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Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures.

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps.

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