Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.
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.
Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass Spectrometry, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Rosie
R
Upton
Covance Laboratories Ltd. , Otley Road, Harrogate, HG3 1PY, United Kingdom.
Leonard
L
Bell
Covance Laboratories Ltd. , Otley Road, Harrogate, HG3 1PY, United Kingdom.
Colin
C
Guy
Covance Laboratories Ltd. , Otley Road, Harrogate, HG3 1PY, United Kingdom.
Paul
P
Caldwell
Covance Laboratories Ltd. , Otley Road, Harrogate, HG3 1PY, United Kingdom.
Sian
S
Estdale
Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass Spectrometry, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
Perdita E
PE
Barran
Covance Laboratories Ltd. , Otley Road, Harrogate, HG3 1PY, United Kingdom.
David
D
Firth
D001426:Bacterial Proteins / Q000737:chemistry; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D003546:Cysteine Endopeptidases / Q000737:chemistry; D005643:Fucose / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D011134:Polysaccharides / Q000032:analysis* / Q000145:classification; D011485:Protein Binding; D011786:Quality Control; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D000068878:Trastuzumab / Q000737:chemistry* / Q000145:classification / Q000378:metabolism / Q000494:pharmacology*
D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D005643:Fucose; D006026:Glycoside Hydrolases; D008358:Mannose; D011134:Polysaccharides; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG; D000068878:Trastuzumab; C442815:Mac-1-like protein, Streptococcus