Combining High-Throughput Screening of Caspase Activity with Anti-Apoptosis Genes for Development of Robust CHO Production Cell Lines Haimanti Dorai, Dawn Ellis, Yun Seung Keung, Marguerite Campbell, Minhong Zhuang, and Chengbin Lin Pharmaceutical Development, Centocor R&D, Radnor, PA 19087 Michael J. Betenbaugh Dept. of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218 DOI 10.1002/btpr.426 Published online May 5, 2010 in Wiley Online Library (wileyonlinelibrary.com). A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improve- ment in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2D (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti- apoptotic genes, e.g., E1B-19K, Aven, and XIAPD. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BD31-1, a stable clone expressing Bcl-2D, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BD31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumu- late as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics. V V C 2010 American Institute of Chemical Engi- neers Biotechnol. Prog., 26: 1367–1381, 2010 Keywords: mammalian cell culture, apoptosis, productivity, sodium butyrate, Chinese hamster ovary, lactate, monoclonal antibody titer, Bcl-2, Bcl-XL, E1B-19K, Aven, XIAP Introduction Mammalian cell culture is the production process of choice for many recombinant proteins due to its capacity to perform complex post-translational modifications. Increasing manufacturing demand for mammalian-derived products such as monoclonal antibodies necessitates a strong motivation to improve process efficiency by increasing product yield and product quality. Both cellular physiology and cell culture processes should be optimized to achieve the desired grams per liter titer for production of today’s biotherapeutics. Moreover, these optimization processes must occur in animal protein free (APF) media. Inherent in current high density, protein-free mammalian cell cultures is the problem of cell death. It has been estimated that apoptosis can account for up to 80% of cell death 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, has been implicated in promot- ing the release of toxic metabolites from dead cells, and potentially decreases heterologous protein yield (Chiang and Sisk, 2005; Figueroa et al., 2001, 2003, 2004; Mastrangelo et al., 2000a,b; Mercille and Massie, 1994). Apoptosis is a result of a complex network of signaling pathways initiating from both inside and outside the cell, culminating in the activation of cysteine aspartate proteases (caspases) that execute the final stages of cell death. Various methods of apoptosis prevention have been used to maintain cell viability during extended production runs in mammalian cell culture (Arden and Betenbaugh, 2004; Mercille and Massie, 1994; Vives et al., 2003). Altering the extracellular environment through media supplementation of growth fac- tors, hydrolysates, and limiting nutrients has led to increased protein production and decreased apoptosis (Burteau et al., 2003; Zhang and Robinson, 2005). Other researchers have turned to chemical and genetic strategies to inhibit the apo- ptotic signaling cascade from within the cell (Sauerwald et al., 2002, 2003). Researchers have found that over- Correspondence concerning this article should be addressed to H. Dorai at hdorai@its.jnj.com. V V C 2010 American Institute of Chemical Engineers 1367