title: 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.
authors.affiliation: Cell Culture Sciences, Tanox, Inc., Houston, Texas, USA.
authors.forename: Bruno
authors.initials: B
authors.lastname: Figueroa
authors.forename: Eric
authors.initials: E
authors.lastname: Ailor
authors.forename: Douglas
authors.initials: D
authors.lastname: Osborne
authors.forename: J Marie
authors.initials: JM
authors.lastname: Hardwick
authors.forename: Mitchell
authors.initials: M
authors.lastname: Reff
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
mesh_terms: D017501:Adenovirus E1B Proteins / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics*; D019149:Bioreactors; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D024861:Genetic Enhancement / Q000379:methods*; D031986:Geum / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D008954:Models, Biological
chemical_list: D017501:Adenovirus E1B Proteins; D000911:Antibodies, Monoclonal; D016895:Culture Media, Serum-Free; D005947:Glucose