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Abstract

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.

Engineering mammalian cells in bioprocessing – current achievements and future perspectives

“…In the majority of studies over‐expressing Bcl‐2 and Bcl‐x L , extended viability and enhanced cell numbers were observed, but results regarding productivity have been mixed. Bcl‐2 over‐expression in hybridoma cells led to increased antibody titre [66,67,72], and an increase in final antibody concentration under sodium butyrate‐induced apoptosis in CHO cells [83]. However, there was no significant improvement in antibody titre under growth arrest conditions in CHO cells when Bcl‐2 was over‐expressed [81], and Tey and Al‐Rubeai [76] observed reduced antibody specific production rates in NS0 perfusion cultures with increasing viable cell numbers.…”

Engineering death resistance in CHO cells for improved perfusion culture

“…The reduction in dead cells and debris would also make purification easier and reduce filter clogging. Despite these very alluring hypotheses and the long history of perfusion culture, only limited forays have been made in this field, 4–7 due to the complexities of cell line development and perfusion bioreactor setup.…”

Stable inhibition of mmu-miR-466h-5p improves apoptosis resistance and protein production in CHO cells

“…Several anti-apoptotic proteins including Bcl-x L , Bcl-2 and Mcl-1 are known to protect the cells from apoptosis onset by maintaining the integrity of mitochondrial membrane. Exogenous over-expression of these proteins protects mammalian cells from apoptosis induction and increases cell density and viability (Fassnacht et al 1999; Majors et al 2008b; Majors et al 2009; Mastrangelo et al 2000). The over-expression of some other anti-apoptotic proteins such as E1B-19K, Aven, or XIAP enhanced the apoptosis resistance in mammalian cell cultures (Figueroa et al 2007; Mercille and Massie 1999; Sauerwald et al 2002).…”