Cassandra L. Pegg1,2, Lucia F. Zacchi1, Ruby Pelingon1, Christopher Howard1, Stephen Mahler1 Benjamin L. Schulz1,2*


1Centre for Biopharmaceutical Innovation at the Australian Institute for Bioengineering and Nanotechnology and 2The School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, QLD, Australia, 4072


Glycosylation is an essential regulatory mechanism of protein function and is associated with a range of biological processes. The impact of glycosylation has been highlighted in pharmaceutical and biotech industries where glycans have been shown to affect the stability, solubility, bioactivity and pharmacokinetic properties of protein therapeutics  1-4. More than two-thirds of marketed protein therapeutics are glycosylated5 and several different approaches have been taken to modify glycan-driven properties, in particular, receptor-mediated serum clearance of theraputics6. These approaches include manipulation of the composition or number of glycan structures or enrichment of specific glycoforms to improve in-vivo bioactivity and pharmacokinetic properties  1,4,7. With this focus on glycoengineering it remains important to have robust analytical methods to characterise and measure glycosylation patterns. Glycan heterogeneity and the potential substoichiometric presence of glycoforms make characterising protein glycosylation a bioanalytical challenge. This challenge can be met by mass spectrometry (MS) and we will show how site-specific glycan heterogeneity can be assessed after digestion of therapeutics with proteolytic enzymes. The resultant glycopeptides (peptide + attached glycan) preserve information about site occupancy, the monosaccharide composition of the glycan and the site of glycosylation, all of which can be elucidated after tandem MS. This workflow enables comprehensive characterisation of protein glycosylation and can be used to investigate glycosylation analogs for the rational design of protein therapeutics.




1  Sinclair, A. M. & Elliott, S. J. Pharm. Sci. 2005, 94, 1626-1635, Glycoengineering: The effect of glycosylation on the properties of therapeutic proteins.

2  Schiestl, M. et al. Nat. Biotechnol. 2011, 29, 310-312, Acceptable changes in quality attributes of glycosylated biopharmaceuticals.

3  Higel, F., Seidl, A., Sorgel, F. & Friess, W. Eur. J. Pharm. Biopharm. 2016, 100, 94-100, N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins.

4  Elliott, S. et al. Nat. Biotechnol. 2003, 21, 414-421, Enhancement of therapeutic protein in vivo activities through glycoengineering.

5  Li, H. & d’Anjou, M. Curr. Opin. Biotechnol. 2009, 20, 678-684, Pharmacological significance of glycosylation in therapeutic proteins.

6  Kontermann, R. E. Curr. Opin. Biotechnol. 2011, 22, 868-876, Strategies for extended serum half-life of protein therapeutics.

7  Liu, L. et al. Pharm. Res. 2013, 30, 803-812, The impact of glycosylation on the pharmacokinetics of a TNFR2:Fc fusion protein expressed in Glycoengineered Pichia Pastoris.


Biographic Details

Name: Cassandra Pegg

Title: Ms

Affiliation, Country: Australia

Phone: +61 7 336 54614 E-mail:

Research interests: glycobiology, glycoproteomics, mass spectrometry


AEB 301