The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions.     

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.     

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.     

Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells

The aim of the present work was to study the effect of ammonia and lactate on growth, metabolism, and productivity of BHK cells producing a recombinant fusion protein. Results show that cell growth was reduced with the increase in ammonia or lactate: k(1/2) of 1.1 mM and 3.5 mM for stirred and stationary cultures, respectively, for ammonia and of 28 mM for both stationary and stirred cultures for lactate, were obtained. The cell-specific consumption rates of both glucose (q(Glc)) and glutamine (q(Gln)) increased, whereas that of oxygen (q(O2)) decreased, with the increase in ammonia or lactate concentrations. The cell-specific production rates of lactate (q(Lac)) increased with an increase in ammonia concentration; similarly for the cell-specific production rates of ammonia (q(Amm)), which also increased with an increase in lactate concentration; on the other hand, both q(Lac) and q(Amm) markedly decreased when lactate or ammonia concentrations were increased, respectively; lactate was consumed at lactate concentrations above 30 mM and ammonia was consumed at ammonia concentrations above 5 mM. In vivo (31)P NMR experiments showed that ammonia and lactate affect the intracellular pH, leading to intracellular acidification, and decrease the content in phosphomonoesters, whereas the cell energy state was maintained. The effect of lactate on cell growth and q(Gln) is partially due to osmolarity, on q(Glc) and q(Amm) is entirely due to osmolarity, but on q(Lac) is mainly due to lactate effect per se. An increase in ammonia from 0 to 20 mM induced a 50% reduction in specific productivity, whereas an increase in lactate from 0 to 60 mM induced a 40% decrease.     

Transient responses of hybridoma metabolism to changes in the oxygen supply rate in continuous culture

Oxygen is an important nutrient that may limit the productivity of commercial cell culture reactors. The transient responses of hybridoma growth and metabolism to step changes in the oxygen supply rate have been examined for dissolved oxygen concentrations (DO) ranging from 0.1% to 10% of air saturation in continuous culture. Metabolic quotients are reported for glucose, lactate, ammonia, oxygen, glutamine, alanine and other amino acids. A majority of the estimated ATP production was due to oxidative phosphorylation under all conditions tested. Decreases in the oxygen supply rate below the value required to maintain 0.5% DO caused the viable cell concentration to decrease. Glycolysis was enhanced at the lower oxygen concentrations, and after an initial decrease, the specific glutamine consumption rate was also higher. High residual glutamine concentrations occurred below 0.5% DO. Oxidation of other amino acids and production of serine were also inhibited. The cells subsequently adapted to low oxygen concentrations. The increase in cell concentration following the return to 10% DO was preceded by increased biosynthetic activity, as evidenced by transiently reduced yields of lactate from glucose, and alanine and ammonia from glutamine.     

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

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

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

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

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.     

Reassessing culture media and critical metabolites that affect adenovirus production

Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell‐specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell‐specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 × 10⁶ cells/mL. In comparison, only 50% of reduction in the cell‐specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 × 10⁶ cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 × 10⁶ cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture

In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (K ^Glc _Lac) was 1.4 ± 0.1 mM and for the dependence of ammonia production on glutamine (K ^Gln _Amm) was 0.25 ± 0.11 mM and 0.10 ± 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This␣metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was␣highly dependent on glucose concentration, the oxygen consumption also increased with the increase in␣glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism. Received: 21 August 1998 / Received revision: 11 November 1998 / Accepted: 17 January 1999     

Cell attachment to microcarriers affects growth, metabolic activity, and culture productivity in bioreactor culture

It is not well understood how changes from suspension to microcarrier cultures affect cell growth, metabolism, and yield of recombinant proteins. To investigate the effects of culture conditions on cell characteristics, fed-batch bioreactor cultures were performed under different culture conditions (suspension cultures, cultures attached to Cytodex 3 and Cytopore 1 microcarriers) using two different Chinese hamster ovary cell lines producing either secreted human placental alkaline phosphatase (TR2-255) or tissue plasminogen activator (CHO 1-15-500). In controlled, agitated bioreactors, suspension cultures reached cell densities and product titers higher than those in microcarrier cultures, in contrast to the results in static flask cultures. Growth and metabolic activities showed similar trends in suspension and microcarrier culture regardless of cell line. However, the responses of the specific productivities to the different culture conditions differed significantly between the cell lines.     

