Skip to main content
SpringerLink
Log in

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Andersen DC, Goochee CF (1994) The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins. Curr Opin Biotechnol 5:546–549

    Article  CAS  Google Scholar 

  • Arakawa F, Kuroki M, Kuwahara M, Senba T, Ozaki H, Matsuoka Y, Misumi Y, Kanda H, Watanabe T (1996) Cloning and sequencing of the VH and V kappa genes of an anti-CD3 monoclonal antibody, and construction of a mouse/human chimeric antibody. J Biochem 120:657–662

    CAS  Google Scholar 

  • Arakawa T, Philo JS, Tsumoto K, Yumioka R, Ejima D (2004) Elution of antibodies from a Protein-A column by aqueous arginine solutions. Protein Expr Purif 36:244–248

    Article  CAS  Google Scholar 

  • Asano R, Watanabe Y, Kawaguchi H, Fukazawa H, Nakanishi T, Umetsu M, Hayashi H, Katayose Y, Unno M, Kudo T, Kumagai I (2007) Highly effective recombinant format of a humanized IgG-like bispecific antibody for cancer immunotherapy with retargeting of lymphocytes to tumor cells. J Biol Chem 282:27659–27665

    Article  CAS  Google Scholar 

  • Asano R, Kawaguchi H, Watanabe Y, Nakanishi T, Umetsu M, Hayashi H, Katayose Y, Unno M, Kudo T, Kumagai I (2008) Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells. J Immunother 31:752–761

    Article  CAS  Google Scholar 

  • Baik JY, Lee GM (2010) A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: effect of Bcl-xL overexpression. Biotechnol Bioeng 105:358–367

    Article  CAS  Google Scholar 

  • Butler M (2006) Optimisation of the cellular metabolism of glycosylation for recombinant proteins produced by mammalian cell systems. Cytotechnology 50:57–76

    Article  CAS  Google Scholar 

  • Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68:283–291

    Article  CAS  Google Scholar 

  • Chung JY, Lim SW, Hong YJ, Hwang SO, Lee GM (2004) Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant Chinese hamster ovary cells. Biotechnol Bioeng 85:539–546

    Article  CAS  Google Scholar 

  • Datta AK, Sinha A, Paulson JC (1998) Mutation of the sialyltransferase S-sialylmotif alters the kinetics of the donor and acceptor substrates. J Biol Chem 273:9608–9614

    Article  CAS  Google Scholar 

  • De Leon GM, Wlaschin KF, Nissom PM, Yap M, Hu WS (2007) Comparative transcriptional analysis of mouse hybridoma and recombinant Chinese hamster ovary cells undergoing butyrate treatment. J Biosci Bioeng 103:82–91

    Article  Google Scholar 

  • Doolan P, Melville M, Gammell P, Sinacore M, Meleady P, McCarthy K, Francullo L, Leonard M, Charlebois T, Clynes M (2008) Transcriptional profiling of gene expression changes in a PACE-transfected CHO DUKX cell line secreting high levels of rhBMP-2. Mol Biotechnol 39:187–199

    Article  CAS  Google Scholar 

  • Drickamer K (1993) A conserved disulphide bond in sialyltransferases. Glycobiology 3:2–3

    Article  CAS  Google Scholar 

  • Dwek RA (1995) Glycobiology: more functions for oligosaccharides. Science 269:1234–1235

    Article  CAS  Google Scholar 

  • Fujiwara M, Tsukada R, Tsujinaga Y, Takagi M (2007) Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum. Appl Microbiol Biotechnol 75:983–987

    Article  CAS  Google Scholar 

  • Fujiyama K, Furukawa A, Katsura A, Misaki R, Omasa T, Seki T (2007) Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human β(1,4)-galactosyltransferase. Biochem Biophys Res Commun 358:85–91

    Article  CAS  Google Scholar 

  • Geremia RA, Harduin-Lepers A, Delannoy P (1997) Identification of two novel conserved amino acid residues in eukaryotic sialyltransferases: implications for their mechanism of action. Glycobiology 7:v–vii

    Article  CAS  Google Scholar 

  • Ghaderi D, Taylor RE, Padler-Karavani V, Diaz S, Varki A (2010) Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol 28:863–867

    Article  CAS  Google Scholar 

  • Gillespie W, Kelm S, Paulson JC (1992) Cloning and expression of the Gal β1, 3GalNAc α2,3-sialyltransferase. J Biol Chem 267:21004–21010

    CAS  Google Scholar 

  • Harduin-Lepers A, Mollicone R, Delannoy P, Oriol R (2005) The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach. Glycobiology 15:805–817

