development.     

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.     

Robust parameter estimation during logistic modeling of batch and fed‐batch culture kinetics

Methods for robust logistic modeling of batch and fed‐batch mammalian cell cultures are presented in this study. Linearized forms of the logistic growth, logistic decline, and generalized logistic equation were derived to obtain initial estimates of the parameters by linear least squares. These initial estimates facilitated subsequent determination of refined values by nonlinear optimization using three different algorithms. Data from BHK, CHO, and hybridoma cells in batch or fed‐batch cultures at volumes ranging from 100 mL–300 L were tested with the above approach and solution convergence was obtained for all three nonlinear optimization approaches for all data sets. This result, despite the sensitivity of logistic equations to parameter variation because of their exponential nature, demonstrated that robust estimation of logistic parameters was possible by this combination of linearization followed by nonlinear optimization. The approach is relatively simple and can be implemented in a spreadsheet to robustly model mammalian cell culture batch or fed‐batch data. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

The paper re-evaluates Verhulst and Monod models. It has been claimed that standard logistic equation cannot describe the decline phase of mammalian cells in batch and fed-batch cultures and in some cases it fails to fit somatic growth data. In the present work Verhulst, population-based mechanistic growth model was revisited to describe successfully viable cell density (VCD) in exponential and decline phases of batch and fed-batch cultures of three different CHO cell lines. Verhulst model constants, K, carrying capacity (VCD/ml or μg/ml) and r, intrinsic growth factor (h⁻¹) have physical meaning and they are of biological significance. These two parameters together define the course of growth and productivity and therefore, they are valuable in optimisation of culture media, developing feeding strategies and selection of cell lines for productivity. The Verhulst growth model approach was extended to develop productivity models for batch and fed-batch cultures. All Verhulst models were validated against blind data (R² > 0.95). Critical examination of theoretical approaches concluded that Monod parameters have no physical meaning. Monod-hybrid (pseudo-mechanistic) batch models were validated against specific growth rates of respective bolus and continuous fed-batch cultures (R² ≈ 0.90). The reduced form of Monod-hybrid model C_L/(K_L + C_L) describes specific growth rate during metabolic shift (R² ≈ 0.95). Verhulst substrate-based growth models compared favourably with Monod-hybrid models. Thus, experimental evidence implies that the constants in the Monod-hybrid model may not have physical meaning but they behave similarly to the biological constants in Michaelis–Menten enzyme kinetics, the basis of the Monod growth model.     

Caspase activation, sialidase release and changes in sialylation pattern of recombinant human erythropoietin produced by CHO cells in batch and fed-batch cultures

The activation of caspases represents a crucial turning point during a batch or a fed-batch culture of mammalian cells. It not only affects the quantity but also the quality of the recombinant glycoprotein produced. In this study, the activation of various caspases, the release of intracellular sialidase and the changes in sialylation pattern of a recombinant product, erythropoietin (EPO), in the culture medium were analyzed in both batch and fed-batch cultures. In both setups, all caspase activities peaked at the culture time point at which decline of cell viability was most pronounced. In addition, the release of intracellular lactate dehydrogenase (LDH) was also tracked during these cultures. The increase in LDH activity in the medium coincided with the increase of intracellular caspase activities, the release of sialidase and the observed decline in cell viability, suggesting that the LDH activity in the medium can be used as an indirect indicator of apoptotic cell death in bioreactors. Isoelectric focusing (IEF) coupled with double blotting was employed to analyze the changes in sialylation pattern of the recombinant EPO. This assay resulted in a prompt resolution of secreted EPO isoforms in a time course format. IEF profile of batch culture showed relatively consistent product sialylation compared to fed-batch culture, which showed gradual band shifts towards the isoforms with fewer sialic acid as the culture progressed. These data provided a guideline for the optimal time point to terminate the culture and collect products in batch and fed-batch cultures.Kok Hwee Chuan and Sing Fee Lim contributed equally to this work.     

Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network

A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc.     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Dynamic model of CHO cell metabolism

Fed-batch cultures are extensively used for the production of therapeutic proteins. However, process optimization is hampered by lack of quantitative models of mammalian cellular metabolism in these cultures. This paper presents a new kinetic model of CHO cell metabolism and a novel framework for simulating the dynamics of metabolic and biosynthetic pathways of these cells grown in fed-batch culture. The model defines a subset of the intracellular reactions with kinetic rate expressions based on extracellular metabolite concentrations and temperature- and redox-dependent regulatory variables. The simulation uses the rate expressions to calculate pseudo-steady state flux distributions and extracellular metabolite concentrations at discrete time points. Experimental data collected in this study for several different CHO cell fed-batch cultures are used to derive the rate expressions, fit the parameters, and validate the model. The simulations accurately predicted the effects of process variables, including temperature shift, seed density, specific productivity, and nutrient concentrations.     

An unstructured kinetic model of macromolecular metabolism in batch and fed-batch cultures of hybridoma cells producing monoclonal antibody

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359–368; E. Suzuki, D.F. Ollis, Biotechnol. Bioeng. 34 (1989) 1398–1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61–69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Phenomenological Background and Some Preliminary Trials of Automated Substrate Supply in pH-Stat Modal Fed-Batch Culture Using a Setpoint of High Limit

First, by considering all possible combination of methanol (as a carbon-energy source), peptone (as an organic carbon-nitrogen source), and ammonium sulfate (as an inorganic nitrogen source), five batch cultures of a methanol-assimilating bacterium, Protomonas extorquens, were done to elucidate the cause(s) of pH variations during the microbial cultivations. The batch cultures have been classified into five types in terms of stoichiometric equations of cell growth which involve the elements, C, H, O, and N. The equations explained the pH variation (drop and rise) of the batch cultures on the basis of the consumption and liberation of ammonium ion. Then, six fed-batch cultures using a setpoint of high limit were done by feeding either methanol only or methanol plus peptone. Growth rates could be controlled by the amount of substrate(s) fed per pulse. Supplying peptone in addition to methanol enhanced cell growth. Characteristic differences between pH-stat modal fed-batch cultures using a low limit and those using a high limit, and advantages of the pH-stat modal fed-batch culture using a setpoint of high limit are discussed.     

Influence of Primatone RL supplementation on sialylation of recombinant human interferon-gamma produced by Chinese hamster ovary cell culture using serum-free media

Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn(25) and Asn(97)) of IFN-gamma with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-gamma by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 1997.     

Fed-batch fermentation for the production of alpha-amylase by Bacillus amyloliquefaciens

Fed-batch cultures were performed to maximize the alpha-amylase activity in a bioreactor. Kinetic equations containing a catabolite repression effect were used to model the enzyme formation from Bacillus amyloliquefaciens. Fed-batch culture experiments were performed using maltose to implement the optimal feeding strategy. Optimal fed-batch culture based on sequential parameter estimation was performed successfully using off-line analysis while the fermentation was in progress. The enzyme activity from the fed-batch culture employing maltose was higher than that of the batch culture by 60%. Enzyme production using starch showed similar trends to those obtained using maltose.     

Growth-rate-independent production of recombinant glucoamylase by Fusarium venenatum JeRS 325

Most recombinant proteins generated in filamentous fungi are produced in fed-batch cultures, in which specific growth rate normally decreases progressively with time. Because of this, such cultures are more suited to the production of products that are produced efficiently at low-growth rates (e.g., penicillin) than to products which are produced more efficiently at high-growth rates (e. g., glucoamylase). Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was detected in the culture supernatant during exponential growth of F. venenatum JeRS 325 in batch culture. In glucose-limited chemostat cultures, GAM concentration increased with decrease in dilution rate, but the specific production rate of GAM (g GAM [g biomass](-1) h(-1)) remained approximately constant over the dilution-rate range 0.05 h to 0.19 h(-1), i.e., the recombinant protein was produced in a growth-rate-independent manner. The specific production rate decreased at dilution rates of 0.04 h(-1) and below. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass](-1) h(-1) were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L(-1). Compared to production in batch culture, and for the same final volume of medium, there was no increase in glucoamylase production when cultures were grown in fed-batch culture. The results suggested that a chemostat operated at a slow dilution rate would be the most productive culture system for enzyme production under this trypsin-like promoter.     

