Cell cycle- and growth phase-dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities

Recombinant adenoviruses became one of the vectors of choice for delivery and expression of foreign proteins for gene therapy and vaccination purposes. Nevertheless, the production of adenovirus is currently limited by the so-called "cell density effect", i.e., a drop in cell specific productivity concomitant with increased cell concentration at infection (CCI). This work describes the characterisation and optimisation of the infection process in order to improve recombinant adenovirus type 5 yields at high cell densities. For that purpose, 293 cells adapted to suspension were grown in 2l bioreactors and infected at different cell concentrations, using different re-feed strategies, while evaluating cell metabolism. The consumption of amino acids is enhanced during infection, although no amino acid limitation was detected for cells infected at concentrations in the range of 2 x 10(6)cell/ml, for which the highest volumetric productivity was obtained in batch mode. Conversely, infecting at cell concentrations in the range of 3 x10(6)cell/ml led to complete depletion of glucose, glutamine and threonine before the optimal harvesting time, a significant decrease in volumetric productivity being observed; the effect of amino acids and glucose addition at infection time on cell specific and volumetric productivity of adenovirus was assessed, no improvement on adenovirus production being achieved. The effect of ammonia, present in high concentrations at 3 x10(6)cell/ml, was evaluated and seem to be detrimental; an 1.8-fold increase on adenovirus volumetric productivity was obtained for infections performed at 3 x10(6)cell/ml when non-ammoniagenic medium was used.     

Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.     

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.     

重组CHO细胞培养过程中氨对细胞代谢的影响

研究了重组CHO细胞批培养过程中,氨浓度对细胞的葡萄糖、谷氨酰胺及其它氨基酸代谢的影响。表明,细胞对葡萄糖和谷氨酰胺的得率系数随着氨浓度的增加而降低,起始氨浓度为566mmol/L的批培养过程与起始氨浓度为021mmol/L的批培养过程相比,细胞对葡萄糖和谷氨酰胺的得率系数分别下降了78%和74%,细胞对其它氨基酸的得率系数也分别下降了50%～70%。氨浓度的增加明显地改变了细胞的代谢途径,葡萄糖代谢更倾向于厌氧的乳酸生成。在谷氨酰胺的代谢过程中,谷氨酸经谷氨酸脱氢酶进一步生成α酮戊二酸的过程受到了氨的抑制,而氨对谷氨酸经谷氨酸转氨酶反应生成α酮戊二酸的过程有促进作用,但总体上谷氨酸进一步脱氨生成α酮戊二酸的反应受到了氨的限制。     

Whole body and skeletal muscle glutamine metabolism in healthy subjects

We measured glutamine kinetics using L-[5-15N]glutamine and L-[ring-2H5]phenylalanine infusions in healthy subjects in the postabsorptive state and during ingestion of an amino acid mixture that included glutamine, alone or with additional glucose. Ingestion of the amino acid mixture increased arterial glutamine concentrations by approximately 20% (not by 30%; P < 0.05), irrespective of the presence or absence of glucose. Muscle free glutamine concentrations remained unchanged during ingestion of amino acids alone but decreased from 21.0 +/- 1.0 to 16.4 +/- 1.6 mmol/l (P < 0.05) during simultaneous ingestion of glucose due to a decrease in intramuscular release from protein breakdown and glutamine synthesis (0.82 +/- 0.10 vs. 0.59 +/- 0.06 micromol x 100 ml leg(-1) x min(-1); P < 0.05). In both protocols, muscle glutamine inward and outward transport and muscle glutamine utilization for protein synthesis increased during amino acid ingestion; leg glutamine net balance remained unchanged. In summary, ingestion of an amino acid mixture that includes glutamine increases glutamine availability and uptake by skeletal muscle in healthy subjects without causing an increase in the intramuscular free glutamine pool. Simultaneous ingestion of glucose diminishes the intramuscular glutamine concentration despite increased glutamine availability in the blood due to decreased glutamine production.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

葡萄糖对重组CHO细胞生长代谢及EPO表达的影响

在CHO细胞批培养中 ,葡萄糖浓度从8.9增加到49.6mmol/L ,最大活细胞密度没有明显的差异 ,乳酸对葡萄糖的得率系数首先随着葡萄糖浓度的增加而增加 ,葡萄糖浓度达到 17.9mmol/L后 ,乳酸对葡萄糖的得率系数基本上维持恒定。在本实验中 ,葡萄糖浓度对谷氨酰胺代谢没有明显的影响。EPO的累积浓度首先随着起始葡萄糖浓度的增加 ( 8.9～ 17.9mmol L)而增加 ,进而又随着葡萄糖浓度的增加 (17.9～ 49.6mmol L)而下降 ,表明存在一最适浓度 ,在此浓度下重组CHO细胞的EPO表达最大。     