    Article  CAS  Google Scholar 

  • Helenius A, Aebi M (2001) Intracellular functions of N-linked glycans. Science 291:2364–2369

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Hong JK, Cho SM, Yoon SK (2010) Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells. Appl Microbiol Biotechnol 88:869–876

    Article  CAS  Google Scholar 

  • Jassal R, Jenkins N, Charlwood J, Camilleri P, Jefferis R, Lund J (2001) Sialylation of human IgG-Fc carbohydrate by transfected rat α2,6-sialyltransferase. Biochem Biophys Res Commun 286:243–249

    Article  CAS  Google Scholar 

  • Jeanneau C, Chazalet V, Auge C, Soumpasis DM, Harduin-Lepers A, Delannoy P, Imberty A, Breton C (2004) Structure-function analysis of the human sialyltransferase ST3Gal I: role of N-glycosylation and a novel conserved sialylmotif. J Biol Chem 279:13461–13468

    Article  CAS  Google Scholar 

  • Jefferis R (2005) Glycosylation of recombinant antibody therapeutics. Biotechnol Prog 21:11–16

    Article  CAS  Google Scholar 

  • Jenkins N, Parekh RB, James DC (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14:975–981

    Article  CAS  Google Scholar 

  • Kageshita T, Hirai S, Kimura T, Hanai N, Ohta S, Ono T (1995) Association between sialyl Lewisa expression and tumor progression in melanoma. Cancer Res 55:1748–1751

    CAS  Google Scholar 

  • Kaneko Y, Nimmerjahn F, Ravetch JV (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313:670–673

    Article  CAS  Google Scholar 

  • Kim SH, Lee GM (2009) Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments. Appl Microbiol Biotechnol 83:639–648

    Article  CAS  Google Scholar 

  • Kim WD, Tokunaga M, Ozaki H, Ishibashi T, Honda K, Kajiura H, Fujiyama K, Asano R, Kumagai I, Omasa T, Ohtake H (2010) Glycosylation pattern of humanized IgG-like bispecific antibody produced by recombinant CHO cells. Appl Microbiol Biotechnol 85:535–542

    Article  CAS  Google Scholar 

  • Kitazume S, Tachida Y, Oka R, Shirotani K, Saido TC, Hashimoto Y (2001) Alzheimer's β-secretase, β-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase. Proc Natl Acad Sci U S A 98:13554–13559

    Article  CAS  Google Scholar 

  • Lee EU, Roth J, Paulson JC (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of β-galactoside α 2,6-sialyltransferase. J Biol Chem 264:13848–13855

    CAS  Google Scholar 

  • Livingston BD, Paulson JC (1993) Polymerase chain reaction cloning of a developmentally regulated member of the sialyltransferase gene family. J Biol Chem 268:11504–11507

    CAS  Google Scholar 

  • Lund J, Takahashi N, Nakagawa H, Goodall M, Bentley T, Hindley SA, Tyler R, Jefferis R (1993) Control of IgG/Fc glycosylation: a comparison of oligosaccharides from chimeric human/mouse and mouse subclass immunoglobulin Gs. Mol Immunol 30:741–748

    Article  CAS  Google Scholar 

  • Monaco L, Marc A, Eon-Duval A, Acerbis G, Distefano G, Lamotte D, Engasser J-M, Soria M, Jenkins N (1996) Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ. Cytotechnology 22:197–203

    Article  CAS  Google Scholar 

  • Nissom PM, Sanny A, Kok YJ, Hiang YT, Chuah SH, Shing TK, Lee YY, Wong KT, Hu WS, Sim MY, Philp R (2006) Transcriptome and proteome profiling to understanding the biology of high productivity CHO cells. Mol Biotechnol 34:125–140

    Article  CAS  Google Scholar 

  • Omasa T, Higashiyama K, Shioya S, Suga K (1992) Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng 39:556–564

    Article  CAS  Google Scholar 

  • Omasa T, Onitsuka M, Kim WD (2010a) Cell engineering and cultivation of Chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol 11:233–240

    Google Scholar 

  • Omasa T, Furuichi K, Iemura T, Katakura Y, Kishimoto M, Suga K (2010b) Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture. Bioproc Biosyst Eng 33:117–125 865

    Google Scholar 

  • Omasa T, Tanaka R, Doi T, Ando M, Kitamoto Y, Honda K, Kishimoto M, Ohtake H (2008) Decrease in antithrombin III fucosylation by expressing GDP-fucose transporter siRNA in Chinese hamster ovary cells. J Biosci Bioeng 106:168–173

