The transient responses of hybridoma cells to nutrient additions in continuous culture: II. Glutamine pulse and step changes

The transient and steady-state responses of hybridoma growth and metabolism to glutamine pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The specific glutamine consumption rate increased rapidly after all glutamine additions, but the responses of the glucose and oxygen consumption rates and the cell concentration were found to depend on the intial feed glutamine concentration. The glucose consumption rate was 1.4-10.9 times that of glutamine, and serine and branched-chain amino acids were consumed in larger amounts at the higher glucose: glutamine uptake ratios. It was estimated that maintenance accounted for ca. 60% of the cellular ATP requirements at specific growth rates ranging from 0.57 to 0.68 day(-1).     

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.     

Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture

Ammonia and lactate are the major byproducts from mammalian cells grown in medium containing glutamine and glucose. Both can be toxic to cells, and may limit the productivity of commercial bioreactors. The transient and steady-state responses of hybridoma growth and metabolism to lactate and ammonia pulse and step changes in continuous suspension culture have been examined. No inhibition was observed at 40 mM lactate. Cell growth was inhibited by 5 mM ammonia, but the cells were able to adapt to ammonia concentrations as high as 8.2 mM. Ammonia production decreased and alanine production increased in response to higher ammonia concentrations. Increased ammonia concentrations also inhibited glutamine and oxygen consumption. The specific oxygen consumption rate decreased by an order of magnitude after an ammonia pulse to 18 mM. Under these conditions, over 90% of the estimated ATP production was due to glycolysis and a large fraction of glutamine was converted to lactate.     

Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: part I. Cell density, viability, and cell-cycle distribution

For the development of optimal perfusion processes the effect of the feed and bleed rate on cell growth in a perfusion bioreactor was studied. The viable-cell density, viability, growth, death, and lysis rate and cell-cycle distribution of a hybridoma cell line producing an IgG1 were studied over a range of specific feed and bleed rates. It was found that the feed and bleed rates applied in the different cultures could be divided into two regions based on the viable-cell density and cell-cycle distribution. The cultures in the first region, low feed rates (0.5 and 1.0 d(-1)) combined with low bleed rates (0.05 and 0.10 d(-1)), were nutrient-limited, as an increase in the feed rate resulted in an increase in the viable-cell density. The cultures in the second region, high feed and bleed rates, were nonnutrient-limited. In this region the viable-cell density decreased more or less linearly with an increase in the bleed rate and was independent of the feed rate. This suggests that the cells were limited by a cell-related factor. Comparison of Trypan-blue dye-exclusion measurements and lactate-dehydrogenase activity measurements revealed that cell lysis was not negligible in this bioreactor set-up. Therefore, lactate-dehydrogenase activity measurements were essential to measure the death rate accurately. The specific growth rate was nearly constant for all tested conditions. The viability increased with an increase of the bleed rate and was independent of the feed rate. Furthermore, the specific productivity of monoclonal antibody was constant under all tested conditions. For the optimal design of a perfusion process it should first be established whether viability is an important parameter. If not, a bleed rate as low as possible should be chosen. If low viabilities are to be avoided, the bleed rate chosen should be higher, with the value depending on the desired viability. Next, the feed rate should be set at such a rate that the cells are just in the nonnutrient-limited region.     

Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media

Two murine hybridoma cell lines (167.4G5.3 and S3H5/gamma2bA2) were adapted to grow in low-serum and serum-free media by a weaning procedure. The changes in cell growth, metabolic, and antibody production rates with adaptation were examined using biochemical and flow cytometric analyses. After adaptation to a particular serum level, the short-term serum response of the cells was experimentally determined. Specific growth rates, glucose and glutamine uptake and lactate and ammonia production rates, and specific antibody production rates were evaluated from the data. For both cell lines, an improvement in cell growth was observed after adaptation, and both higher growth rates and higher cell concentrations were obtained. The specific glucose and glutamine uptake rates and the lactate and ammonia production rates changed insignificantly with adaptation. Conversely, changes in the specific antibody production rate of the two cell lines differed. Cell line 167.4G5.3 showed a loss in antibody productivity at low serum levels, while the S3H5/gamma2bA2 kept its original productivity in low-serum-containing media. The intracellular antibody content for S3H5/gamma2bA2 cells remained unaltered by adaptation, but a low antibody containing cell population appeared in the 167.4G5.3 culture. The loss of specific antibody productivity in this cell line was due to the appearance of this population.     

