Fed-batch Culture Of Hybridoma Cells Research Articles

FROM MFA TO FBA: LEGITIMATING OBJECTIVE FUNCTION AND LINEAR CONSTRAINTS

A careful analysis of the admissible metabolic flux intervals (determined from linear programs) obtained with detailed (input and output) or limited (only input) flux measurements is proposed in a case study made of a fed-batch culture of hybridoma cells. It is shown how a cost function in FBA and additional linear constraints on the fluxes can be legitimated and efficiently introduced so as to recover admissible flux intervals based on limited external measurements which are similar to the ones based on all the available measurements. A way to model overflow metabolism on both glucose and glutamine thanks to only two inequality constraints is proposed.

State estimation and predictive control of fed-batch cultures of hybridoma cells

Fed-batch cultures of hybridoma cells are commonly used for the production of monoclonal antibodies (MAb). In this study, a simple macroscopic model of the cell culture is used, which is based on the overflow metabolism paradigm. This allows to specify optimal culture conditions, and the natural formulation of a nonlinear model predictive control strategy (NMPC). As not all the component concentrations are available for measurement, system observability is analyzed, and an unscented Kalman filter (UKF) is designed, which provides satisfactory estimates of glucose and glutamine concentrations. Robustness of the NMPC scheme is investigated, as well as the combined UKF+NMPC scheme, through a minimax robust version and the closed-loop system.

Optimization and robustness analysis of hybridoma cell fed-batch cultures using the overflow metabolism model

The maximization of biomass productivity in fed-batch cultures of hybridoma cells is analyzed based on the overflow metabolism model. Due to overflow metabolism, often attributed to limited oxygen capacity, lactate and ammonia are formed when the substrate concentrations (glucose and glutamine) are above a critical value, which results in a decrease in biomass productivity. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder-Mead simplex optimization algorithm. The optimal multi exponential feed rate trajectory improves the biomass productivity by 10 % as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture so as to control the hybridoma cells at the critical metabolic state, which allows maximizing the biomass productivity. The robustness analysis of optimal feeding profiles obtained with different optimization strategies is considered, first, with respect to parameter uncertainties and, finally, to model structure errors.

Optimal Operation of Hybridoma Cell Fed-Batch Cultures Using the Overflow Metabolism Model: Numerical and Analytical Approach

The maximization of biomass productivity in fed-batch cultures of hybridoma cells is analyzed based on the overflow metabolism model. Due to overflow metabolism, often attributed to limited oxygen capacity, lactate and ammonia are formed when the substrate concentrations (glucose and glutamine) are above a critical value, which results in a decrease in biomass productivity. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder-Mead simplex optimization algorithm. The optimal multi exponential feed rate trajectory improves the biomass productivity by 10% as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture such as to control the hybridoma cells at the critical metabolism state, which allows maximizing the biomass productivity.

Macroscopic Modelling of Overflow Metabolism in Fed-batch Cultures of Hybridoma Cells

AbstractA macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Model parameters and confidence intervals are obtained via a non linear least squares identification. This model, on the one hand, allows quantitatively describing overflow metabolism in mammalian cell cultures and, one the other hand, will be valuable for monitoring and control of fed-batch cultures in order to optimize the process.

A serum substitute for fed-batch culturing of hybridoma cells

We developed a substitute for serum to produce fed-batch cultures of hybridoma cells in serum-free medium and confirmed that the cells could be successfully cultivated this way. Our substitute consisted of 12 components. The specific production rates of lactate and ammonia, which are harmful byproducts from the cells, were significantly reduced compared with a conventional serum-containing batch culture. This reduction led to a higher cell concentration and a longer production lifetime. As a result, the final concentration of monoclonal antibody was 400 mg/L, or five times greater than that in the conventional serum-containing batch culture. The developed substitute is expected to enable fed-batch cultivation in a serum-free condition.

On-line identification and optimization of feed rate profiles for high productivity fed-batch culture of hybridoma cells using genetic algorithms

Abstract In this paper, an on-line identification and optimization method based on genetic algorithms (GAs) has been used to optimize the productivity of a seventh-order nonlinear model of fed-batch culture of hybridoma cells. The parameters of the seventh-order nonlinear model are assumed to be unknown. The intention of this paper is to use GAs for (1) identifying the parameters of a seventh-order nonlinear model of fed-batch culture of hybridoma cells, and (2) determining the best feed rate control profiles for glucose and glutamine. The final level of monoclonal antibodies obtained by this method is then compared with the case where all the parameters are assumed to be known. It is found that the final level of monoclonal antibodies obtained by the on-line identification and optimization method is only about 3% less than the final level of monoclonal antibodies obtained by the case where all the parameters are assumed to be known. GAs proved to be a good alternative method for solving on-line identification and optimization problems.

Optimisation of fed-batch culture of hybridoma cells using genetic algorithms

In this paper, a program describing a genetic algorithm is used for optimising fed-batch culture hybridoma cells to obtain the highest yield over certain time period. Optimal feed rate trajectories for a single feed stream containing both glucose and glutamine, and separate feed streams of glucose and glutamine are determined via the genetic algorithm. As compared to the optimal constant feed rate regime, optimal varying feed rate trajectories improve the final monoclonal antibodies concentration by 10% for the single feed rate case and by 39% for the multi feed rate case in this simulation. In comparsion with a dynamic programming, GA calculated feed trajectories yield a much higher level of monoclonal antibodies concentration.

