Abstract
The effect of ammonia on Chinese hamster ovary (CHO) cell growth and galactosylation of recombinant immunoglobulin (rIgG) was investigated using shaking flasks with serum free media containing 0–15 mM NH4Cl. The elevated ammonia inhibited cell growth and negatively affected the galactosylation of rIgG. At 15 mM NH4Cl, the proportions of monogalactosylated glycan with fucosex (monogalactosylated glycan with fucose) and digalactosylated glycan with fucose (G2F) were 23.9% and 6.3% lower than those at 0 mM NH4Cl, respectively. To reduce ammonia formation by cells, glutamate was examined as a substitute for glutamine. The use of glutamate reduced the accumulation of ammonia and enhanced the production of rIgG while depressing cell growth. At 6 mM glutamate, ammonia level did not exceed 2 mM, which is only one third of that at 6 mM glutamine. Also, a 1.7-fold increase in the titer of rIgG and specific rIgG productivity, q rIgG, was achieved at 6 mM glutamate. The galactosylation of rIgG was favorable at 6 mM glutamate. The proportion of galactosylated glycans, G1F and G2F, at 6 mM glutamate was 59.8%, but it was 50.4% at 6 mM glutamine. The use of glutamate also increased complement-dependent cytotoxicity activity, one of the effector functions of rIgG. Taken together, substitution of glutamine by glutamate can be considered relevant for the production of rIgG in CHO cells since glutamate not only enhances q rIgG but also generates a higher galactosylation essential for the effector function of rIgG.
Similar content being viewed by others
References
Altamirano C, Paredes C, Cairó JJ, Gòdia F (2000) Improvement of CHO cell culture medium formulation: Simulataneous substitution of glucose and glutamine. Biotechnol Prog 16:69–75
Altamirano C, Illanes A, Casablancas A, Gámez X, Cairó JJ, Gòdia F (2001a) Analysis of CHO cells metabolic redistribution in a glutamate-based defined medium in continuous culture. Biotechnol Prog 17:1032–1041
Altamirano C, Cairó JJ, Gòdia F (2001b) Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control. Biotechnol Bioeng 76:351–360
Altamirano C, Paredes C, Illanes A, Cairó JJ, Gòdia F (2004) Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. J Biotechnol 110:171–179
Anderson DC, Goochee CF (1995) The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by chinese hamster ovary cells. Biotechnol Bioeng 47:96-105
Boyd PN, Lines AC, Patel AK (1995) The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol Immunol 32:1311–1318
Butler M, Christie A (1994) Adaptation of mammalian cells to non-ammoniagenic media. Cytotechnology 15:87–94
Capiaumont J, Legrand C, Carbonell D, Dousset B, Belleville F, Nabet P (1995) Methods for reducing the ammonia in hybridoma cell-cultures. J Biotechnol 39:49–58
Chen P, Harcum SW (2006) Effects of elevated ammonium on glycosylation gene expression in CHO cells. Metabolic Eng 8:123–132
Christie A, Butler M (1994) Glutamine-based dipeptides are utilized in mammalian cell culture by extracellular hydrolysis catalyzed by a specific peptidase. J Biotechnol 37:277–290
Christie A, Butler M (1999) The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium. Biotechnol Bioeng 64:298–309
Gawlitzek M, Ryll T, Lofgren L, Sliwkowski MB (2000) Ammonium alters N-glycan structures of recombinant TNFR-IgG: Degradative versus biosynthetic mechanisms. Biotechnol Bioeng 68:637–646
Gillmeister MP, Tomiya N, Jacobia SJ, Lee YC, Gorfien SF, Betenbaugh MJ (2009) An HPLC-MALDI MS method for N-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions. Glycoconj J 26:1135–1149
Hamako J, Matsui T, Ozeki Y, Mizouchi T, Titani K (1993) Comparative studies of asparagines-linked sugar chains of immunoglobulin G from eleven mammalian species. Comp Biochem Physiol B106:949–954
Hansen HA, Emborg C (1994) Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture. Biotechnol Prog 10:121–124
Hodoniczky J, Zheng YZ, James DC (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21:1644–1652
Jefferis R (2009) Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 8:226–234
Kurano N, Leist C, Messi F, Kurano S, Fiechter A (1990) Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor: 1. Effects of physical and chemical environments. J Biotechnol 15:101–111
Lao M-S, Toth D (1997) Effects of ammonium and lactate on growth and metabolism of a recombinant Chinese hamster ovary cell culture. Biotechnol Prog 13:688–691
Newsholme P, Procopio J, Lima MMR, Pithon-Curi TC, Curi R (2003) Glutamine and glutamate-their central role in cell metabolism and function. Cell Biochem 21:1–9
Ozkan P, Mutharasan R (2002) A rapid method for measuring intracellular pH using BCECF-AM. Biochimica et Biophysica Acta 1572:143–148
Park H-S, Kim I-H, Kim I-Y, Kim K-H, Kim H-J (2000) Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr- cells decreases accumulation of ammonium ion in culture mediua. J Biotechnol 81:129–140
Raju TS (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20:471–478
Raju TS, Briggs JB, Borge SM, Jones AJ (2000) Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10:477–486
Wang M-D, Yang M, Huzel N, Butler M (2002) Erythropoietin production from CHO cells grown by continuous culture in a fludized-bed bioreactor. Biotechnol Bioeng 77:194–203
Werner RG (2007) Glycosylation of therapeutic proteins in different production systems. Acta Paediatrica 96:17–22
Wong DCF, Wong KTK, Goh LT, Heng CK, Yap MGS (2005) Impact of dynamic online feb-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89:164–177
Yang M, Butler M (2000) Effect of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol Bioeng 68:370–380
Yang M, Butler M (2002) Effect of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms. Biotechnol Prog 18:129-138
Zanghi JA, Mendoza TP, Knop RH, Miller WM (1998) Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells. J Cell Physiol 177:248–263
Acknowledgements
The authors thank Jin Sook Lee for her assistance with the CDC assay of rIgG.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hong, J.K., Cho, S.M. & Yoon, S.K. Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells. Appl Microbiol Biotechnol 88, 869–876 (2010). https://doi.org/10.1007/s00253-010-2790-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-010-2790-1