BATCH PRODUCTION KINETICS OF HYBRIDOMAS: PULSE-EXPERIMENTS

Abstract Pulse-experiments, avoiding changes in the cellular physiology, have been conducted, in order to establish, (a) wether the secretion of monoclonal antibodies (mAbs) by mouse hybridomas occurs mainly by a de novo synthesis or by a release of intracellularly stored IgG/M, and (b) to verify if the calculated specific productivity (pg produced IgG/cellxh) gives a real indication about the cellular mAb-production-activity or not. The results, obtained from two hybridoma cell lines (10/8/20, I.13.17), indicate that during the growth phase (lag and/or exponential phase) the calculated specific productivity correlated well with the incorporation of S-35-methionine or H-3-leucine into cytoplasmic IgG. However, hybridoma cultures, showing a second peak of calculated specific productivity during the death phase (so called dead production), did not show any incorporation of S-35-methionine or H-3-leucine into cytoplasmic IgG. This may be interpreted as mAbs secreted during the death phase were not synthesised de novo, but were released from an intracellular pool. By conducting a second set of pulse-experiments using cell line I.13.17, we were able to show, that this non-correlation between the effective production during the death phase (Merten et al. Cytotechnology (1990) in press) and the incorporation of radiolabeled amino acid (H-3-leucine) into IgG was due to (a) the development of the intracellular leucine-concentration (strong reduction); and (b) the profound reduction of the incorporation and utilisation of leucine during the stationary and death phase. The results indicate, that both pulse-methods, ours, which avoids most of the physiological changes during the pulse-periode, and the classical one, which follows the incorporation/synthesis kinetics of a new batch culture after a complete medium change, are not applicable for this type of study, because both are influenced by, or influence the actual state, of cellular physiology. The results will be discussed with those obtained from pulse-chase experiments.