Batch production and secretion kinetics of hybridomas: pulse-chase experiments.
Pulse chase experiments of two mouse hybridoma lines were conducted in order to elucidate the kinetics of monoclonal antibody (mAb) production and secretion during different stages of batch cultures. The results indicate that a stock of cytoplasmic IgG exists in hybridoma cells and that the concentration of this stored IgG depends on the cell line used and the stage of the culture. This stored IgG can be released by dying cells, and a certain quantity of the secreted IgG is derived from this source. However, only between 0.3 and 9.3% of the released IgG of U0208 (average: 2.08%) and between 2.08 and 25.8% of the IgG, released from I.13.17 (average: 6.95%), were of storage origin, calculated on culture viability and intracellular IgG-stock. Comparing the accumulation of radio-labelled IgG (IgG*) in the supernatant with the reduction of cytoplasmic IgG* during the chase experiments, the percentages range between 14 and 50%, somewhat higher values probably caused by changes in the culture conditions. These changes led to a release of IgG during the chase experiments, which accounts for about 20-25% of the totally secreted IgG. It could be established that during the logarithmic growth phase of batch cultures a certain percentage of synthesized IgG was not released but stored within the cells: for U0208: 0.3-4.5%, for I.13.17: 1-7.6%. During the stationary and death phase, this percentage ranged between 1.5 and 20% for U0208 and between 0.5 and 8.1% for I.13.17. Finally, the chase experiments also revealed that the time of synthesis, assembly, and secretion of mAbs does not vary much during the different phases of batch cultures, and is within the range of 1.5 and 3 hrs.
Institut Pasteur, Laboratoire de Technologie Cellulaire, Paris, France.
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Merten
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Keller
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Cabanié
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Leno
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Hardefelt
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002455:Cell Division / Q000276:immunology; D003593:Cytoplasm / Q000276:immunology; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D051379:Mice
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G