Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.     

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.     

Growth characteristics and nutrient depletion of Miscanthus × ogiformis Honda ‘Giganteus’ suspension cultures

The growth characteristics and nutrient depletion in suspension cultures of Miscanthus × ogiformis Honda ‘Giganteus’ grown in media containing either Murashige and Skoog or N6 basal nutrient salts were studied during a culture period of 15 days. Proline was added to both media in concentrations from 0 to 300 mM. The fresh and dry weights of the suspension aggregates and the concentrations of ammonium, nitrate, proline and sugar remaining in the medium were measured at different points in time during the culture period. The results showed an almost total depletion of ammonium but a limited nitrate depletion of only 15 mM in both media. Proline at 12.5 to 50 mM was totally depleted during the culture period whereas approximately half the concentration remained in media with 100 to 300 mM proline. Higher fresh weight increases were obtained by the proline additions to the N6 as compared to the MS suspension cultures. Sucrose was hydrolysed into its monosaccharide components in the culture medium. Glucose was depleted faster than fructose indicating a preference for glucose as a carbohydrate source of the M. × ogiformis cultures. The high water uptake by the suspension aggregates 12 to 15 days after subculture indicate that the cultures should be subcultured more frequently to prevent the formation of nonembryogenic cells in the cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improving an on-line feeding strategy for fed-batch cultures of hybridoma cells by dialysis and `Nutrient-Split'-feeding

The concept of the feeding strategy was to minimise the formation of inhibiting metabolites and to increase the yield of monoclonal antibodies in fed-batch cultures of hybridoma cells by a balanced supply of substrates. A process control system based on fieldbus technology was used for monitoring and control. External program routines were implemented to control dissolved oxygen (DO) and to calculate the oxygen uptake rate (OUR) and cumulative oxygen consumption (COC) simultaneously. A concentrated feed solution was supplied according to the off-line estimated stoichiometric ratio between oxygen and glucose consumption (GC). Feeding was initiated automatically when the OUR decreased due to substrate limitation. The antibody concentration increased three-fold compared to the conventional batch culture by applying this strategy. But it was not possible to avoid inhibition by ammonia during the fed-batch phase. This was accomplished by the use of a dialysis membrane. Dialysis fed-batch cultures were performed in a membrane dialysis reactor with a `nutrient-split' feeding strategy, where concentrated medium is fed to the cells and toxic metabolites are removed into a buffer solution. This resulted in a ten-fold increase of the antibody concentration compared to the batch. Amino acid concentrations were analysed to identify limiting conditions during the cultivation and to analyse the performance of the nutrient supply in the fed-batch and dialysis fed-batch.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

Regulation and utilization of microsin promoter turn on in a chromosomal fusion

Prior work has demonstrated that the microsin antibiotics are produced by enteric bacteria when the growth medium is depleted of nutrients. Because the control loci could have biotechnical potential, and general stress-response phenomena are of importance to understanding how bacteria survive in natural and bioreactor environments, we examined further the growth rate dependence of gene expression under the control of the microsin B17 promoter. This work entailed performing batch and chemostat growth experiments with a strain of E. coli K-12 containing a mcbA-lacZ gene fusion in the chromosome. Our results indicate that when a culture is presented with excess respiratory substrate, a well defined growth rate exists, below which a significant induction event occurs. However, cultures that are fermenting or highly glycolytic tend to express poorly. Additionally, the utility of the fusion strain was examined by performing fed-batch cultivation experiments. We found that sustained production in a fed-batch reactor can be accomplished by using a straightforward, exponential nutrient feeding profile.     

Application of hypoosmolar medium to fed-batch culture of hybridoma cells for improvement of culture longevity

