The relationship between intracellular nucleotides and hybridoma cell culture productivity and viability

Abstract

Monoclonal antibody (Mab) production is dependent upon the metabolic state of hybridomas. The objective of this research project was to determine if the intracellular nucleotide pool could be used as an indicator of specific antibody productivity (qMab) or of cell viability. Serum-free cultures of the murine hybridoma (CC9C10) which secretes Mab against bovine insulin were used as a model in these studies. Changes in qMab were monitored in relation to the measured intracellular nucleotide pool. Under certain culture conditions the qMab increased significantly (65-97%) and was shown to be concomitant with an increase (13-65%) in UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine (UDP-GNac) and concomitant with a decrease in growth rate (22-59%). Hybridomas were then grown in conditions favorable for sugar-nucleotide accumulation. Supplementation of cultures with tunicamycin or NH$\sb4$Cl caused a x5 increase in intracellular UDP-GNac but without significantly affecting the qMab. Tunicamycin, but not NH$\sb4$Cl, inhibited Mab glycosylation. However, non-glycosylated Mab was secreted at the same rate as the glycosylated form. Therefore, neither the availability of UDP-GNac nor glycosylation appear to limit productivity in these cells. The data suggests that a reduction in growth rate rather than UDP-GNac accumulation may cause an increased qMab. From a study of the relationship between cell viability and intracellular nucleotides, it was found that programmed cell death (apoptosis) was preceded by an intracellular decrease in CTP and UTP. These nucleotides may act as mediators of apoptosis and may be used as predictors of imminent cell death. The level of intracellular nucleotides depends on the availability of nutrients. Increasing medium glucose (0-25 mM) resulted in the proportional increase in NTP levels ($>$45%), although further enhancement was not observed at glutamine $>$0.5 mM. The relationships between intracellular nucleotides levels and cell viability are potentially useful for monitoring bioreactor cultures for large scale production processes. Manipulating nucleotide levels by culture supplementation may prove to be an important tool for the control of cell growth and the prevention cell death.