Metabolism of PER.C6 cells cultivated under fed-batch conditions at low glucose and glutamine levels

This is the first study to examine PER.C6 cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6(TM) cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced q(Gln) and q(NH(4)(+)) by approximately 30%, q(Ala) 85%, and q(NEAA) 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced q(Glc) about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y(O(2)/Glc) of approximately 6 mol/mol. However, despite no increase in q(Gln), an increased ammonium ion accumulation and Y(NH(4)(+)/Gln) were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6 cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The q(Gln) value was again reduced and, despite an increased q(NH(4)(+)) compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6 cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6 cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism.     

The cause of "acid-crash" and "acidogenic fermentations" during the batch acetone-butanol-ethanol (ABE-) fermentation process

Experiments were performed to determine the cause of "acid crash", a phenomenon which occasionally occurs in pH-uncontrolled batch fermentations resulting in premature cessation of ABE (acetone butanol) production. The results indicate that "acid crash" occurs when the concentration of undissociated acids in the broth exceeds 57 - 60 mmol/l. Prevention can be achieved by introducing some limited pH control to minimize the concentration of undissociated acids or by slowing the metabolic rate, and thus the rate of acid production, by, for example, lowering the fermentation temperature. "Acidogenic fermentations", which occur when batch fermentations are performed at pH values close to neutrality, are due to rapid production of acids followed by inhibition of solventogenesis when the total acid concentration reaches 240 - 250 mmol/l. Solventogenesis can be achieved at these pH values by lowering the glucose uptake rate / acid production rate by use of e.g. elevated glucose or lowered yeast extract concentrations in the growth medium.     

Nicotinamide influence on pancreatic cells viability

The study was undertaken to investigate the modulating effect of nicotinamide (NAm) in different concentrations and under different glucose concentrations on the viability and oxidative stress induced by streptozotocin (STZ, 5 mmol/l) and hydrogen peroxide (H2O2, 100 micromol/l) on isolated rat pancreatic cells of the Langerhans islets in vitro. Cell viability did not depend on the concentration of glucose in the range of 5-20 mmol/l, and in subsequent studies we used glucose in concentration of 10 mmol/l to protect cells against its hypo- and hyperglycemic action. Cytoprotective effect of NAm in concentrations from 5 to 20 mmol/l on cells survival was the same. It was found that the destructive action of STZ and H2O2 during 24 hours on isolated cells of the pancreas resulted in the significant cell death. It was revealed that NAm in concentration of 5 mmol/l not only had cytoprotective effects against STZ and H2O2 but also partially reduced the level of oxidative stress in the investigated cells induced by these compounds. High concentration of NAm, 35 mmol/l, causes cytotoxic effect on the viability of pancreatic islet cells and increase of oxidative stress induced by STZ and H2O2. Most likely these effects could be associated with direct modulatory action of NAm on important effector mechanisms involved in cell death, including PARP-dependent processes, or/and indirectly, through metabolic and antioxidant effects of the compound.     

Growth Kinetics of Thiobacillus thiooxidans on the Surface of Elemental Sulfur

The growth kinetics of Thiobacillus thiooxidans on elemental sulfur in batch cultures at 30(deg)C and pH 1.5 was studied by measuring the time courses of the concentration of adsorbed cells on sulfur, the concentration of free cells suspended in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate ions, the surface concentration of adsorbed cells per unit mass of sulfur approached a maximum value (maximum adsorption capacity of sulfur particles) whereas the concentration of free cells continued to increase with time. There was a close relationship between the concentrations of free and adsorbed cells during the microbial sulfur oxidation, and the two cell concentrations were well correlated by the Langmuir isotherm with adsorption equilibrium constant K(infA) and maximum adsorption capacity X(infAm) of 2.10 x 10(sup-9) ml per cell and 4.57 x 10(sup10) cells per g, respectively. The total concentration of free and adsorbed cells increased in parallel with the amount of sulfate formed. The total growth on elemental sulfur gave a characteristic growth curve in which a linear-growth phase followed the period of an initial exponential phase. The batch rate data collected under a wide variety of inoculum levels (about 10(sup5) to 10(sup8) cells per ml) were consistent with a kinetic model assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration, X(infA), of adsorbed cells and the fraction, (theta)(infV), of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were estimated from the experimental data, and the specific growth rate, (mu)(infA), and growth yield, Y(infA), were 2.58 day(sup-1) and 2.05 x 10(sup11) cells per g, respectively. The proposed model and the parameter values allowed us to predict quantitatively the surface attachment of T. thiooxidans cells on elemental sulfur and the bacterial growth in both initial exponential and subsequent linear phases. The transition from exponential to linear growth was a result of two competing factors: an increase in the adsorbed-cell concentration, X(infA), permitted a decrease in the unoccupied-site fraction, (theta)(infV).     

