, Volume 32, Issue 1, pp 11–19 | Cite as

Nickel and cobalt affect galactosylation of recombinant IgG expressed in CHO cells

  • Anuja Prabhu
  • Mugdha GadgilEmail author


Glycosylation is an important product quality attribute of antibody biopharmaceuticals. It involves enzymatic addition of oligosaccharides on proteins by sequential action of glycosyltransferases and glycosidases in the endoplasmic reticulum and golgi. Some of these enzymes like galactosyltransferase and N-acetylglucosaminyltransferase-I require trace metal cofactors. Variations in trace metal availability during production can thus affect glycosylation of recombinant glycoproteins such as monoclonal antibodies. Variability in trace metal concentrations can be introduced at multiple stages during production such as due to impurities in raw materials for culture medium and leachables from bioreactors. Knowledge of the effect of various trace metals on glycosylation can help in root-cause analysis of unintended variability in glycosylation. In this study, we investigated the effect of nickel and cobalt on glycosylation of recombinant IgG expressed in Chinese hamster ovary cells. Nickel concentrations below 500 µM did not affect glycosylation, but above 500 µM it significantly decreases galactosylation of IgG. Cobalt at 50 µM concentration causes slight increase in G1F glycans (mono galactosylated) as previously reported. However, higher concentrations result in a small increase in G0F (non galactosylated) glycans. This effect of nickel and cobalt on galactosylation of recombinant IgG can be reversed by supplementation of uridine and galactose which are precursors to UDP-Galactose, a substrate for the enzymatic galactosylation reaction.


Glycosylation Trace metals Process variability Nickel Cobalt Galactosylation 



MG acknowledges funding from the Department of Biotechnology, Government of India. The authors are thankful to the MALDI-MS facility at CSIR-NCL and to Dr. Gadre for help with HPLC-based galactosyltransferase assay.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10534_2018_152_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)


