Antibody production kinetics

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. 

Suzuki E, Ollis DF (1989), Cell cycle model for antibody production kinetics, Biotechnol. Bioeng. 34 1398-1402. 

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. 

Renard JM, Spagnoli R, Mazier C, Salles MF Mandine E (1988) Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotechnology Letters 10 91-96. 

Cells are inoculated, grown up to a desired cell or product concentration and harvested. The duration of batch culture depends on inoculation density, cell line and characteristics such as growth rate and antibody-production kinetics of the cell line. After every run the bioreactor must be cleaned and autoclaved for the next run. 

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. 

Linardos, T., "Kinetics of Monoclonal Antibody Production in Chemostat Hybrid-oma Cultures", Ph D. thesis, Dept, of Chemical Petroleum Engineering, University of Calgary, AB, Canada, 1991. 

Many of the 60 known reactions catalyzed by monoclonal antibodies involve kinetically favored reactions e.g., ester hydrolysis), but abzymes can also speed up kinetically disfavored reactions. Stewart and Benkovic apphed transition-state theory to analyze the scope and limitations of antibody catalysis quantitatively. They found the observed rate accelerations can be predicted from the ratio of equilibrium binding constants of the reaction substrate and the transition-state analogue used to raise the antibody. This approach permitted them to rationalize product selectivity displayed in antibody catalysis of disfavored reactions, to predict the degree of rate acceleration that catalytic antibodies may ultimately afford, and to highlight some differences between the way that they and enzymes catalyze reactions. 

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. 

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. 

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)-... 

Batt BC, Kompala DS (1989), A structured kinetic modeling framework for the dynamics of hibrodoma growth and monoclonal antibody production in continuous suspension cultures, Biotechnol. Bioeng. 34 515-531... 

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

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. 

The technique described above was used to characterize several monoclonal (14) as well as recombinant (15) antibodies. In the case of polyclonal antibodies, exact kinetic parameters are not obtained (Note 8) nevertheless, the quantitative comparison of different products is possible. Moreover, fast determination of affinities is useful during screening and development of antibodies. The kinetic parameters enable to select the most suitable antibody for the planned purpose when several monoclonals are available (Note 9). 

This definition of productivity disregards any time invested in preparing the fermenter prior to inoculation of the batch. Lower values of productivity result when the additional preparatory time is taken into account. Often, the product of a fermenter is not the biomass per se, but a compound produced by the cells. Examples of such products are antibiotics and monoclonal antibodies. The kinetics of product formation may mirror those of the cell growth, or they may be quite different. Products known as secondary... 

The antibodies also catalyzed retro-aldol reactions of secondary [9, 11, 16] and tertiary aldols [10]. In these retro-aldol reactions antibodies 38C2 and 33F12 processed hydroxyketones whose stereochemistry was the same as that of the aldol reaction product. Kinetic resolution by the retro-aldol reaction therefore provided the opposite enantiomer from the forward aldol reaction (Scheme 6.5 and Table 6.2). For example, (K)-14 (> 99% ee) was obtained by the 38C2-catalyzed kinetic resolution of ( )-14 (Table 6.2) whereas... 

ESTEVE-GASSENT, M.D., NIELSEN, M.E. and AMARO, c. (2003) The kinetics of antibody production in mucus and serum of European eel (Anguilla angujilla L.) after vaccination against Vibrio vulnificus development of a new method for antibody quantification in skin mucus. Fish Shellfish Immunology 15,51-61. 

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 control of kinetic vs thermodynamic product formation can often be achieved by suitable modification of reaction conditions. A far more difficult task is to switch from the formation of a favoured major product to a disfavoured minor product, especially when the transition states for the two processes share most features in common. This challenge has been met by antibodies with considerable success, both for reaction pathways differing in regioselectivity and also for ones differing in stereoselectivity. In both situations, control of entropy in the transition state must hold the key. 

Table 4 Kinetic parameters for those antibodies raised against phosphonates [88-91] which effect the resolution of the fluorinated alcohols [84-87]. The configuration of the disastereoisomerically pure product from each antibody-catalysed process was shown to correspond to that of the antibody-inducing hapten.

The formation of J/-H2A.X occurs rapidly after the production of double strand breaks by ionizing radiation with detectable amounts present in seconds and maximal levels reached in about 10 min [57]. y-H2A.X disappears with kinetics consistent with the repair of the double stranded break. Western blots with antibodies that specifically recognize the phosphorylated SQ motif indicate that phosphorylation of this motif occurs in response to double breaks in many organisms, including Drosophila and S. cerevisiae [58]. 

Many biopharmaceutical preparations are heterogeneous and may be difficult to fully characterise. Certain fractions of a preparation may have different biological activity or kinetics than the intended product. It is important that such fractions are appropriately qualified. The proportions of these fractions may be altered when production changes are made or they may be different between similar products produced by different manufacturers. Because of their proteinaceous nature and their novel mechanisms of action, all preclinical and clinical development steps must be re-evaluated. For pharmacokinetic studies, blood concentrations should be measured by specific analytical techniques (most often ELISA), which quantify the active protein and not one of its fragments or inactive forms, such as antigen-antibody complexes. For PK-PD studies of monoclonal antibodies, relevant biomarkers are most often circu-... 

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