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Highly sensitive glycosylamine labelling of O-glycans using non-reductive β-elimination

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Abstract

When developing biopharmaceuticals, glycans are the most important posttranslational protein modifications that must be addressed because they affect the between-protein interactions that maintain homeostasis. The glycan profile may be defined as a critical quality attribute of a biopharmaceutical. Comprehensive analysis of protein glycosylation must overcome challenges such as the release, labelling, separation and detection of O-glycans. In contrast, N-glycans can be readily released non-reductively from peptide backbones using an enzyme such as peptide N-glycosidase F. We developed a highly sensitive protocol using RapiFluor-MS to label glycosylamines for O-glycan analysis combined with a non-enzyme treatment for efficient release of the reduced O-glycans from the glycoproteins. Here we used the cytotoxic T lymphocyte associated protein 4-immunoglobulin G (Ig) fusion protein and fetuin as models for O-glycan analysis and compared the analytical methods glycopeptide mapping, 2-AB labelling and RapiFluor-MS labelling. The structures of major O-glycans and low-abundance O-glycans were successfully identified using the third technique, which detected the O-glycans with high sensitivity.

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Abbreviations

2-AB:

2-Aminobenzamide

CQA:

Critical quality attribute

CTLA4-Ig:

The cytotoxic T lymphocyte associated protein 4-immunoglobulin G fusion protein

DMF:

Dimethylformamide

HILIC:

Hydrophilic interaction liquid chromatography

MS:

Mass spectrometry

NMR:

Nuclear magnetic resonance

PNGaseF:

Peptide N-glycosidase F

SPE:

Solid-phase extraction

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Authors and Affiliations

  1. Process Lab II, Biotechnology Labs, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki, 300-2698, Japan

    Kenichiro Furuki

  2. Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan

    Kenichiro Furuki & Toshimasa Toyo’oka

  3. Biotechnology Labs, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki, 300-2698, Japan

    Kazutoshi Ban

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  1. Kenichiro Furuki
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  2. Toshimasa Toyo’oka
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  3. Kazutoshi Ban
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Furuki, K., Toyo’oka, T. & Ban, K. Highly sensitive glycosylamine labelling of O-glycans using non-reductive β-elimination. Anal Bioanal Chem 409, 2269–2283 (2017). https://doi.org/10.1007/s00216-016-0171-z

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