Comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers

Optimization of culture conditions such as the dissolved O₂ (DO) concentration, temperature and pH was attempted regarding both cell growth and the production of tissue plasminogen activator (TPA) in a microcarrier cell culture of human embryo lung cells. The growth rate was suppressed at a DO concentration below 30% saturation. From the pH range 7.2–7.6, both the specific growth rate and maximal cell concentration decreased. At a lower temperature than 37°C, although both the specific growth rate and the maximal cell concentration decreased, the cell concentration was maintained for a longer time during the production period, high TPA productivity being maintained. As the optimal conditions for culture growth, a DO concentration of 30% saturation or over, temperature of 37°C and pH of 7.4 are recommended. However, for TPA production after cell culture growth, the DO concentration should be in the range 20–30% O₂ saturation, and the temperature and pH should be lowered to 33°C and 6.8, respectively.     

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.     

An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture

An oxygen supply strategy involving agitation speed and aeration method for the large-scale production of tissue plasminogen activator (TPA) by a microcarrier cell culture was investigated by small-scale model experiments. A preliminary calculation indicated that diffusion limitation of dissolved oxygen (DO) could be caused in a microcarrier sedimentation layer more than 0.5 mm in thickness. Within an agitation speed range above 70 rpm, which was the critical speed for all of the microcarrier beads to remain suspended and thus for avoiding a deficiency of DO, the TPA productivity was higher at a lower agitation speed, while the cell concentration was not affected by the agitation speed. The addition of soluble starch to the culture medium prevented sedimentation of the microcarrier beads, even at the low agitation speed of 20 rpm, resulting in a TPA productivity higher than that at 70 rpm, which was the optimum speed without soluble starch. Use of an air spray system with an optimized air flow rate resulted in a k_La 2.35 times higher than that with simple surface aeration. Increasing the internal pressure of the culture from 0.2 kg/cm² (1209 hPa) to 1.5 kg/cm² (2483 hPa) had no effect on the cell growth but slightly increased the TPA production rates. However, based on the glucose consumption, both the cell and TPA yields were much improved by pressurization. As an optimum mixing and oxygen supply strategy for the production of TPA on a large scale, it is recommended that soluble starch be added to the culture medium to allow the microcarrier suspension to be maintained at a low agitation speed, while keeping a high oxygen transfer rate by means of an air spray system and pressurization.     

Maximum virginiamycin production by optimization of cultivation conditions in batch culture with autoregulator addition

A strategy for optimization of non-growth-associated production in batch culture employing an empirical approach was developed through the study of virginiamycin production. The strategy is formulated with two aims: attaining a high cell concentration at the beginning of the production phase without decrease in production activity; and enhancing the production activity during the production phase. As a practical example, the goal of a maximum virginiamycin (M and S) production in the batch culture of Streptomyces virginiae was set. To attain a high cell concentration in the production phase of the batch culture, that is, to extend the growth phase for as long as possible, the optimum composition and concentration of the complex medium, especially the yeast extract (YE) concentration, were first investigated. Dissolved oxygen (DO) concentration control was also a parameter considered in maintaining the production activity during the production phase. In addition, to enhance the production activity, an optimum addition strategy of an autoregulator, virginiae butanolide-C (VB-C), was investigated. Combining these measures, the optimum cultivation conditions were found to be an initial YE concentration in the complex medium of 45 g/L, the shot addition of 300 mug/L of VB-C 11.5 h after the start of the batch culture, and a DO concentration maintained above 2 mg/L. The maximum concentrations of virginiamycin M and S were about ninefold those obtained under nonoptimum cultivation conditions. Nonoptimum cultivation conditions consisted of an initial YE concentration one sixth (7.5 g/L) that of the optimum cultivation conditions, and no VB-C addition. These conditions were used as representative of the standard cultivation of virginiamycin in this study. The strategy developed here will be applicable to the production of other antibiotics, especially to the cultivation of Streptomyces species, in which a hormonelike signal material (an autoregulator) plays an important role in antibiotic production. (c) 1996 John Wiley & Sons, Inc.     

Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase, improves adenovirus production

Hyperosmotic stress has been widely explored as a means of improving specific antibody productivity in mammalian cell cultures. In contrast, a decrease in cell-specific productivity of adenovirus production has been reported in several studies in which virus production in HEK 293 cell cultures was conducted under hyperosmotic conditions. However, production of viral vectors and, in particular, adenoviral vectors is the result of two consecutive phases: the growth phase and the virus production phase. In this study, the singular and combined effects of osmolality on the phases of cell growth and virus production were evaluated in culture media with osmolalities ranging from 250 to 410 mOsm. A two-factor, five-level full factorial design was used to investigate the effect of osmotic stress on cell physiology, as determined through the characterization of cell growth, cell metabolism, cell viability, cell cycle, cell RNA and total protein content, and total virus yield/cell-specific virus productivity. Overall, the results show that the growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410 mOsm during the cell growth phase. Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. Optimal virus productivity was achieved by growing cells in media with an osmolality of 370 mOsm or greater, followed by a virus production phase at an osmolality of 290 mOsm. Compared to standard culture and production conditions in isotonic media, the shift from high to low osmolality between the two phases resulted in a two- to three-fold increase in virus yields. This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media.     

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein

Gene expression in insect cells is an advantageous system for recombinant protein production, mainly because of its capacity to produce complex proteins with correct post-translational modifications. Recently, we identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of rabies virus glycoprotein, expressed in Drosophila melanogaster cells, by about 60%. In this work, the kinetic parameters for cell growth and recombinant rabies virus glycoprotein production were determined in cultures of transfected Drosophila melanogaster Schneider 2 (S2) cells expressing recombinant rabies virus glycoprotein (rRVGP), enriched and non-enriched with the hemolymph of Lonomia obliqua (Hb). The highest concentration of rRVGP was achieved at the beginning of the culture enriched with Hb, indicating that the cells produce greater amounts of rRVGP per cell (specific rRVGP concentration) at the early exponential growth phase. After day 8, a decrease in the concentration of rRVGP (ng/mL) was observed, probably due to protein decomposition. The average specific rRVGP production rate (μ_rRVGP) was 30 ng rRVGP/10⁷cell.day, higher than that observed in the non-enriched culture.     

Dynamics and modeling on fermentative production of poly (β-hydroxybutyric acid) from sugars via lactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus

The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly β-hydroxybutyrate (PHB) by Alcaligenes eutrophus in one fermentor. For the modeling of the effect of NH₃ concentration on the cell growth of A. eutrophus and PHB production rates, metabolic flux distributions were computed at two culture phases of cell growth and PHB production periods. It was found that the NADPH, generated through isocitrate dehydrogenate in TCA cycle, was predominantly utilized for the reaction from α-ketoglutalate to glutamate when NH₃ was abundant, while it tended to be utilized for the PHB production through acetoacetyl CoA reductase as NH₃ concentration decreased. This phenomenon was reflected in the development of mathematical model. In the mixed culture experiments, the two phases were observed, namely the lactate production phase due to L. delbrueckii and the lactate consumption phase due to A. eutrophus. The lactate concentration could be estimated on-line by the amount of NaOH solution and HCl solution supplied to keep the culture pH at constant level. Several mixed culture experiments were conducted to see the dynamics of the system. Finally, a mathematical model which can describe the dynamic behavior of the present mixed culture was developed and the model parameters were tuned for fitting the experimental data. The model may be used for several purposes such as control, optimization, and understanding process dynamics etc.     

Application of process mass spectroscopy to the detection of metabolic changes in plant tissue culture

There is a clear need in the area of plant cell culture for methods of on-line estimation of culture parameters. The introduction of plant cells into culture can result in a loss of their photoautotrophic character so that they are largely heterotrophic. As a result, fermentation off-gas analysis may not be confounded by photosynthetically-related O₂ production. In this study performance of a suspension culture of Syringa vulgaris, in a pneumatically agitated bioreactor of in-house design, was investigated. The effect of light on growth, carbohydrate metabolism and the respiratory quotient (RQ), determined by process mass spectroscopy, was studied. Yield coefficients for cells grown in the light and dark were similar although the patterns of carbohydrate uptake were quite different. Maximum biomass yields were higher in this bioreactor than normally observed in shake flasks. The RQ was dynamic during the course of the fermentation, peaking during the transition from the lag phase to the growth phase. It is suggested that the RQ may prove useful as an on-line parameter for monitoring transitions in cellular metabolism during plant cell culture fermentations.Abbreviations RQ respiratory quotient - v.v.m. volume of gas fed to fermenter per unit volume per minute - Y_X/S growth yield coefficient based on total carbohydrate     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17–20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.     

