Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.

[1]  Mei Liu,et al.  Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody , 2013, Cytotechnology.

[2]  W. Miller,et al.  Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality. , 2002, Biotechnology and bioengineering.

[3]  P A Marichal-Gallardo,et al.  State‐of‐the‐art in downstream processing of monoclonal antibodies: Process trends in design and validation , 2012, Biotechnology progress.

[4]  Yuanxing Zhang.  Approaches to optimizing animal cell culture process: substrate metabolism regulation and protein expression improvement. , 2009, Advances in biochemical engineering/biotechnology.

[5]  M M Alvarez,et al.  Use of a Plackett–Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells , 2011, Biotechnology progress.

[6]  Yuanxing Zhang,et al.  INSIGHT INTO METABOLISM OF CHO CELLS AT LOW GLUCOSE CONCENTRATION ON THE BASIS OF THE DETERMINATION OF INTRACELLULAR METABOLITES , 2005 .

[7]  Antonio Aliaga Navarro,et al.  Metabolic reaction network approach for CHO modelling culture , 2010 .

[8]  Christopher H Contag,et al.  Integrated studies of biology: multiplexed imaging assays from molecules to man and back. , 2009, Current opinion in biotechnology.

[9]  P. Lio,et al.  Monitoring Chinese hamster ovary cell culture by the analysis of glucose and lactate metabolism. , 2005, Journal of biotechnology.

[10]  F. Blanchard,et al.  Combination of yeast hydrolysates to improve CHO cell growth and IgG production , 2013, Cytotechnology.

[11]  H. Mayer,et al.  Characterization of amino acid profiles of culture media via pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization and ultra performance liquid chromatography. , 2011, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[12]  Peifeng Chen,et al.  Effects of amino acid additions on ammonium stressed CHO cells. , 2005, Journal of biotechnology.

[13]  R. Goodacre,et al.  Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production. , 2011, Biotechnology and bioengineering.

[14]  N. Dohmae,et al.  Amino acid analysis of sub-picomolar amounts of proteins by precolumn fluorescence derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. , 2011, Bioscience trends.

[15]  Gyun Min Lee,et al.  CHO cells in biotechnology for production of recombinant proteins: current state and further potential , 2012, Applied Microbiology and Biotechnology.

[16]  Katie F Wlaschin,et al.  Recombinant protein therapeutics from CHO cells : 20 years and counting , 2007 .

[17]  Hervé Broly,et al.  Cell culture medium improvement by rigorous shuffling of components using media blending , 2012, Cytotechnology.

[18]  Alan T. Bull,et al.  Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells , 1992, Applied Microbiology and Biotechnology.

[19]  Gary Walsh,et al.  Biopharmaceutical benchmarks 2010 , 2010, Nature Biotechnology.

[20]  Y. Ahn,et al.  Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design. , 1999, Journal of biotechnology.

[21]  Andrés Illanes,et al.  Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production , 2006 .

[22]  Michael C. Borys,et al.  Optimizing amino acid composition of CHO cell culture media for a fusion protein production , 2011 .

[23]  Hervé Broly,et al.  Lactate metabolism shift in CHO cell culture: the role of mitochondrial oxidative activity. , 2013, New biotechnology.

[24]  Leonard A. Smith,et al.  Developement of serum-free media in CHO-DG44 cells using a central composite statistical design , 2007, Cytotechnology.

[25]  Z. Gerdtzen,et al.  Comparative metabolic analysis of lactate for CHO cells in glucose and galactose , 2011 .

[26]  C. Demangel,et al.  Factors controlling cell proliferation and antibody production in mouse hybridoma cells: I. Influence of the amino acid supply , 1991, Biotechnology and bioengineering.

[27]  F Gòdia,et al.  Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. , 2004, Journal of biotechnology.

[28]  H. Bonarius.  Metabolic-flux analysis of mammalian-cell culture , 1998 .

[29]  Yuanxing Zhang,et al.  Fed-batch culture of hybridoma cells in serum-free medium using an optimized feeding strategy , 2004 .

[30]  Tim Tressel,et al.  Current Therapeutic Antibody Production and Process Optimization , 2006 .

[31]  H. Broly,et al.  A high-throughput media design approach for high performance mammalian fed-batch cultures , 2013, mAbs.

[32]  Michael Butler,et al.  Expression systems for therapeutic glycoprotein production. , 2009, Current opinion in biotechnology.

[33]  Ashraf Amanullah,et al.  Automated dynamic fed‐batch process and media optimization for high productivity cell culture process development , 2013, Biotechnology and bioengineering.