    Article  CAS  Google Scholar 

  • Patel RY, Balaji PV (2006) Identification of linkage-specific sequence motifs in sialyltransferases. Glycobiology 16:108–116

    Article  CAS  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(Pt 3):865–870

    Google Scholar 

  • Radaev S, Motyka S, Fridman WH, Sautes-Fridman C, Sun PD (2001) The structure of a human type III Fcγ receptor in complex with Fc. J Biol Chem 276:16469–16477

    Article  CAS  Google Scholar 

  • Scallon BJ, Tam SH, McCarthy SG, Cai AN, Raju TS (2007) Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol Immunol 44:1524–1534

    Article  CAS  Google Scholar 

  • Schwarzkopf M, Knobeloch KP, Rohde E, Hinderlich S, Wiechens N, Lucka L, Horak I, Reutter W, Horstkorte R (2002) Sialylation is essential for early development in mice. Proc Natl Acad Sci U S A 99:5267–5270

    Article  CAS  Google Scholar 

  • Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278:3466–3473

    Article  CAS  Google Scholar 

  • Sondermann P, Huber R, Oosthuizen V, Jacob U (2000) The 3.2-Å crystal structure of the human IgG1 Fc fragment–Fc gammaRIII complex. Nature 406:267–273

    Article  CAS  Google Scholar 

  • Sugimoto I, Futakawa S, Oka R, Ogawa K, Marth JD, Miyoshi E, Taniguchi N, Hashimoto Y, Kitazume S (2007) β-Galactoside α2,6-sialyltransferase I cleavage by BACE1 enhances the sialylation of soluble glycoproteins. A novel regulatory mechanism for α2,6-sialylation. J Biol Chem 282:34896–34903

    Article  CAS  Google Scholar 

  • Terada S, Nishimura T, Sasaki M, Yamada H, Miki M (2002) Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma. Cytotechnology 40:3–12

    Article  CAS  Google Scholar 

  • Thaisuchat H, Baumann M, Pontiller J, Hesse F, Ernst W (2011) Identification of a novel temperature sensitive promoter in CHO cells. BMC Biotechnol 11:51

    Article  CAS  Google Scholar 

  • Valley U, Nimtz M, Conradt HS, Wagner R (1999) Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins. Biotechnol Bioeng 64:401–417

    Article  CAS  Google Scholar 

  • Varki A (1997) Sialic acids as ligands in recognition phenomena. FASEB J 11:248–255

    CAS  Google Scholar 

  • Weinstein J, Lee EU, McEntee K, Lai PH, Paulson JC (1987) Primary structure of β-galactoside α 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor. J Biol Chem 262:17735–17743

    CAS  Google Scholar 

  • Wong NS, Yap MG, Wang DI (2006) Enhancing recombinant glycoprotein sialylation through CMP–sialic acid transporter over expression in Chinese hamster ovary cells. Biotechnol Bioeng 93:1005–1016

    Article  CAS  Google Scholar 

  • Wright A, Morrison SL (1997) Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol 15:26–32

    Article  CAS  Google Scholar 

  • Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S, Andersen MR, Neff N, Passarelli B, Koh W, Fan HC, Wang J, Gui Y, Lee KH, Betenbaugh MJ, Quake SR, Famili I, Palsson BO, Wang J (2011) The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol 29:735–741

    Article  CAS  Google Scholar 

  • Yang M, Butler M (2002) Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms. Biotechnol Prog 18:129–138

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work is partially supported by grants from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS).

Author information

Authors and Affiliations

  1. Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima, 770-8506, Japan

    Masayoshi Onitsuka & Takeshi Omasa

  2. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Wook-Dong Kim, Hiroyuki Ozaki, Akira Kawaguchi, Kohsuke Honda, Hisao Ohtake & Takeshi Omasa

  3. International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Hiroyuki Kajiura & Kazuhito Fujiyama

  4. Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-11-606 Aoba-yama, Aramaki, Aoba-ku, Sendai, 980-8579, Japan

    Ryutaro Asano & Izumi Kumagai

Authors
  1. Masayoshi Onitsuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wook-Dong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroyuki Ozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Akira Kawaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kohsuke Honda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiroyuki Kajiura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kazuhito Fujiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ryutaro Asano
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Izumi Kumagai
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hisao Ohtake
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Takeshi Omasa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takeshi Omasa.

Additional information

M.O. and W-D.K. contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Onitsuka, M., Kim, WD., Ozaki, H. et al. Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells. Appl Microbiol Biotechnol 94, 69–80 (2012). https://doi.org/10.1007/s00253-011-3814-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-011-3814-1

Keywords

Search

Navigation