Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions

PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions. Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8. The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates. Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions. It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced. Adenovirus infection resulted in profound changes in cell growth and metabolism. Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection. Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH. The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study. Deviations from this optimum resulted in significant reductions of virus productivity. The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production.     

The production of monoclonal antibody in growth-arrested hybridomas cultivated in suspension and immobilized modes.

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutamine, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH. Reprinted from Biotechnology and Bioengineering, Vol. 32, Pp 947-965 (1988)

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.     

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.     

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.     

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.     

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.     

Dichloroacetate increases cell and antibody yields in batch cultures of a hybridoma cell line

We have studied the effect of the pyruvate dehydrogenase (PDH) activator, dichloroacetate (DCA), on the growth, metabolism, and productivity of the PQXB (1/2) hybridoma cell line. In control batch cultures, cessation of growth and the onset of decline phase coincided with the time at which the media became exhausted of glutamine. Supplementation of the media with DCA (1 mM) extended the growth phase of this cell line by approximately 20 h without affecting its growth rate. This prolonged period of growth resulted in an increased maximum cell density (16%) and final antibody yield (55%). Repeat experiments showed these effects to be reproducible, with the increases in antibody yield being between 50 and 60%. DCA did not affect the specific rates of glucose utilization and lactate production. However, it decreased the specific glutamine consumption rate. This characteristic of DCA action appeared, at least in part, to provide an explanation for the extended growth phase exhibited by DCA-treated cultures, since it delayed the time at which the media became depleted of glutamine. The consumption and production kinetics for various nutrients and their metabolites in both control and DCA-treated cultures suggested that: (1) glutamine catabolism proceeded by a pathway involving conversion to glutamate by glutaminase followed by subsequent transamination by alanine aminotransferase, and (2) DCA decreased the specific glutamine consumption rate by directly or indirectly inhibiting the transamination. It is expected that the routine inclusion of DCA in media used for hybridoma cultivation will be valuable for enhancement of monoclonal antibody (Mab) yields on a laboratory scale. (c) 1996 John Wiley & Sons, Inc.     

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.     

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

Summary Recombinant human interferon- production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation. Offprint requests to: P. M. Hayter     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells

Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.     

Feed development for fed‐batch CHO production process by semisteady state analysis

Semisteady state cultures are useful for studying cell physiology and facilitating media development. Two semisteady states with a viable cell density of 5.5 million cells/mL were obtained in CHO cell cultures and compared with a fed‐batch mode control. In the first semisteady state, the culture was maintained at 5 mM glucose and 0.5 mM glutamine. The second condition had threefold higher concentrations of both nutrients, which led to a 10% increase in lactate production, a 78% increase in ammonia production, and a 30% reduction in cell growth rate. The differences between the two semisteady states indicate that maintaining relatively low levels of glucose and glutamine can reduce the production of lactate and ammonia. Specific amino acid production and consumption indicated further metabolic differences between the two semisteady states and fed‐batch mode. The results from this experiment shed light in the feeding strategy for a fed‐batch process and feed medium enhancement. The fed‐batch process utilizes a feeding strategy whereby the feed added was based on glucose levels in the bioreactor. To evaluate if a fixed feed strategy would improve robustness and process consistency, two alternative feeding strategies were implemented. A constant volume feed of 30% or 40% of the initial culture volume fed over the course of cell culture was evaluated. The results indicate that a constant volumetric‐based feed can be more beneficial than a glucose‐based feeding strategy. This study demonstrated the applicability of analyzing CHO cultures in semisteady state for feed enhancement and continuous process improvement. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Metabolic and kinetic studies of hybridomas in exponentially fed-batch cultures using T-flasks