Analysis of the ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line Huh 7

Ammonia metabolism of ratprimary hepatocytes and a human hepatocyte cell line,Huh 7, at different concentrations of glutamine,glucose and ammonia was examined. During theincubation of the primary hepatocyte cells, glutamineand ammonia concentrations decreased, that of ureaincreased, and that of glucose remained the same. Inthe case of Huh 7 cells, glucose was consumed rapidly,the concentration of ammonia increased and that of urearemained the same. The major energy sources amongmedium components were glutamine for the primary cellsand glucose for Huh 7 cells, although the primaryhepatocytes may utilize intracellular glycogen asenergy source. As the glutamine concentration in theincubation medium increased, the specific rates of notonly glutamine consumption, but also ammonia productionby the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primarycells increased then. Increase of glucoseconcentration had no effect on glutamine and ammoniametabolism by both cells, although it increased glucoseconsumption by Huh 7 cells. The incubation of theprimary cells with higher ammonia concentrationincreased all specific rates of glutamine consumption,ammonia consumption and urea production. An increasein the ammonia concentration to 5 mM changed theammonia metabolism from production to consumption andincreased the specific glucose consumption rate. Consequently, increases in the glutamine and ammoniaconcentrations were revealed to have negative andpositive effects, respectively, on decreasing ammoniaconcentration by both of rat primary hepatocytes andHuh 7 cells.     

The use of a metabolically structured model in the study of growth, nitrification, and denitrification by Thiosphaera pantotropha

Thiosphaera pantotropha is capable of aerobic heterotrophic nitrification and both aerobic and anaerobic denitrification. These phenomena have been studied in acetate-limited aerobic and anaerobic continuous cultures supplied with ammonia and nitrate. The internal reaction rates were defined, based on biochemical knowledge. The observable external conversion rates are related through a linear equation on the basis of the specified internal reaction rates. The linear equation is a Pirt relation extended for microbial systems with multiple electron donors (acetate and ammonia) and electron acceptors (oxygen and nitrate). The coefficients in this equation were estimated from the continuous culture measurements, and are composed of parameters involved in ATP production and consumption by the microorganism. It is shown that with realistic values for these parameters, the metabolically structured model describes the aerobic as well as the anaerobic experiments.     

Transient responses of hybridoma cells to nutrient additions in continuous culture: I. Glucose pulse and step changes

Glucose and glutamine are the main nutrients used by mammalian cells in culture. Each provides unique biosynthetic precursors but are complementary for production of other metabolites and energy. The transient and steady-state responses of hybridoma growth and metabolism to glucose pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The glucose consumption rate increased by 100-200% immediately after glucose was added to the reactor, and the increased glycolytic ATP production appears to be responsible for the concurrent rapid decrease in the oxygen consumption rate. The effects on glutamine consumption were delayed, probably due to buffering by the TCA cycle and interrelated pathways. A period of increased biosynthetic activity, as evidenced by an increase in the estimated specific ATP production rate and lower by-product yields from glutamine, preceded the increase in cell concentration after the glucose step change. The biosynthetic yield of cells from ATP was calculated, and it was estimated that maintenance accounted for about 60% of the energy used by the cells at a specific growth rate of 0.66 day(-1). The estimated 22% ATP production due to glycoysis was twice as great as that before the step change.     

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products.

Effects of biochemical factors, i.e., medium components and metabolic byproducts, on growth of Chinese hamster ovary (CHO) cells were investigated. Glucose and ammonia were found to inhibit the growth. Kinetic analysis gave the inhibition constants, 0.14 g l-1 for ammonia and 5.0 g l-1 for glucose. Since glutamine was unstable and was a main source of ammonia, precise studies on glutamine degradation and ammonia formation process were done. By evaluating the spontaneous reactions, net glutamine utilization and net ammonia production by the cells could be estimated. It became evident that asparagine could support the growth of CHO cells as a stable substitute for glutamine. Then, a glucose fed-batch culture was grown on a glutamine free and asparagine supplemented medium. Because of (1) low glucose concentration, but (2) no glucose limitation and (3) low ammonia accumulation, the maximum total cell concentration reached 3.4 x 10(6) ml-1, which was 1.8 times greater than that in the control experiment (initial 1.15 g l-1 glucose and 0.29 g l-1 glutamine, and no glucose feed).     

Continuous hybridoma suspension cultures with and without cell retention: kinetics of growth, metabolism and product formation.