Adaptive fuzzy control of nutrients concentration in fed-batch culture of mammalian cells

An adaptive fuzzy controller was developed to control the glucose and glutamine concentrations in the reactor constant at the desired level. The parameter values of the controller change during the cultivation according to the culture phase which was detected by the lactate concentration. Cultivations with different glucose and glutamine set point concentrations of a recombinant BHK anchorage-dependent cell line were performed in a fed-batch reactor on-line connected with an HPLC system. Glucose and glutamine concentrations were satisfactorily controlled at each set point during all cultivation periods. Ammonia had a determining effect on productivity since it inhibited cell growth and protein specific production. Ammonia production increased with an increase of glutamine or a decrease of glucose set point concentrations, indicating the importance of glucose to glutamine ratio for the optimization of productivity in mammalian cell cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells

The present work aims at characterizing the regulatory mechanisms of metabolism and product formation of BHK cells producing a recombinant antibody/cytokine fusion protein. This work was carried out through the achievement of several steady-states in chemostat cultures, corresponding to different glucose and glutamine levels in the feed culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on residual glucose and glutamine concentrations, respectively. Similar dependence was also observed for lactate and ammonia productions. K(Glc)(Glc) and K(Gln)(Gln) were estimated to be 0.4 and 0.15 mM, respectively, while q(max)(Glc) and q(max)(Gln) were estimated to be 1.8 and 0.55 nmol 10(-6)cells min(-1), respectively. At very low glucose concentrations, the glucose-to-lactate yield decreased markedly showing a metabolic shift towards lower lactate production; also, the glucose-to-cells yield was increased. At very low-glutamine concentrations, the glutamine-to-ammonia and glutamine-to-cells yields increased, showing a more efficient glutamine metabolism. Overall, amino acid consumption was increased under low glucose or glutamine concentrations. Metabolic-flux analysis confirmed the metabolic shifts by showing increases in the fluxes of the more energetically efficient pathways, at low-nutrient concentrations. No effect of glucose or glutamine concentrations on the cell-specific productivity was observed, even under metabolically shifted metabolism; therefore, it is possible to confine the cells to a more efficient metabolic state maintaining the productivity of the recombinant product of interest, and consequently, increasing final product titers by increasing cell concentration and culture length. This work is intended to be a model approach to characterize cell metabolism in an integrated way; it is highly valuable for the establishment of operating strategies in mammalian cell fermentations in which cell metabolism is to be confined to a desired state.     

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     

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.     

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.     

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.     

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.     

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.     

Modeling kinetics of a large‐scale fed‐batch CHO cell culture by Markov chain Monte Carlo method

Markov chain Monte Carlo (MCMC) method was applied to model kinetics of a fed‐batch Chinese hamster ovary cell culture process in 5,000‐L bioreactors. The kinetic model consists of six differential equations, which describe dynamics of viable cell density and concentrations of glucose, glutamine, ammonia, lactate, and the antibody fusion protein B1 (B1). The kinetic model has 18 parameters, six of which were calculated from the cell culture data, whereas the other 12 were estimated from a training data set that comprised of seven cell culture runs using a MCMC method. The model was confirmed in two validation data sets that represented a perturbation of the cell culture condition. The agreement between the predicted and measured values of both validation data sets may indicate high reliability of the model estimates. The kinetic model uniquely incorporated the ammonia removal and the exponential function of B1 protein concentration. The model indicated that ammonia and lactate play critical roles in cell growth and that low concentrations of glucose (0.17 mM) and glutamine (0.09 mM) in the cell culture medium may help reduce ammonia and lactate production. The model demonstrated that 83% of the glucose consumed was used for cell maintenance during the late phase of the cell cultures, whereas the maintenance coefficient for glutamine was negligible. Finally, the kinetic model suggests that it is critical for B1 production to sustain a high number of viable cells. The MCMC methodology may be a useful tool for modeling kinetics of a fed‐batch mammalian cell culture process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010