Characterization of antibody products obtained through enzymatic and nonenzymatic glycosylation reactions with a glycan oxazoline and preparation of a 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

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Glycan engineering of antibodies has received considerable attention. Although various endo-β-N-acetylglucosaminidase mutants have been developed for glycan remodeling, a 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 nonenzymatic reactions with the aim of maximizing the efficiency of the glycosylation reaction with fewer side products. The reactions were monitored by an ultraperformance 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 a combination of an azide-carrying glycan transfer reaction under optimized conditions and a bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with a drug-to-antibody ratio of 4. The prepared antibody-drug conjugate exhibited cytotoxicity against HER2-expressing cells.

Original languageEnglish
Pages (from-to)1343-1355
Number of pages13
JournalBioconjugate chemistry
Volume30
Issue number5
DOIs
Publication statusPublished - 2019 May 15
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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