Exponentially fed-batch cultures (EFBC) of a murine hybridoma in T-flasks were explored as a simple alternative experimental tool to chemostats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode using an exponentially increasing and predetermined stepwise feeding profile of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution rates below the maximum growth rate. In particular, steady-state for growth rate and concentration of viable cells, glucose, and lactate was attained at different dilution rates between 0.005 and 0.05 h^–1. For such a range, the glucose and lactate metabolic quotients and the steady-state glucose concentration increased, whereas total MAb, volumetric, and specific MAb production rates decreased 65-, 6-, and 3-fold, respectively, with increasing dilution rates. The lactate from glucose yield remained relatively constant for dilution rates up to 0.03 h^–1, where it started to decrease. In contrast, viability remained above 80% at high dilution rates but rapidly decreased at dilution rates below 0.02 h^–1. No true washout occurred during operation above the maximum growth, as concluded from the constant viable cell number. However, growth rate decreased to as low as 0.01 h^–1, suggesting the requirement of a minimum cell density, and concomitant autocrine growth factors, for growth. Chemostat operation drawbacks were avoided by EFBC in T-flasks. Namely, simple and stable operation was obtained at dilution rates ranging from very low to above the maximum growth rate. Furthermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.Abbreviations EFBC exponentially-fed batch culture - CSC continuous suspended culture - MAb monoclonal antibody - D dilution rate - q _i metabolic quotient or specific rate of consumption or production of i     

Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.     

Control of redox potential in hybridoma cultures: effects on MAb production, metabolism, and apoptosis

Culture redox potential (CRP) has proven to be a valuable monitoring tool in several areas of biotechnology; however, it has been scarcely used in animal cell culture. In this work, a proportional feedback control was employed, for the first time, to maintain the CRP at different constant values in hybridoma batch cultures for production of a monoclonal antibody (MAb). Reducing and oxidant conditions, in the range of -130 and +70 mV, were maintained in 1-l bioreactors through automatic control of the inlet gas composition. Cultures at constant DOT, in the range of 3 and 300 %, were used for comparison. The effect of constant CRP on cell concentration, MAb production, metabolism of glucose, glutamine, thiols, oxygen consumption, and programmed cell death, was evaluated. Reducing conditions resulted in the highest viable cell and MAb concentrations and thiols production, whereas specific glucose and glutamine consumption rates remained at the lowest values. In such conditions, programmed cell death, particularly apoptosis, occurred only after nutrient exhaustion. The optimum specific MAb production rate occurred at intermediate CRP levels. Oxidant conditions resulted in a detrimental effect in all culture parameters, increasing the specific glucose, glutamine, and oxygen consumption rates and inducing the apoptotic process, which was detected as early as 24 h even when glutamine and glucose were present at non-limiting concentrations. In most cases, such results were similar to those obtained in control cultures at constant DOT.     

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

A peculiar phenomenon, differing from the response of mammalian cells, occurred when Chinook salmon embryo (CHSE) cells were passaged in the medium lacking of both glucose and glutamine. To elucidate metabolic mechanism of CHSE cells, the metabolism parameters, key metabolic enzymes, and ATP levels were measured at different glucose and glutamine concentrations. In the glutamine-free culture, hexokinase activity kept constant, and lactate dehydrogenase (LDH) activity decreased. This indicated that lack of glutamine did not expedite glucose consumption but made it shift to lower lactate production and more efficient energy metabolism. The results coincided with the experimental results of unaltered specific glucose consumption rate and decreased yield coefficients of lactate to glucose. In the glucose-free culture, simultaneous increase of glutaminase activity and of specific ammonia production rate suggested an increased flux into the glutaminolysis pathway, and increases of both glutamate dehydrogenase activity and yield coefficient of ammonia to glutamine showed an increased flux into deamination pathway. However, when glucose and glutamine were both lacking, the specific consumption rates of most of amino acids increased markedly, together with decrease of LDH activity, indicating that pyruvate derived from amino acids, away from lactate production, remedied energy deficiency. When both glucose and glutamine were absent, intracellular ATP contents and the energy charge remained virtually unaltered.Revisions requested 16 December 2004; Revisions received 24 January 2005     

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor

In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed. Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b). In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model. Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies. Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model. The formation of toxic byproducts, such as ammonia and lactate (Hassellet al., 1991), was greatly reduced. The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model. Ammonia formation was also decreased compared with both the batch and fed-batch cultures. Most importantly, the monoclonal antibody concentration reached 900 mg l^?1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.