Chemoenzymatic Glyco-engineering of Monoclonal Antibodies

  • John P. Giddens
  • Lai-Xi Wang
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)


Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications.

Key words

Monoclonal antibody Fc glycosylation Glyco-engineering Chemoenzymatic synthesis Transglycosylation ADCC 



This work was supported by the National Institutes of Health (NIH grant R01GM096973).


  1. 1.
    Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23:1147–1157PubMedCrossRefGoogle Scholar
  2. 2.
    Hogarth PM, Pietersz GA (2012) Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond. Nat Rev Drug Discov 11:311–331PubMedCrossRefGoogle Scholar
  3. 3.
    Jeffery R (2005) Glycosylation of recombinant antibody therapeutics. Biotechnol Prog 21:11–16CrossRefGoogle Scholar
  4. 4.
    Jeffery R (2009) Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 8:226–234CrossRefGoogle Scholar
  5. 5.
    Kaneko Y, Nimmerjahn F, Ravetch JV (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313:670–673PubMedCrossRefGoogle Scholar
  6. 6.
    Anthony RM, Nimmerjahn F, Ashline DJ et al (2008) Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320:373–376PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Karsten CM, Pandey MK, Figge J et al (2012) Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcgammaRIIB and dectin-1. Nat Med 18:1401–1406PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Frenzel A, Hust M, Schirrmann T (2013) Expression of recombinant antibodies. Front Immunol 4:217PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Huang W, Giddens J, Fan SQ et al (2012) Chemoenzymatic glyco-engineering of intact IgG antibodies for gain of functions. J Am Chem Soc 134:12308–12318PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Zou G, Ochiai H, Huang W et al (2011) Chemoenzymatic synthesis and Fcgamma receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcgammaIIIa receptor. J Am Chem Soc 133:18975–18991PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Wei Y, Li C, Huang W et al (2008) Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation. Biochemistry 47:10294–10304PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Collin M, Olsen A (2001) EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. EMBO J 20:3046–3055PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Wang LX, Amin MN (2014) Chemical and chemoenzymatic synthesis of glycoproteins for deciphering functions. Chem Biol 21:51–66PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Collin M, Svensson MD, Sjoholm AG et al (2002) EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis. Infect Immun 70:6646–6651PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Seko A, Koketsu M, Nishizono M et al (1997) Occurrence of a sialylglycopeptide and free sialylglycans in hen’s egg yolk. Biochim Biophys Acta 1335:23–32PubMedCrossRefGoogle Scholar
  16. 16.
    Umekawa M, Huang W, Li B et al (2008) Mutants of Mucor hiemalis endo-beta-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J Biol Chem 283:4469–4479PubMedCrossRefGoogle Scholar
  17. 17.
    Noguchi M, Tanaka T, Gyakushi H et al (2009) Efficient synthesis of sugar oxazolines from unprotected N-acetyl-2-amino sugars by using chloroformamidinium reagent in water. J Org Chem 74:2210–2212PubMedCrossRefGoogle Scholar
  18. 18.
    Huang W, Yang Q, Umekawa M et al (2010) Arthrobacter endo-beta-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C. Chem Bio Chem 11:1350–1355PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • John P. Giddens
    • 1
  • Lai-Xi Wang
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of MarylandCollege ParkUSA

Personalised recommendations