  1. Boeggeman E, Qasba PK (2002) Studies on the metal binding sites in the catalytic domain of β1,4-galactosyltransferase. Glycobiology 12:395–407CrossRefGoogle Scholar
  2. Campbell C, Stanley P (1984) A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc: glycopeptide beta-4-N-acetylglucosaminyltransferase III activity. J Biol Chem 259:13370–13378Google Scholar
  3. Clincke M-F, Guedon E, Yen FT, Ogier V, Goergen J-L (2011) Effect of iron sources on the glycosylation macroheterogeneity of human recombinant IFN-γ produced by CHO cells during batch processes. In: BMC proceedings. BioMed Central, London, p P114Google Scholar
  4. Crowell CK, Grampp GE, Rogers GN, Miller J, Scheinman RI (2007) Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system. Biotechnol Bioeng 96:538–549CrossRefGoogle Scholar
  5. Dionne B, Mishra N, Butler M (2017) A low redox potential affects monoclonal antibody assembly and glycosylation in cell culture. J Biotechnol 246:71–80CrossRefGoogle Scholar
  6. Dorival-García N et al (2018) Large-scale assessment of extractables and leachables in single-use bags for biomanufacturing. Anal Chem. Google Scholar
  7. Fujiyama K et al (2001) Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides. J Biosci Bioeng 92:569–574CrossRefGoogle Scholar
  8. Gao Y et al (2016) Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale-up to a 5000-liter CHO bioprocess. Biotechnol J 11:1190–1200CrossRefGoogle Scholar
  9. Goh JB, Ng SK (2017) Impact of host cell line choice on glycan profile. Crit Rev Biotechnol 38:1–17Google Scholar
  10. Grainger RK, James DC (2013) CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation. Biotechnol Bioeng 110:2970–2983CrossRefGoogle Scholar
  11. Gramer MJ et al (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108:1591–1602CrossRefGoogle Scholar
  12. Ha TK, Kim Y-G, Lee GM (2014) Effect of lithium chloride on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture. Appl Microbiol Biotechnol 98:9239–9248. CrossRefGoogle Scholar
  13. Ha TK, Hansen AH, Kol S, Kildegaard HF, Lee GMC (2017) Baicalein reduces oxidative stress in CHO cell cultures and improves recombinant antibody productivity. Biotechnol J 13:1700425. CrossRefGoogle Scholar
  14. Hills AE, Patel A, Boyd P, James DC (2001) Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnol Bioeng 75:239–251CrossRefGoogle Scholar
  15. Hossler P, Racicot C, McDermott S (2014) Targeted shifting of protein glycosylation profiles in mammalian cell culture through media supplementation of cobalt. J Glycobiol 3:108Google Scholar
  16. Kucharzewska P, Christianson HC, Belting M (2015) Global profiling of metabolic adaptation to hypoxic stress in human glioblastoma cells. PLoS ONE 10:e0116740CrossRefGoogle Scholar
  17. Kuhn NJ, Ward S, Leong WS (1991) Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase). FEBS J 195:243–250Google Scholar
  18. Kunkel JP, Jan DCH, Jamieson JC, Butler M (1998) Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody. J Biotechnol 62:55–71CrossRefGoogle Scholar
  19. Maekawa H, Inagi R (2017) Stress signal network between hypoxia and ER stress in chronic kidney disease. Front Physiol 8:74CrossRefGoogle Scholar
  20. Malhotra JD, Kaufman RJ (2007) Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal 9:2277–2294CrossRefGoogle Scholar
  21. Mitchelson FG, Mondia JP, Hughes EH (2017) Effect of copper variation in yeast hydrolysate on C-terminal lysine levels of a monoclonal antibody. Biotechnol Prog 33(2):463–468CrossRefGoogle Scholar
  22. Miyoshi E et al (1999) The α1-6-fucosyltransferase gene and its biological significance. Biochim Biophys Acta (BBA) 1473:9–20CrossRefGoogle Scholar
  23. O’Keeffe ET, Hill RL, Bell JE (1980) Active site of bovine galactosyltransferase: kinetic and fluorescence studies. Biochemistry 19:4954–4962CrossRefGoogle Scholar
  24. Powell JT, Brew K (1976) Metal ion activation of galactosyltransferase. J Biol Chem 251:3645–3652Google Scholar
  25. Prabhu A, Gadre R, Gadgil M (2018) Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells. Appl Microbiol Biotechnol. Google Scholar
  26. Ramakrishnan B, Balaji PV, Qasba PK (2002) Crystal structure of β1,4-galactosyltransferase complex with UDP-Gal reveals an oligosaccharide acceptor binding site. J Mol Biol 318:491–502CrossRefGoogle Scholar
  27. Reusch D, Tejada ML (2015) Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 25:1325–1334CrossRefGoogle Scholar
  28. Rijcken WRP, Overdijk B, Van den Eijnden DH, Ferwerda W (1995) The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes. Biochem J 305:865–870CrossRefGoogle Scholar
  29. Salnikow K, Su W, Blagosklonny MV, Costa M (2000) Carcinogenic metals induce hypoxia-inducible factor-stimulated transcription by reactive oxygen species-independent mechanism. Cancer Res 60:3375–3378Google Scholar
  30. Salnikow K, Davidson T, Costa M (2002) The role of hypoxia-inducible signaling pathway in nickel carcinogenesis. Environ Health Perspect 110:831CrossRefGoogle Scholar
  31. Shen R, Wang S, Ma X, Xian J, Li J, Zhang L, Wang P (2010) An easy colorimetric assay for glycosyltransferases. Biochemistry (Moscow) 75:944–950CrossRefGoogle Scholar
  32. St Amand MM, Radhakrishnan D, Robinson AS, Ogunnaike BA (2014) Identification of manipulated variables for a glycosylation control strategy. Biotechnol Bioeng 111:1957–1970CrossRefGoogle Scholar
  33. Surve T, Gadgil M (2014) Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: implications for use of alternate sugars. Biotechnol Prog 31:460–467CrossRefGoogle Scholar
  34. Villiger TK et al (2016) High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation. J Biotechnol 229:3–12CrossRefGoogle Scholar
  35. Wentz AE, Hemmavanh D, Matuck JG (2015) Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins. US Patent 9,598,667, B2Google Scholar
  36. Williamson J, Miller J, McLaughlin J, Combs R, Chu C (2018) Scale-dependent manganese leaching from stainless steel impacts terminal galactosylation in monoclonal antibodies. Biotechnol Prog 100:101. Google Scholar
  37. Wong NSC, Wati L, Nissom PM, Feng HT, Lee MM, Yap MGS (2010) An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding. Biotechnol Bioeng 107:321–336CrossRefGoogle Scholar
  38. Yuk IH et al (2015) Effects of copper on CHO cells: cellular requirements and product quality considerations. Biotechnol Prog 31:226–238CrossRefGoogle Scholar
  39. Zhou S, Schöneich C, Singh SK (2011) Biologics formulation factors affecting metal leachables from stainless steel. AAPS PharmSciTech 12:411–421CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Chemical Engineering and Process Development DivisionCSIR-National Chemical LaboratoryPuneIndia

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