High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor

A simple feeding strategy was developed and successfully employed for nutritional control in a 2-L fed-batch culture of hybridoma cells. A previously developed stoichiometric model for animal cell growth was used to design a supplemental medium for feeding. Undialyzed fetal bovine serum and trace metals (Fe(2+), SeO(3) (2-), Li(+), Zn(2+), and Cu(2+)) were fed to the cells periodically in addition to the automatic feeding of other nutrients in the supplemental medium. In this study, the maximum viable cell density was increased from 6.3 x 10(6) to 1.7 x 10(7) cells/mL, and the culture span was extended from 340 to 550 hours. The final monoclonal antibody titer achieved was 2400 mg/L. The specific production rates for ammonia and lactate were further reduced from 0.0045 and 0.0048 in our previous fed-batch experiments to 0.0028 and 0.0036 mmol/10(9) cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional 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.     

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor

In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed. Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b). In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model. Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies. Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model. The formation of toxic byproducts, such as ammonia and lactate (Hassellet al., 1991), was greatly reduced. The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model. Ammonia formation was also decreased compared with both the batch and fed-batch cultures. Most importantly, the monoclonal antibody concentration reached 900 mg l^?1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.     

Influence of Primatone RL supplementation on sialylation of recombinant human interferon-gamma produced by Chinese hamster ovary cell culture using serum-free media

Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn(25) and Asn(97)) of IFN-gamma with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-gamma by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 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.     

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.     

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.     

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.     

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.     

Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-gamma glycosylation during batch and fed-batch cultures of CHO cells

Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.     

Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network

A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc.     

High density culture of insect cells using rational medium design and feeding strategy

The objective of this study is to achieve high density cell culture by a rational medium design and feeding strategy. Insect cell/baculovirus expression system is one of the widely used methods for the production of heterologous proteins in the cell culture domain. Insect cell Spodoptera frugiperda Sf-21 and a recombinant baculovirus with encoded gene for human interleukin-5 were chosen as the model system in this study. A stoichiometric model was established to study the demand of nutrients, including glucose, 20 amino acids, and yeastolate, for the synthesis of cell mass. The coefficients for individual nutrients in the stoichiometric equation governing insect cell growth were determined from the information of cell mass and compositions. Based on the stoichiometric coefficients, the initial and supplemental media for fed-batch cell cultures were designed. The experiments began with the inoculation of Sf-21 cells into a spinner flask with the initial medium, which provided a starting environment for achieving optimum cell growth. This was followed by the periodic feeding of supplemental medium designed by utilizing the stoichiometric equation that governs insect cell growth. With this strategy, it was demonstrated that the Sf-21 cell culture reached a cell density in excess of 1.9᎒⁷ cells/ml. During the cultivation process, the utilization of various nutrients and the production of metabolites were also monitored. Further experiments proved that high concentration of recombinant product (such as human interleukin-5) could be achieved by infecting the high density cells (resulting from the designed medium) with recombinant baculoviruses.     

Enhanced productivity of Protein C by recombinant human cells in automated fed-batch cultures

Recombinant human kidney cells, 293, were cultivated in serum-free fed-batch cultures for the production of Protein C. By coupling the feeding of concentrated medium to pH control based on established stoichiometric relations, the titer of Protein C increased by more than ten fold as compared to batch culture, even though the total cell concentration increased only by less than two fold. Such a fed-batch culture is a simple system for enhancing the productivity of mammalian cells in culture.     

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.     

Modeling of <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial growth kinetics and optimal fed-batch fermentation for beauvericin production

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20–50 g l⁻¹ glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l⁻¹) than in the batch culture (194 mg l⁻¹). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.     

Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells

Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.     

Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.     

293SF metabolic flux analysis during cell growth and infection with an adenoviral vector

Metabolic flux quantification of cell culture is becoming a crucial means to improve cell growth as well as protein and vector productions. The technique allows rapid determination of cell culture status, thus providing a tool for further feeding improvements. Herein, we report on key results of a metabolic investigation using 293 cells adapted to suspension and serum-free medium (293SF) during growth and infection with an adenoviral vector encoding the green fluorescence protein (GFP). The model developed contains 35 fluxes, which include the main fluxes of glycolysis, glutaminolysis, and amino acids pathways. It requires specific consumption and production rate measurements of amino acids, glucose, lactate, NH(3), and O(2), as well as DNA and total proteins biosynthesis rate measurements. Also, it was found that extracellular protein concentration measurement is important for flux calculation accuracy. With this model, we are able to describe the 293SF cell metabolism, grown under different culture conditions in a 3-L controlled bioreactor for batch and fed-batch with low glucose. The metabolism is also investigated during infection under two different feeding strategies: a fed-batch starting at the end of the growth phase and extending during infection without medium change and a fed-batch after infection following medium renewal. Differences in metabolism are observed between growth and infection, as well as between the different feeding strategies, thus providing a better understanding of the general metabolism.     

Dynamic optimization of hybridoma growth in a fed-batch bioreactor

This study addressed the problem of maximizing cell mass and monoclonal antibody production from a fed-batch hybridoma cell culture. We hypothesized that inaccuracies in the process model limited the mathematical optimization. On the basis of shaker flask data, we established a simple phenomenological model with cell mass and lactate production as the controlled variables. We then formulated an optimal control algorithm, which calculated the process-model mismatch at each sampling time, updated the model parameters, and re-optimized the substrate concentrations dynamically throughout the time course of the batch. Manipulated variables were feed rates of glucose and glutamine. Dynamic parameter adjustment was done using a fuzzy logic technique, while a heuristic random optimizer (HRO) optimized the feed rates. The parameters selected for updating were specific growth rate and the yield coefficient of lactate from glucose. These were chosen by a sensitivity analysis. The cell mass produced using dynamic optimization was compared to the cell mass produced for an unoptimized case, and for a one-time optimization at the beginning of the batch. Substantial improvements in reactor productivity resulted from dynamic re-optimization and parameter adjustment. We demonstrated first that a single offline optimization of substrate concentration at the start of the batch significantly increased the yield of cell mass by 27% over an unoptimized fermentation. Periodic optimization online increased yield of cell mass per batch by 44% over the single offline optimization. Concomitantly, the yield of monoclonal antibody increased by 31% over the off-line optimization case. For batch and fed-batch processes, this appears to be a suitable arrangement to account for inaccuracies in process models. This suggests that implementation of advanced yet inexpensive techniques can improve performance of fed-batch reactors employed in hybridoma cell culture.     

Heuristic optimization of antibody production by Chinese hamster ovary cells

Large-scale production of monoclonal antibodies necessitates the development of a commercially viable process using the appropriate bioreactors, culture medium, and optimal feeding strategies. In the development of feeding strategies for higher antibody titers it is critical to assess the effects of limiting substrates on cell culture longevity and antibody production. In this study, glucose and L-glutamine were identified as limiting substrates and their effects on culture longevity and antibody production were evaluated in small-scale experiments. The results suggested that an optimal feeding strategy should account for the osmolality profile of the culture. The heuristic approach taken to optimize the antibody production showed that the fed-batch cultivation is superior to batch culture and maintaining low osmolality during growth phase increases cumulative viable cell density and thus leads to higher final antibody titer.