Cell culture longevity in fed-batch culture of hybridomas is often limited by elevated medium osmolality caused by repeated nutrient feeding. Shotwise feeding of 10x Dulbecco's modified Eagle's medium (DMEM) concentrates elevated the osmolality of medium up to 540 mOsm/kg at the end of fed-batch culture of S3H5/gamma2bA2 hybridoma which is known to be lethal to most hybridomas. S3H5/gamma2bA2 hybridoma has been shown to grow without significant growth depression at 219 mOsm/kg in DMEM supplemented with 10% fetal bovine serum. To improve culture longevity in fed-batch cultures of S3H5/gamma2bA2 hybridoma, a hypoosmolar medium (223 mOsm/kg) was used as an initial basal medium. The use of hypoosmolar medium delayed the onset of severe cell death resulting from elevated osmolality and allowed one more addition of 10x DMEM concentrates to the culture. As a result, a final antibody concentration obtained was 121.5 microg/mL which is approximately 1.5-fold higher compared to fed-batch culture using a standard medium (335 mOsm/kg). When compared to batch culture, a more than 5-fold increase in the final antibody concentration was achieved. Taken together, the use of hypoosmolar medium as an initial medium in fed-batch culture improved culture longevity of S3H5/gamma2bA2 hybridoma, resulting in a substantial increase in the final antibody concentration.     

Defined protein and animal component-free NS0 fed-batch culture

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.     

Production of thuringiensin by fed-batch culture of Bacillus thuringiensis subsp. darmstadiensis 032 with an improved pH-control glucose feeding strategy

A improved pH-control fed-batch strategy for Bacillus thuringiensis subsp. darmstadiensis 032 producing thuringiensin was developed based on the analysis of the batch culture, constant rate fed-batch cultures and the original pH-control fed-batch. Having considered the pH variation and the glucose consumption status, the pH was adjusted from 6.5 to 7.0 by adding base in the late cultivation period of batch culture, and then the pH was kept at 7.0 by glucose feeding. The feeding was terminated when the pH could not be controlled by glucose feeding anymore. The proposed fed-batch strategy effectively avoided underfeeding or overfeeding, and it increased the thuringiensin yield and Y_P/X by 89.51% and 103.2% compared to that of the batch culture, respectively.     

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.     

High-end pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO fed-batch cultures

A simple method for control of lactate accumulation in suspension cultures of Chinese hamster ovary (CHO) cells based on the culture's pH was developed. When glucose levels in culture reach a low level (generally below 1 mM) cells begin to take up lactic acid from the culture medium resulting in a rise in pH. A nutrient feeding method has been optimized which delivers a concentrated glucose solution triggered by rising pH. We have shown that this high-end pH-controlled delivery of glucose can dramatically reduce or eliminate the accumulation of lactate during the growth phase of a fed-batch CHO cell culture at both bench scale and large scale (2,500 L). This method has proven applicable to the majority of CHO cell lines producing monoclonal antibodies and other therapeutic proteins. Using this technology to enhance a 12-day fed-batch process that already incorporated very high initial cell densities and highly concentrated medium and feeds resulted in an approximate doubling of the final titers for eight cell lines. The increase in titer was due to additional cell growth and higher cell specific productivity.     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies

A fed-batch cell culture process was developed that has general applicability to all evaluated Sp2/0 (n = 8) and NS0 (n = 1) antibody-producing cell lines. The two key elements of this generic process were a protein-free concentrated feed medium, and a robust, metabolically responsive feeding strategy based on the off-line measurement of glucose. The fed-batch process was shown to perform equivalently at the 15 L development scale and 750 L manufacturing scale. Compared to batch cultures, the fed-batch process yielded a 4. 3 fold increase in the average integral of viable cell concentration and a 1.7 fold increase in average specific antibody production rate, equivalent to a 7.6 fold increase in average final antibody concentration. The highest producing cell line reached a peak viable cell concentration of 1.0 x 10(7) cell mL(-1) and a final antibody concentration of 750 mg L(-1) in a 10 day process. For all lines evaluated, reducing bioreactor pH set point from 7.2 to 7.0 resulted in an additional 2.4 fold increase in average final antibody concentration. The optimized fed-batch process consistently yielded a volumetric productivity exceeding 50 mg L(-1) day(-1). This generic, high-yielding fed-batch process significantly decreased development time, and increased manufacturing efficiency, thereby facilitating the clinical evaluation of numerous recombinant antibodies.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca²⁺ and Mg²⁺ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl₂ and MgCl₂ concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca²⁺/Mg²⁺ feeding to 117 h with Ca²⁺/Mg²⁺ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 10⁶ and 9.12 × 10⁶ cells ml⁻¹, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.     

Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies

In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end-products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL-1606) was cultivated in T-flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed-batch culture and by 26-fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed-batch culture and by 50-fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five- and tenfold respectively, compared with conventional batch culture. (c) 1994 John Wiley & Sons, Inc.