Abstract
The majority of therapeutic proteins are expressed in mammalian cells, predominantly in Chinese Hamster Ovary cells. While cell culture media and feed supplements are crucial to protein productivity, medium optimization can be labor intensive and time-consuming. In this chapter, we describe some basic concepts in medium development and introduce a rational and rapid workflow to screen and optimize media and feeds. The major goal of medium screening is to select a base formulation as the foundation for further optimization, but ironically, the most conventional screening method may actually rule out ideal chemically defined medium candidates. Appropriate cell adaptation is the key to identifying an optimal base medium, particularly when cells were originally cultured in serum-free medium containing recombinant proteins and/or undefined hydrolysates. The efficient workflow described herein integrates the optimization of both medium and feed simultaneously using a Design-of-Experiment (DOE) approach. The feasibility of the workflow is then demonstrated with a case study, in which chemically defined medium and feed were optimized in a single fed-batch study using a high-throughput microbioreactor system (SimCell™), which resulted in improving protein titers three- to sixfold.
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References
Merten OW (2006) Introduction to animal cell culture technology-past, present and future. Cytotechnology 50(1–3):1–7
Ozturk SS, Hu W-S (2006) Cell culture technology-an overview. Biotechnology and bioprocessing, 1–14
Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398
Jayme DW, Smith SR (2000) Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture. Cytotechnology 33(1–3):27–36
Jayme DW, Epstein DA, Conrad DR (1988) Fetal bovine serum alternatives. Nature 334(6182):547–548
Wong VV et al (2006) Zinc as an insulin replacement in hybridoma cultures. Biotechnol Bioeng 93(3):553–563
Burteau CC et al (2003) Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line. In Vitro Cell Dev Biol Anim 39(7):291–296
Zhang J et al (2003) Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process. Biotechnol Bioeng 82(6):640–652
Gorfien SF et al (2003) Optimized nutrient additives for fed-batch cultures. BioPharm Int April: 34–40
Fike R (2009) Nutrient supplementation strategies for biopharmaceutical production, part 1: Identifying a formulation. BioProcess Int 7(10):44–51
Fletcher T (2005) Designing culture media for recombinant protein production: a rational approach. BioProcess Int 3(1):2–6
Wlaschin KF, Hu WS (2006) Fed batch culture and dynamic nutrient feeding. Adv Biochem Eng Biotechnol 101:43–74
Chun C et al (2003) Application of factorial design to accelerate identification of CHO growth factor requirements. Biotechnol Prog 19(1):52–57
Parampalli A et al (2007) Development of serum-free media in CHO-DG44 cells using a central composite statistical design. Cytotechnology 54(1):57–68
De Alwis DM et al (2007) Statistical methods in media optimization for batch and fed-batch animal cell culture. Bioprocess Biosyst Eng 30(2):107–113
Hacker DL, De Jesus M, Wurm FM (2009) 25 years of recombinant proteins from reactor-grown cells—where do we go from here? Biotechnol Adv 27(6):1023–1027
Betts JI, Baganz F (2006) Miniature bioreactors: current practices and future opportunities. Microb Cell Fact 5:21
De Jesus M et al (2004) TubeSpin satellites: a fast track approach for process development with animal cells using shaking technology. Biochem Eng J 17(3):217–223
Chen A et al (2009) Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development. Biotechnol Bioeng 102(1):148–160
Kensy F et al (2005) Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates. Bioprocess Biosyst Eng 28(2):75–81
Funke M et al (2010) Microfluidic biolector-microfluidic bioprocess control in microtiter plates. Biotechnol Bioeng 107(3):497–505
Silk NJ et al (2010) Fed-batch operation of an industrial cell culture process in shaken microwells. Biotechnol Lett 32(1):73–78
Amanullah A et al (2010) Novel micro-bioreactor high throughput technology for cell culture process development: reproducibility and scalability assessment of fed-batch CHO cultures. Biotechnol Bioeng 106(1):57–67
Legmann R et al (2009) A predictive high-throughput scale-down model of monoclonal antibody production in CHO cells. Biotechnol Bioeng 104(6):1107–1120
Xiao Z et al (2010) Rapid creation of platform fed-batch process using a high throughput (SimCell) system. In: Cell culture engineering XII, 2010. Banff, Canada
Ma N et al (2009) A single nutrient feed supports both chemically defined NS0 and CHO fed-batch processes: improved productivity and lactate metabolism. Biotechnol Prog 25(5):1353–1363
Paul WC et al (2009) Maintaining product titer while replacing undefined components in a CHO culture system. BioProcess Int 7(8):30–38
Crowell CK et al (2007) Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system. Biotechnol Bioeng 96(3):538–549
Delorme E et al (1992) Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 31(41):9871–9876
Gnoth S et al (2008) Control of cultivation processes for recombinant protein production: a review. Bioprocess Biosyst Eng 31(1):21–39
Acknowledgements
The authors wish to acknowledge the contribution of scientists at Life Technologies, particularly Erica Wehling, Ekta Goel, Shyam Kumar, and Yuan Wen, for their execution of the studies presented here. The authors also wish to acknowledge the contribution of Lu Ren, Jaime Clark, Eric Burden, and Brian Benoit from Seahorse Bioscience for their engineering and application support on the SimCell™ system.
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Xiao, Z., Sabourin, M., Piras, G., Gorfien, S.F. (2014). Screening and Optimization of Chemically Defined Media and Feeds with Integrated and Statistical Approaches. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Molecular Biology, vol 1104. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-733-4_9
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DOI: https://doi.org/10.1007/978-1-62703-733-4_9
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