Enhancement of haloacetate dehalogenase production by strain mutation and condition optimization

Enhancement of the activity of an inducible chloroacetate dehalogenase was carried out by efficient and safe mutation with UV and microwave irradiation along with optimization of culture conditions. First, a stable mutant of Pseudomonas sp. CGMCC 3267-MW6 with chloroacetate dehalogenase activity of 2.77 U/mL (3-fold higher activity than the wild strain) was produced by mutation. The maximum activity of this inducible enzyme was measured as 29.41 U/mL when Pseudomonas sp. CGMCC 3267-MW6 was cultured with 4 g/L 3-hydroxybutyrate for 12 h followed by 40 mM 3-chlorobutyrate for an additional 20 h. Production of the enzyme was found to be associated with growth of the bacterium. According to these results, we determined that the optimum inducer of chloroacetate dehalogenase activity would be a hard degradable substrate. The optimum auxiliary carbon source would be the primary metabolite of the substrate or the precursor of the metabolite. The optimum time of inducer supplementation would be during the middle stage of exponential phase. The optimum concentration of substrate would be sufficient but would not induce inhibition. Finally, the optimum collection time would be at the later stage of exponential phase. This work provides further knowledge of chloroacetate dehalogenase and the optimization of inducible enzyme production.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Effects of light/dark cycle, mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5

The effects of light/dark cycle, mixing pattern and partial pressure of H₂ on the growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated. The results from light/dark cycle culture showed that little or no hydrogen production was observed during the dark periods, and the hydrogen production immediately recovered once illumination was resumed. Also, it was found that the optimum condition of shaking velocity was 120 rpm for hydrogen photo-fermentation. Meanwhile, shaking during H₂ production phase (i.e., cell growth stationary phase) of photo-fermentation played a crucial role on effectively enhancing the phototrophic hydrogen production, rather than that during cell exponential growth phase. The other factor evaluated was hydrogen partial pressure in the culture system. The substrate conversion efficiency increased from 86.07% to 95.56% along with the decrease of the total pressure in the photobioreactor from 1.082 × 10⁵ to 0.944 × 10⁵ Pa, which indicated that reduction of H₂ partial pressure by lowering the operating pressure substantially improved H₂ production in an anaerobic, photo-fermentation process.     

Improved production of polygalacturonate lyase by combining a pH and online methanol control strategy in a two-stage induction phase with a shift in the transition phase

Polygalacturonate lyase is a kind of enzyme that is abundantly used in the textile industry for cotton scouring. Previously, we reconstructed the polygalacturonate lyase gene in Pichia pastoris for the expression of this enzyme. To enhance the production of polygalacturonate lyase (PGL), a combined strategy was formulated by combining online methanol control and two-stage pH control strategies. For the two-stage pH control strategy during the growth phase, the pH was controlled at 5.5, and in the induction phase different pH levels were investigated for the optimum enzyme production. During the online methanol control strategy, the different levels of methanol (v/v) were investigated for the best enzyme production at pH 5.5. These two strategies were combined together for enhanced PGL productivity, and the induction phase was divided into two stages in which methanol concentrations were maintained at different levels online. The transition phase was introduced during the induction phase instead of introducing it after the growth phase. The two-stage combination strategy was formulated on the bases of methanol consumption of cells, optimal pH, cell viability and the production of polygalacturonate lyase by P. pastoris. By using this strategy, the production was doubled compared with common conditions, and the highest polygalacturonate lyase activity reached 1,631 U/ml. This strategy proved to be very useful for the enhancement of polygalacturonate lyase production by achieving higher cell viability, alcohol oxidase activity and phosphate-related compounds of the cells during the induction phase.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: -α-Difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4–8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4–8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE₂ or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor -α-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis ([³H]leucine incorporation), and the cell cycle progression from G₂/M to G₁, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G₂/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.     

Relationship between hybridoma growth and monoclonal antibody production.

Factors affecting cell growth and antibody production in a mouse hybridoma were investigated. Antibody was produced during the growth and decline phases of a batch culture with an increase in the specific rate of antibody production during the decline phase. The specific rate of antibody production was also increased in cells arrested by 2 mM thymidine, suggesting that cell proliferation and antibody production can be uncoupled. Reduced serum concentrations resulted in lower cell growth rates but increased antibody production rates. However, this trend was reversed in hybridomas which had been arrested by thymidine, since the highest antibody production rate was associated with high serum concentrations. Likewise, in proliferating cells, the optimum pH for antibody production (pH 6.8) was lower than the optimum pH for cell growth (pH 7.2), whereas in thymidine-blocked cells, the highest antibody production rate was at pH 7.2. High antibody production rates and product yields were also associated with low growth rates in continuous cultures. The possibility that antibody was under cell cycle control was investigated in synchronized hybridoma cultures. Antibody production occurred during G1 and G2 with a decline in the M phase and evidence of a further decline in the S phase. Thus antibody production was not restricted to the G1 and S phase in this hybridoma.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).     

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.