谷氨酰胺对微囊化重组CHO细胞生长代谢及内皮抑素表达的影响

微囊化技术是一种有发展潜力的生物技术,在细胞移植和药物控释等方面具有广泛的应用。然而由于目前微囊化细胞规模化培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。为了了解微囊化重组CHO细胞的生长代谢特性为今后规模化培养优化提供技术参考,考察了主要氮源物质谷氨酰胺对微囊化重组CHO细胞生长代谢及内皮抑素表达的影响。结果显示:当谷氨酰胺起始浓度从2.69mmolL增加到9.05mmolL时最大活细胞密度并没有增高,细胞增殖没有显著差异。当谷氨酰胺起始浓度较低(2.69mmolL)时,葡萄糖的比消耗速率较大;当谷氨酰胺起始浓度增高时(7.91mmolL～9.05mmolL)葡萄糖和谷氨酰胺的比消耗速率增大,但细胞对葡萄糖和谷氨酰胺的利用率降低。谷氨酰胺对产物表达有显著影响,起始浓度为4.97mmolL时的内皮抑素累积浓度最高,达546.36ngmL,过低和过高谷氨酰胺起始浓度下内皮抑素的累积浓度均较低。     

Human 293 cell metabolism in low glutamine-supplied culture: interpretation of metabolic changes through metabolic flux analysis

Metabolic flux analysis is a useful tool to analyze cell metabolism. In this study, we report the use of a metabolic model with 34 fluxes to study the 293 cell, in order to improve its growth capacity in a DMEM/F12 medium. A batch, fed-batch with glutamine feeding, fed-batch with essential amino acids, and finally a fed-batch experiment with both essential and nonessential amino acids were compared. The fed-batch with glutamine led to a maximum cell density of 2.4x10(6) cells/ml compared to 1.8x10(6) cells/ml achieved in a batch mode. In this fed-batch with glutamine, it was also found that 2.5 mM ammonia was produced compared to the batch which had a final ammonia concentration of 1 mM. Ammonia was found to be growth inhibiting for this cell line at a concentration starting at 1 mM. During the fed-batch with glutamine, the flux analysis shows that a majority of amino acid fluxes and Kreb's cycle fluxes, except for glutamine flux, are decreased. This observation led to the conclusion that the main nutrient used is glutamine and that during the batch there is an overflow in the Kreb's cycle. Thus, a fed-batch with glutamine permits a better utilization of this nutrient. A fed-batch with essential amino acid without glutamine was also assayed in order to reduce ammonia production. The maximum cell density was increased further to 3x10(6) cells/ml and ammonia production was reduced below 1 mM. Flux analysis shows that the cells could adapt to a medium with low glutamine by increasing the amino acid fluxes toward the Kreb's cycle. Adding nonessential amino acids during this feeding strategy did not improve growth further and the nonessential amino acids accumulated in the medium.     

Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production

The effects of NH(4)Cl addition on batch hybridoma cell growth at different external pH values (pH(e)) were investigated in a bioreactor at constant pH and dissolved oxygen concentration. In agreement with measurements in flasks, changes in pH(e) over the range 6.8-7.6 had minor effects on growth. Addition of 3 mM NH(4)Cl had little effect on cell growth while 10 mM NH(4)Cl caused a substantial growth inhibition, Measurements of the effects of pH(e) and NH(4)Cl concentration on cell metabolism gave similar results for cells grown in flasks in an incubator and in the bioreactor. As pH(e) decreases, the integral cell yield on glucose increases. There is a correlation between the effects of pH(e) on glycolysis and previous measurements of its effects on intracellular pH (pH(i)). Increases in NH(4)Cl concentration were previously determined to decrease pH(i) and are shown here to decrease the integral cell yield on glucose. At all pH(e) values in the absence of NH(4)Cl, glutamine is depleted at the time the maximum cell density is reached. Both pH(e) decreases and NH(4)Cl concentration increases lead to decreases in the integral cell yield on glutamine. Changes in pH(e) and in the NH(4)Cl concentration that cause growth inhibition have no effect on the specific antibody production rate for cells grown in flasks in an incubator or in the bioreactor. Changes in the NH(4)Cl concentration have no effect on the quality of the antibody produced, to a first level of characterization.     

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.     

Influence of different ammonium,lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.     

Growth characteristics in fed-batch culture of hybridoma cells with control of glucose and glutamine concentrations

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 x 10(6) cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation. (c) 1994 John Wiley & Sons, Inc.