Enhanced hyaluronic acid production by a two-stage culture strategy based on the modeling of batch and fed-batch cultivation of Streptococcus zooepidemicus

This study aimed to enhance hyaluronic acid (HA) production by a two-stage culture strategy based on the modeling of batch and fed-batch culture of Streptococcus zooepidemicus. Batch culture had higher specific HA synthesis rate while fed-batch culture had higher specific cell growth rate. The lower specific HA synthesis rate in fed-batch culture resulted from the competition of cell growth for the common precursors at a low substrate concentration. Based on the modeling of batch and fed-batch culture of S. zooepidemicus, a two-stage culture strategy was proposed to enhance HA production. S. zooepidemicus were cultured in a fed-batch mode with sucrose concentration maintained at 1.0+/-0.2g/L during 0-8h and then batch culture was performed during 8-20h with an initial sucrose concentration of 15g/L. With the proposed two-stage culture strategy, HA production was increased to 6.6g/L compared with 5.0g/L in batch culture with the same total sucrose. The enhanced HA production by the proposed two-stage culture strategy resulted from the decreased inhibition of cell growth and the increased transformation rate of sucrose to HA.     

Analysis of kinetic,stoichiometry and regulation of glucose and glutamine metabolism in hybridoma batch cultures using logistic equations

Batch cultures were carried out to study the kinetic, stoichiometry, and regulation of glucose and glutamine metabolism of a murine hybridoma line. Asymmetric logistic equations (ALEs) were used to fit total and viable cell density, and nutrient and metabolite/product concentrations. Since these equations were analytically differentiable, specific rates and yield coefficients were readily calculated. Asymmetric logistic equations described satisfactorily uncontrolled batch cultures, including death phase. Specific growth rate showed a Monod-type dependence on initial glucose and glutamine concentrations. Yield coefficients of cell and lactate from glucose, and cell and ammonium from glutamine were all found to change dramatically at low residual glucose and glutamine concentrations. Under stoichiometric glucose limitation, the glucose-to-cell yield increased and glucose-to-lactate yield decreased, indicating a metabolic shift. Under stoichiometric glutamine limitation the glutamine-to-cell and glutamine-to-ammonium yields increased, but also glucose-to-cell yield increased and the glucose-to-lactate yield decreased. Monoclonal antibody production was mainly non-growth associated, independently of glucose and glutamine levels.     

Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.     

Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris

To improve the specific production rate of Rhizopus oryzae lipase (ROL) in Pichia pastoris, a protein that triggers the unfolded protein response in P. pastoris, the effect of sorbitol/methanol mixed substrates was tested in batch and fed-batch cultures. Remarkably, a different substrate consumption behaviour was observed depending on the host's phenotype (Mut(+) or Mut(s)) in batch cultures: when the methanol assimilation capacity is genetically reduced (Mut(s) phenotype), both substrates were consumed simultaneously, allowing not only a higher specific growth rate but also higher lipase levels (8.7-fold) compared to those obtained by cells growing on methanol as a sole carbon source in batch culture. This effect was not observed in Mut(+) phenotype, where the two substrates were consumed sequentially and the levels of heterologous product were only slightly higher (1.7-fold). A mixed substrate strategy was also applied to a Mut(s) fed-batch culture at a low methanol concentration set-point (0.5 gl(-1)). This resulted in a 2.2-fold increase in the heterologous protein level achieved, compared with the methanol-only feeding strategy. In addition, sorbitol co-feeding permitted the achievement of higher specific growth rates, and avoided the drastic decrease of the specific production rate observed after the start of the induction phase when methanol was used as sole carbon source This resulted in a significant increase in the overall bioprocess volumetric productivity (2.2-fold) and specific productivity (1.7-fold). Moreover, whereas increased ROL gene dosage in Mut(s) strains have been previously reported to be deleterious for P. pastoris cells growing on methanol, sorbitol co-feeding allowed for sustained cell growth and lipase production.