Hybridomas kinetics

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Shirai, Y., Yamaguchi, M., Kobayashi, A., Nishi, A., Nakamura, H., and Murakami, H., Change in Growth Kinetics of Hybridoma Cells Entrapped in Collagen Gel Affected by Alkaline Supply, Cytotechnol., 14 129 (1994)... 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

The effects of elevated lactic acid concentration on the cell cycle kinetics of hybridoma cell growth and antibody production in batch culture were studied by Kromenaker and Srienc (1994). When 33 mM lactic acid was initially present, the specific growth rate was reduced by 37% and the cell-specific antibody production rate increased by a factor of 2.6 relative to a control culture with no additional lactic acid. 

Glacken MW, Adema E, Sinskey AJ (1988), Mathematical descriptions of hybridoma culture kinetics I. Initial metabolic rates, Biotechnol. Bioeng. 28 1376-1389. 

Kromenaker SJ, Srienc F (1994), Effect of acid lactic on the kinetics of growth and antibody production in a murine hybridoma secretion patterns during the cell cycle, J. Biotechnol. 34 13-34. 

Ozturk SS, Palssom BO (1990), Effects of dissolved oxygen on hybridoma cell growth, metabolism and antibody production kinetics on continuous culture, Biotechnol. Prog. 6 437-446. 

Typical kinetic profiles (hybridoma). (A) Cell concentration and viability (B) glucose consumption (GLC) and lactate production (LAC) (C) monoclonal antibody production (mAb) (D) glutamine consumption (GLN) and ammonium production (NH4+) (E) specific growth rate (px) (F) alanine (ALA) and glycine (GLY) production. Adapted from Lee (2003). Symbols correspond to the experimental data and the lines to the manual curve fitting. Vertical lines indicate the instant at which exponential growth phase ended (gx < Px.max)-... 

Kinetics models for cell growth and death, as well as for substrate consumption and product and byproduct synthesis, are presented here. Most of these were developed for hybridomas in continuous processes. Although these models are representative of animal cell systems, it is important to understand that the cellular response to an environmental stimulus is highly dependent on the specific cell line. The review published by Porter and Schafer (1996) illustrated this variability through the comparison of experimental data and models from different groups of cell lines. Besides this, the lack of proper knowledge to explain experimentally observed phenomena also accounts for the variability of model structures. 

Table 8.1 Kinetic equations for specific hybridoma growth rates...

Table 8.5 Kinetic parameters for specific uptake rates in hybridoma systems equations are valid for both substrates - GLC and GLN...

Bree MA, Dhurjati P, Geoghegan R, Robnett B (1988), Kinetic modeling of hybridoma cell growth and immunoglobulin production in a large-scale suspensions culture, Biotechnol. Bioeng. 32 1067—1072. 

Frame KK, Hu WS (1991a), Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cell, Biotechnol. Bioeng. 37 55-64. 

Gaertner JG, Dhurjati P (1993), Fractional factorial study of hybridoma behavior. 2. Kinetics of nutrient uptake and waste production, Biotechnol. Prog. 9 309-316. 

Hiller GW, Aeschlimann AD, Clark DS, Blanch HW (1991), A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum-free medium, Biotechnol. Bioeng. 38 733-741. 

Portner R, Schilling A, Liidemann I, Markl H (1996), High density fed-batch cultures for hybridoma cells performed with the aid of a kinetic model, Bioprocess. Eng. 15 117-124. 

Ozturk SS (1990), Kinetics and Metabolism of Antibody Production by Hybridoma Cells, Doctoral Thesis, Michigan University, USA. 

Ozturk SS, Palsson BO (1991), Growth, metabolic, and antibody production kinetics of hybridoma cell culture 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor, Biotechnol. Prog. 7 481-494. 

Miller WM, Blanch HW Wilke CR (1988) A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture effect of nutrient concentration, dilution rate, and pH. Biotechnology and Bioengineering 32 947-965. 

The construction of a kinetic model for an animal cell culture involves several steps a kinetic analysis of the experimental results with the formulation of hypotheses on the nature of the rate-limiting steps the choice of rate expressions describing the influence of these phenomena on the cellular processes evaluation of parameter values and validation of the model with different experimental results. In this section a general methodology is described for the modelling of cell cultures, and the procedure is illustrated on the kinetics of a hybridoma cell. (For a summary of terms used, see Table 4.3.3.)... 

The procedure of kinetic data analysis and model construction is illustrated for a hybridoma culture (cell line VO 208) in a batch system. The medium used was RPMI 1640 + 5% (v/v) foetal calf serum (PCS) + 2% (v/v) minimum essential medium (MEM) amino acids + 1% non-essential amino acids and initial glucose and glutamine concentrations of 13 mM and 4.5 mM, respectively. 

Batt BC Kompala DS (1989) A structured kinetic modeling framework for the dynamics of hybridoma growth and monoclonal antibody production in continuous suspension culture. Biotechnology and Bioengineering 3A 515-531. 

Goergen JL, Marc A Engasser JM (1993) Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release. Cytotechnology 11 189-195. 

Louren o da Silva A, Marc A, Engasser JM Goergen JL (1996) Kinetic model of hybridoma cultures for the identification of rate limiting factors and process optimisation. Mathematics Computers in Simulation 1277 1-9. 

Martial A, Engasser J-M Marc A (1990) Influence of inoculum age on hybridoma culture kinetics. Cytotechnology 5 165-171. 

Seamans TC Hu WS (1990) Kinetics of growth and antibody production by a hybridoma cell line in a perfusion culture. Journal of Fermentation Bioengineering 70 241-245. 

Shaughnessy TS Kargi F (1990a) Growth and product inhibition kinetics of T-cell hybridomas producing lymphokines in batch and continuous culture. Enzyme and Microbial Technology 12 669-675. 

Merten OW (1988b) Batch production and growth kinetics of hybridomas. Cytotechnology 1 113-121. 

Fig. 4 Typical batch kinetics of a hybridoma culture showing characteristic growth curve and growth-associated production of lactate and nongrowth-associated production of antibodies.

Canziani G., Klakamp S., and Myszka D. G., Kinetic screening of antibodies from crude hybridoma samples using BlAcore, Anal. Biochem., 325, 301-307, 2004. 

Modeling of the growth kinetics of yeast and hybridoma cells... 

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