Skip to main content
SpringerLink
Log in

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

BioMetals volume 32, pages 11–19 (2019)Cite this article

Abstract

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.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Boeggeman E, Qasba PK (2002) Studies on the metal binding sites in the catalytic domain of β1,4-galactosyltransferase. Glycobiology 12:395–407

    Article  CAS  PubMed  Google Scholar 

  • 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 259:13370–13378

    CAS  PubMed  Google Scholar 

  • 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 P114

  • 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 96:538–549

    Article  CAS  PubMed  Google Scholar 

  • Dionne B, Mishra N, Butler M (2017) A low redox potential affects monoclonal antibody assembly and glycosylation in cell culture. J Biotechnol 246:71–80

    Article  CAS  PubMed  Google Scholar 

  • Dorival-García N et al (2018) Large-scale assessment of extractables and leachables in single-use bags for biomanufacturing. Anal Chem. https://doi.org/10.1021/acs.analchem.8b01208

    Article  PubMed  Google Scholar 

  • 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 92:569–574

    Article  CAS  PubMed  Google Scholar 

  • 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 11:1190–1200

    Article  CAS  PubMed  Google Scholar 

  • Goh JB, Ng SK (2017) Impact of host cell line choice on glycan profile. Crit Rev Biotechnol 38:1–17

    Google Scholar 

  • Grainger RK, James DC (2013) CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation. Biotechnol Bioeng 110:2970–2983

    Article  CAS  PubMed  Google Scholar 

  • Gramer MJ et al (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108:1591–1602

    Article  CAS  PubMed  Google Scholar 

  • 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. https://doi.org/10.1007/s00253-014-6012-0

    Article  CAS  PubMed  Google Scholar 

  • 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. https://doi.org/10.1002/biot.201700425

    Article  CAS  Google Scholar 

  • Hills AE, Patel A, Boyd P, James DC (2001) Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnol Bioeng 75:239–251

    Article  CAS  PubMed  Google Scholar 

  • Hossler P, Racicot C, McDermott S (2014) Targeted shifting of protein glycosylation profiles in mammalian cell culture through media supplementation of cobalt. J Glycobiol 3:108

    Google Scholar 

  • Kucharzewska P, Christianson HC, Belting M (2015) Global profiling of metabolic adaptation to hypoxic stress in human glioblastoma cells. PLoS ONE 10:e0116740

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kuhn NJ, Ward S, Leong WS (1991) Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase). FEBS J 195:243–250

    CAS  Google Scholar 

  • 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 62:55–71

    Article  CAS  PubMed  Google Scholar 

  • Maekawa H, Inagi R (2017) Stress signal network between hypoxia and ER stress in chronic kidney disease. Front Physiol 8:74

    Article  PubMed  PubMed Central  Google Scholar 

  • Malhotra JD, Kaufman RJ (2007) Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal 9:2277–2294

    Article  CAS  PubMed  Google Scholar 

  • 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):463–468

    Article  CAS  PubMed  Google Scholar 

  • Miyoshi E et al (1999) The α1-6-fucosyltransferase gene and its biological significance. Biochim Biophys Acta (BBA) 1473:9–20

    Article  CAS  Google Scholar 

  • O’Keeffe ET, Hill RL, Bell JE (1980) Active site of bovine galactosyltransferase: kinetic and fluorescence studies. Biochemistry 19:4954–4962

    Article  PubMed  Google Scholar 

  • Powell JT, Brew K (1976) Metal ion activation of galactosyltransferase. J Biol Chem 251:3645–3652

    CAS  PubMed  Google Scholar 

  • Prabhu A, Gadre R, Gadgil M (2018) Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-018-9064-8

    Article  PubMed  Google Scholar 

  • 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 318:491–502

    Article  CAS  PubMed  Google Scholar 

  • Reusch D, Tejada ML (2015) Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 25:1325–1334

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 305:865–870

    Article  Google Scholar 

  • 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 60:3375–3378

    CAS  PubMed  Google Scholar 

  • Salnikow K, Davidson T, Costa M (2002) The role of hypoxia-inducible signaling pathway in nickel carcinogenesis. Environ Health Perspect 110:831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shen R, Wang S, Ma X, Xian J, Li J, Zhang L, Wang P (2010) An easy colorimetric assay for glycosyltransferases. Biochemistry (Moscow) 75:944–950

    Article  CAS  Google Scholar 

  • St Amand MM, Radhakrishnan D, Robinson AS, Ogunnaike BA (2014) Identification of manipulated variables for a glycosylation control strategy. Biotechnol Bioeng 111:1957–1970

    Article  CAS  PubMed  Google Scholar 

  • 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 31:460–467

    Article  CAS  PubMed  Google Scholar 

  • Villiger TK et al (2016) High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation. J Biotechnol 229:3–12

    Article  CAS  PubMed  Google Scholar 

  • 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, B2

  • 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. https://doi.org/10.1002/btpr.2662

    Article  CAS  Google Scholar 

  • 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 107:321–336

    Article  CAS  PubMed  Google Scholar 

  • Yuk IH et al (2015) Effects of copper on CHO cells: cellular requirements and product quality considerations. Biotechnol Prog 31:226–238

    Article  CAS  PubMed  Google Scholar 

  • Zhou S, Schöneich C, Singh SK (2011) Biologics formulation factors affecting metal leachables from stainless steel. AAPS PharmSciTech 12:411–421

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Chemical Engineering and Process Development Division, CSIR-National Chemical Laboratory, Pune, 411008, India

    Anuja Prabhu & Mugdha Gadgil

Authors
  1. Anuja Prabhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mugdha Gadgil
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mugdha Gadgil.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 17 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Prabhu, A., Gadgil, M. Nickel and cobalt affect galactosylation of recombinant IgG expressed in CHO cells. Biometals 32, 11–19 (2019). https://doi.org/10.1007/s10534-018-0152-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10534-018-0152-0

Keywords

search

Navigation