Enhanced cell culture performance using inducible anti‐apoptotic genes E1B‐19K and Aven in the production of a monoclonal antibody with Chinese hamster ovary cells

Abstract

Mammalian cells are used for the production of numerous biologics including monoclonal antibodies. Unfortunately, mammalian cells can lose viability at later stages in the cell culture process. In this study, the effects of expressing the anti-apoptosis genes, E1B-19K and Aven, separately and in combination on cell growth, survival, and monoclonal antibody (MAb) production were investigated for a commercial Chinese Hamster Ovary (CHO) mammalian cell line. CHO cells were observed to undergo apoptosis following a model insult, glucose deprivation, and at later stages of batch cell culture. The CHO cell line was then genetically modified to express the anti-apoptotic proteins E1B-19K and/or Aven using an ecdysone-inducible expression system. Stable transfected pools induced to express Aven or E1B-19K alone were found to survive 1-2 days longer than the parent cell line following glucose deprivation while the expression of both genes in concert increased cell survival by 3 days. In spinner flask batch studies, a clonal isolate engineered to express both anti-apoptosis genes exhibited a longer operating lifetime and higher final MAb titer as a result of higher viable cell densities and viabilities. Interestingly, survival was increased in the absence of an inducer, most likely as a result of leaky expression of the anti-apoptosis genes confirmed in subsequent PCR studies. In fed-batch bioreactors, the expression of both anti-apoptosis genes resulted in higher growth rates and cell densities in the exponential phase and significantly higher viable cell densities, viabilities, and extended survival during the post-exponential phase. As a result, the integral of viable cells (IVC) was between 40 and 100% higher for cell lines engineered to express both Aven and E1B-19K in concert, and the operational lifetime of the fed-batch bioreactors was increased from 2 to 5 days. The maximum titers of MAb were also increased by 40-55% for bioreactors containing cells expressing Aven and E1B-19K. These increases in volumetric productivity arose primarily from enhancements in viable cell density over the course of the fed-batch culture period since the specific productivities for the cells expressing anti-apoptosis genes were comparable or slightly lower than the parental hosts. These results demonstrate that expression of anti-apoptosis genes can enhance culture performance and increase MAb titers for mammalian CHO cell cultures especially under conditions such as extended fed-batch bioreactor operation.

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

“…Nivitchanyong et al (2007) found that co-expression of Aven and E1B-19K enhanced BHK cell survival and cell growth and delayed apoptosis relative to the cell lines expressing E1B-19K alone. Also in agreement with our finding is the fact that co-expression of Aven with E1B-19K enhanced protection relative to the expression of either gene alone in batch and fed-batch CHO DHFR cultures (Figueroa et al, 2007). The current study extends these findings in CHO K1 by showing that the expression of two antiapoptosis genes leads to a significant reduction in caspase activity and improved mitochondrial membrane potential in the presence of insults including staurosporine and extended batch cultures.…”

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

“…(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).…”

Dynamics of unfolded protein response in recombinant CHO cells

“…Increasing cell density by either reducing apoptosis (Meents et al 2002;Figueroa et al 2007) or altering cell metabolism (Chen et al 2001) can lead to increase in product titers. Alternatively, the protein synthesis and secretion machinery can be targeted to increase the specific productivity of a cell.…”