title: Optimal Synthetic Glycosylation of a Therapeutic Antibody.
abstract: Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Parsons
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Weston B
authors.initials: WB
authors.lastname: Struwe
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Joseph
authors.initials: J
authors.lastname: Gault
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Taylor
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Ritu
authors.initials: R
authors.lastname: Raj
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.|Department of Pharmacology, University of Oxford, Oxford, OX1 3QT, UK.
authors.forename: Kim
authors.initials: K
authors.lastname: Wals
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK. carol.robinson@chem.ox.ac.uk.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Justin L P
authors.initials: JL
authors.lastname: Benesch
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, UK. ben.davis@chem.ox.ac.uk.
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Davis
mesh_terms: D000906:Antibodies / Q000378:metabolism* / Q000627:therapeutic use; D006031:Glycosylation; D000068878:Trastuzumab / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D000906:Antibodies; D000068878:Trastuzumab
keywords: antibodies; endoglycosidases; glycoengineering; glycosylation; native mass spectrometry