ARTICLE Effects of Solution Environment on Mammalian Cell Fermentation Broth Properties: Enhanced Impurity Removal and Clarification Performance Matthew Westoby, 1 James Chrostowski, 2 Philippe de Vilmorin, 2 John Paul Smelko, 3 Jonathan K. Romero 2 1 Process Biochemistry Technical Development Biogen Idec 5200 Research Place, San Diego, California 2 Process Biochemistry Technical Development Biogen Idec, 15 Cambridge Center, Cambridge, Massachusetts 02142, USA; telephone: 617-914-5829; fax: 617-678-3408; e-mail: jonathan.romero@biogenidec.com 3 Cell Culture Development Technical Development Biogen Idec, Research Triangle Park, North Carolina Received 7 June 2010; revision received 25 August 2010; accepted 26 August 2010 Published online 1 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.22923 ABSTRACT: The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prema- turely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process-related impu- rities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was exam- ined to determine if enhanced impurity removal and clar- ification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade-offs between pro- duct recovery and purification and improved clarification at manufacturing-scale production. A reduction in cell culture broth pH to 4.7–5.0 induced flocculation and impurity precipitation which increased the average feed particle-size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further opti- mized by controlling ionic composition along with pH to improve product recovery from high cell density/high pro- duct titer cell cultures. Biotechnol. Bioeng. 2011;108: 50–58. ß 2010 Wiley Periodicals, Inc. KEYWORDS: enhanced mammalian cell clarification; im- purity removal; cell flocculation Introduction The recent drive to generate increased titer cell culture processes for large-scale production of therapeutic proteins has required bioreactors to operate at high mammalian cell densities (>1  10 7 cells/mL) introducing elevated levels of nucleic acids, host cell proteins (HCP), and complex media and nutrient feed components. Consequently, these com- plex cell culture suspensions have placed increased demands on both the separation (clarification) and purification operations to remove cells, cell debris, and increased levels of impurities. The clarification of recombinant proteins such as monoclonal antibodies and Fc-fusion proteins from manufacturing-scale bioreactors containing mammalian cells is usually performed using either filtration or centrifugation. The filtration process is typically operated in a tangential-flow mode using microfiltration (MF) membranes although depth filtration in a dead-end mode is also common (Chandler and Zydney, 2005). In addition, traditional downstream processing of therapeutic proteins has been designed to place nearly all of the purification capabilities on chromatography steps with the clarification steps designed exclusively for cell and cell debris removal. Previous work has shown successful flocculation of cells and cellular debris and precipitation of contaminants in both mammalian and bacterial systems using various flocculants such as anionic and cationic polymers (Aunins and Wang, 1989; Baran, 1988; Riske et al., 2006). In addition, studies have also shown the benefits of flocculation on improved clarification via tangential flow filtration and centrifugation (Belfort et al., 1994; Gasner and Wang, 1970; Peuchot and Ben Aim, 1992; Roush and Lu, Correspondence to: Jonathan K. Romero 50 Biotechnology and Bioengineering, Vol. 108, No. 1, January 1, 2011 ß 2010 Wiley Periodicals, Inc.