Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action

Research output: Contribution to journalArticle

  • External authors:
  • Rosie Upton
  • Leonard Bell
  • Colin Guy
  • Paul Caldwell
  • Sian Estdale
  • David Firth


In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

Bibliographical metadata

Original languageEnglish
Pages (from-to)10259-10265
Number of pages7
JournalAnalytical Chemistry
Issue number20
Early online date13 Sep 2016
StatePublished - 18 Oct 2016