A laboratory scale bioreactor was constructed from glass and polycarbonate materials whereby a track-etch membrane (3 microns pore diameter) was integrated into its two-part bottom flange. The reactor performance was evaluated for continuous hybridoma suspension cultures under various conditions of cell retention. A total retention experiment demonstrated that this type of stirred tank reactor cannot be operated at near zero growth rate conditions. Instead, at steady viable cell concentrations of congruent to 3 x 10(6) cells per ml, specific growth and death rates were estimated at 0.60 +/- 0.06 d-1. Specific substrate (glucose, glutamine, O2, amino acids) consumption, by-product (ammonia, alanine, amino acids) and product (antibody) production rates as well as various apparent molar yield coefficients were obtained and are compared to metabolic quotients and yield coefficients previously calculated from standard continuous culture experiments, i.e., without cell retention, at specific growth rates of 0.63 and 1.24 d-1. Furthermore, steady-state data on viable cell and antibody concentrations, spec. mAb productivities, and space-time yields determined before and after a step change (2.5-fold increase) in dilution rate at identical specific growth rates mu are presented.     

Ammonia inhibition of hybridomas propagated in batch, fed-batch, and continuous culture

The nature and temporal development of ammonia inhbition were investigated in batch, fed-batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 --> 3.7 --> 4.0 --> 4.9 --> 7.7 --> 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady-state specific growth kinetics or specific antibody productivity. (c) 1994 John Wiley & Sons, Inc.     

In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro (13)C NMR spectroscopy were used, with a [2-(13)C]glucose or [5-(13)C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re-analysis of (13)C NMR data acquired during a [1-(13)C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux ( approximately 0.20 micromol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19-26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia ( approximately 32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.     

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.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

On-line monitoring of human prostate cancer cells in a perfusion rotating wall vessel by near-infrared spectroscopy

PC-3 human prostate cancer cells have been cultivated in a rotating wall vessel in which glucose, lactate, and glutamine profiles were monitored noninvasively and in real time by near-infrared (NIR) spectroscopy. The calibration models were based on off-line spectra from tissue culture experiments described previously (Rhiel et al., Biotechnol Bioeng 77:73-82). Monitoring performance was improved by Fourier filtering of the spectra and initial off-set adjustment. The resulting standard errors of predictions were 0.95, 0.74, and 0.39 mM for glucose, lactate, and glutamine, respectively. The concentration of ammonia could not be accurately measured from the same spectra. In addition, metabolite uptake and production rates were determined for PC-3 prostate cancer cells during exponential growth in batch-mode cultivation. Cells grew with a doubling time of 21 h and consumed glucose and glutamine at rates of 6.8 and 1.8 x 10(-17) mol/cell.s, respectively. This resulted in lactate and ammonia production rates of 11.9 and 1.3 x 10(-17) mol/cell.s, respectively. Compared with other monitoring technologies, this technology has many advantages for spaceflights and stand-alone units; for instance, calibration can be performed at one time and then applied in a reagentless, low-maintenance way at a later time. The resulting concentration information can be incorporated into closed-loop control schemes, thereby leading to better in vitro models of in vivo behavior.     

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     

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.     

Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.     

Effects of glutamine supply on growth and metabolism of mammalian cells in chemostat culture

Glutamine is a major source of energy, carbon, and nitrogen for mammalian cells. The amount of glutamine present in commercial mammalian cell media is, however, not necessarily balanced with cell requirements. Therefore, the effects of glutamine limitation on the physiology of two mammalian cell lines were studied in steady-state chemostat cultures fed with IMDM medium with 5% serum. The cell lines used were MN12, a mouse-mouse hybridoma, and SP2/0-Ag14, a mouse myeloma often used in hybridoma fusions. Cultures, grown at a fixed dilution rate of 0.03 h(-1), were fed with media containing glutamine concentrations ranging from 0.5 to 4 mmol L(-1). Biomass dry weight and cell number were linearly proportional to the glutamine concentrations fed, between 0.5 and 2 mmol L(-1), and glutamine was completely consumed by both cell lines. From this it was concluded that glutamine was the growth-limiting substrate in this concentration range and that the standard formulation of IMDM medium contains a twofold excess of glutamine. In glutamine-limited cultures, the specific rates of ammonia and alanine production were low compared to glutamine-excess cultures containing 4 mmol L(-1) glutamine in the feed medium. The specific consumption rates of nearly all amino acids decreased with increasing glutamine feed, indicating that, in their metabolic function, they may partially be replaced by glutamine. Both cell lines reacted similarly to differences in glutamine feeding in all aspects investigated, except for glucose metabolism, In SP2/0-Ag14 glutamine feed concentrations did not affect the specific glucose consumption, whereas in MN12 this parameter increased with increasing amounts of glutamine fed. This systematic study using controlled culture conditions together with a detailed analysis of culture data shows that, although cells may react similarly in many aspects, cell-line-specific characteristics may be encountered even with respect to fundamental physiological responses like the interaction of the glutamine and glucose metabolism. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 272-286, 1997.     

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

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