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.

An unstructured kinetic model of macromolecular metabolism in batch and fed-batch cultures of hybridoma cells producing monoclonal antibody

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359–368; E. Suzuki, D.F. Ollis, Biotechnol. Bioeng. 34 (1989) 1398–1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61–69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture.

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.

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

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

Comparison of optimization methods for fed-batch cultures of hybridoma cells

Two methods for the calculation of optimal trajectories for the input variables of a fed-batch culture of hybridoma cells are compared. It pointed out that a gradient method based on Pontryagins' minimum principle based yields a significant better performance with respect to computational effort and the calculated minimum than a dynamic programming approach which has been presented in a previous paper [1] as the most suitable method.

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 (Fe2+, SeO, Li+, Zn2+, and Cu2+) 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 × 106 to 1.7 × 107 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/109 cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional batch culture.

Interactive dual control of glucose and glutamine feeding in hybridoma cultivation

With the aim of developing an advanced computer control system for hybridoma cell cultures, on-line monitoring and control strategies for nutrient feeding in a fed-batch culture of a hybridoma cell line were investigated. Good correlation was obtained between the on-line monitoring and the off-line analysis of growth, and between the uptake rates of glucose, oxygen and glutamine, respectively. Using the variables monitored on-line, control of the glucose and glutamine concentrations in fed-batch culture was achieved. The characteristics of the glucose-limited fed-batch culture of hybridoma cells were also investigated. In combination with further research on cell physiology, the results of this study should accelerate the development of an advanced and reliable control system for hybridoma cell cultures.

Fed-batch culture of hybridoma cells: comparison of optimal control approach and closed loop strategies

Control of fed-batch culture of hybridoma cells was investigated based on two approaches optimal control theory and feedback control. Experiments were conducted for both approaches-with a feed enriched in glutamine. The optimal feed trajectory, a decreasing one, yielded a final monoclonal antibody (MAb) concentration of 170 mg/l, a three-fold increase compared to a typical batch operation.

Optimization of fed-batch culture of hybridoma cells using dynamic programming: single and multi feed cases

The optimization of fed-batch culture of hybridoma cells is accomplished on a mathematical model using dynamic programming. Optimal feed trajectories are found using a seventh order model for a single feed stream containing both glucose and glutamine and for two separate feed streams of glucose and glutamine. Compared to a constant feed rate, optimal trajectories can improve the final MAb concentration by 11 % for the single feed case and by 20% for the multifeed case. Higher MAb concentrations can be expected for fed-batch optimization with feed enriched in nutrients.

Kinetic modelling of hybridoma cell growth and immunoglobulin production in a large‐scale suspension culture

Biotechnology and BioengineeringVolume 32, Issue 8 p. 1067-1072 Communications to the EditorFree Access Kinetic modelling of hybridoma cell growth and immunoglobulin production in a large-scale suspension culture Michele A. Bree, Michele A. Bree Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716Search for more papers by this authorPrasad Dhurjati, Corresponding Author Prasad Dhurjati Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716Search for more papers by this authorRichard F. Geoghegan Jr., Richard F. Geoghegan Jr. Research and Development, E. I. duPont de Nemours, Inc., Glasgow, Delaware 19702Search for more papers by this authorBarbara Robnett, Barbara Robnett Research and Development, E. I. duPont de Nemours, Inc., Glasgow, Delaware 19702Search for more papers by this author Michele A. Bree, Michele A. Bree Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716Search for more papers by this authorPrasad Dhurjati, Corresponding Author Prasad Dhurjati Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716Search for more papers by this authorRichard F. Geoghegan Jr., Richard F. Geoghegan Jr. Research and Development, E. I. duPont de Nemours, Inc., Glasgow, Delaware 19702Search for more papers by this authorBarbara Robnett, Barbara Robnett Research and Development, E. I. duPont de Nemours, Inc., Glasgow, Delaware 19702Search for more papers by this author First published: 5 October 1988 https://doi.org/10.1002/bit.260320814Citations: 62AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 G. Kohler and C. Milctein, Nature, 256, 495 (1975). 2 J. R. Birch, P. W. Phompson, K. Lambert, and R. Boraston, “ The Large Scale Cultivation of Hybridoma Cells Producing Monoclonal Antibodies,” American Chemical Society Meeting, Philadelphia, 1984. 3 A. Macario, J. L. deMacario, and E. C. deMacario, Monoclonal Anti bodies against Bacteria, I (Academic, Orlando, FL, 1985). 4 M. W. Glacken and A. J. Sinskey, “ Modeling and Optimization of FedBatch Cultures of Hybridoma Cells,” AIChE Annual Meeting, Chicago, 1985. 5 Harry Eagle, Science, 122 (Sept. 16, 1955). 6 T. Imamura, C. Crespi, W. Thilly, and H. Brunengraber, Anal. Biochem., 124, 353 (1982). 7 J. Bailey and D. Ollis, Biochemical Engineering Fundamentals (McGraw-Hill, New York, 1977). Citing Literature Volume32, Issue85 October 1988Pages 1067-1072 ReferencesRelatedInformation