Toey Nivitchanyong1, Amanda Martinez2, Adiba Ishaque2, John E. Murphy2, Michael J. Betenbaugh1, and John C. Thrift2. (1) Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, (2) Bayer Health Care, 800 Dwight Way, Berkeley, CA 94710
The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression and improved product quality. In this study, a Baby Hamster Kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In small-scale batch culture evaluations, eleven clonal cell lines expressing either E1B-19K or a combination of Aven/E1B-19K showed improved survival compared to both parental and three blank vector cell line controls. In addition, during this evaluation these cell lines exhibited lower caspase-3 activation and lower Annexin-V binding compared to the controls. Further studies with the apoptosis inducers camptothecin and thapsigargin demonstrated the E1B-19K and Aven/E1B-19K cell lines were more resistant to these insults. These results were used to select the best Aven/E1B-19K candidate cell line, and subsequently its performance was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue that depends on cell line performance and other operating conditions. In this study, the perfusion rates were varied for both the parental and engineering BHK cells lines. The bioreactors were operated at sequentially lower specific perfusion rates, known to negatively effect the parental cell line in this media formulation, while maintaining a cell density of 2x107 viable cells/mL. The Aven/E1B-19K cell line was shown to exhibit higher cell viability and enhanced specific productivity compared to the parental control when grown under these stressful reduced perfusion rates. Such a finding has significant implications for the expression of anti-apoptosis genes in cell lines used in a perfusion bioreactor environment.