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Characterization of antibody products obtained through enzymatic and non-enzymatic glycosylation reactions with a glycan oxazoline and preparation of homogeneous antibody-drug conjugate via Fc N-glycan

Shino Manabe, Yoshiki Yamaguchi, Kana Matsumoto, Hirobumi Fuchigami, Taiji Kawase, Kenji Hirose, Ai Mitani, Wataru Sumiyoshi, Takashi Kinoshita, Junpei Abe, Masahiro Yasunaga, Yasuhiro Matsumura, Yukishige Ito
Bioconjugate Chemistry 2019 April 2
Glycan engineering of antibody has received considerable attention. Although various endo-β-N-acetylglucosaminidase mutants have been developed for glycan remodeling, side-reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and non-enzymatic reactions with the aim of maximizing the efficiency of glycosylation reaction with fewer side products. The reactions were monitored by an ultra-performance liquid chromatography system using an amide-based wide pore column. The products were characterized by liquid chromatography coupled with tandem mass spectrometry. The side-reactions were suppressed by adding glycan oxazoline in a stepwise manner under slightly acidic conditions. Through combination of azide carrying glycan transfer reaction under optimized conditions and bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with drug-to-antibody ratio of 4. The prepared ADC exhibited cytotoxicity against Her2-expressing cells.


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