Bcl‐x_L mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells

Abstract

Enhanced product yields, reduction in throughput time, improved cost-effectiveness and product quality are examples of benefits gained by delaying apoptotic cell death in bioreactors. To examine the effect on recombinant protein production, bcl-x(L) was overexpressed in a CHO cell line secreting humanized monoclonal antibody directed against the alpha1beta1 integrin. When cell lines overexpressing bcl-x(L) were compared to the parent, cell viability was increased by 20% and titers by 80%. Total viable cell densities were similar and specific productivities were enhanced by almost two-fold on scale-up to bioreactors. Comparison in a chemically defined media demonstrated an even greater sustained viability in bcl-x(L) expressing cells by 50% and up to 90% increase in titer with no impact on product quality. Caspase 3 activities were monitored as a marker for apoptotic cell death. In the presence of Bcl-x(L), caspase activities were reduced to background levels. The role of Bcl-x(L) in protecting cells from premature death was further examined in studies performed in the presence of NaBu, at concentrations known to trigger cell death. Results demonstrated that cells expressing bcl-x(L) retained 88% cell viability with >2 fold increase in titer. Bcl-x(L) was similarly overexpressed in a different CHO cell line producing a humanized mAb against the chemokine MCP1. Once again, production titer was increased by 80% and viability by 75%. Together the studies have shown that overexpression of bcl-x(L) in production cell lines was able to significantly increase the titer by enhancing both the specific activity and total cell viability while maintaining product quality.

MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells

“…In general most researchers have observed increased maximum cell number, enhanced viability, extended culture duration, protection from viral and sodium butyrate mediated apoptosis and in certain cases increased titers following expression of Bcl family members. (Fassnacht et al 1999;Tey et al 2000;Goswami et al 1999;Simpson et al 1999;Fussenegger et al 2000;Mastrangelo et al 2000a, b;Meents et al 2002;Chiang and Sisk 2005;Kim and Lee 2002;Sung and Lee 2005;Sauerwald et al 2006;Ifandi and Al-Rubeai 2005). Viral homologues of Bcl-2 including E1B19K have also been used for the inhibition of apoptosis in mammalian cell cultures (Wong et al 2006;Figueroa et al 2006), where E1B19K inhibits the apoptotic activity of p53 (Debbas and White 1993;Lowe and Ruley 1993).…”

Expression of anti‐apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture

“…Inherent in current high density, protein-free mammalian cell cultures is the problem of cell death of which apoptosis can account for up to 80% in a typical fed-batch bioreactor, induced in response to insults such as nutrient and growth factor deprivation, oxygen depletion, toxin accumulation, and shear stress (Goswami et al, 1999). Apoptosis limits the maximum viable cell density, accelerates the onset of the death phase and potentially decreases heterologous protein yield (Chiang and Sisk, 2005;Figueroa et al, 2001Figueroa et al, , 2003Figueroa et al, , 2004Mastrangelo and Betenbaugh, 1998;Mercille and Massie, 1994).…”

Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines

“…V79020) or pcDNA3.1+neo-bcl-x L construct lacking 3'UTR of bcl-x L gene. 68 Transfections were performed as described above, except DharmaFECT Duo transfection reagent and corresponding manufacturer's protocols were used (Dharmacon, Cat. No.…”