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.

Global N-Glycan Site Occupancy of HIV-1 gp120 by Metabolic Engineering and High-Resolution Intact Mass Spectrometry

“…High-resolution MS has been applied to glycoproteins with only one 30 or two glycan sites 31, 32 , but not to highly glycosylated proteins due to overlapping glycoforms.…”

“…Native high-resolution mass spectrometry holds enormous promise for glycoprotein characterization, but has only been demonstrated on relatively simple systems and has failed for more complex targets 30–32 . We have chosen one of the most heavily glycosylated glycoproteins in nature which is currently under investigation for clinical use 37 .…”

GDCh Innovation Prize in Medicinal/Pharmaceutical Chemistry: D. Schade and A. Koeberle / Feodor Lynen Lectureship: D. Hilvert / Roy L. Whistler Award: B. G. Davis

“…Benjamin G. Davis (University of Oxford) has been announced as the winner of the Roy L. Whistler International Award in Carbohydrate Chemistry 2016, which is presented by the International Carbohydrate Organization. Davis, who was featured here when he was elected a Fellow of the Royal Society, has recently reported in Angewandte Chemie on the glycosylation of a therapeutic antibody . Davis is on the Editorial Advisory Board of ChemBioChem .…”

Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody–drug conjugates

“…Since the original method was published, we and other scientists have made substantial improvements, including the development of one-pot chemoenzymatic procedures 26 , use of new endoglycosidases 25 and improved fucosidases 23,25 , identification of various N -glycan substrates 23,26 , the introduction of different types of modification chemistry 24,37,38 , and an optimized procedure for N -glycan extraction 37 , to broaden the adaptation of this method. We report here the updated protocol describing all required steps of this approach.…”