Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column

Masato Kiyoshi, Jose M.M. Caaveiro, Minoru Tada, Hiroko Tamura, Toru Tanaka, Yosuke Terao, Koldo Morante, Akira Harazono, Noritaka Hashii, Hiroko Shibata, Daisuke Kuroda, Satoru Nagatoishi, Seigo Oe, Teruhiko Ide, Kouhei Tsumoto, Akiko Ishii-Watabe

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Abstract

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

Original languageEnglish
Article number3955
JournalScientific Reports
Volume8
Issue number1
DOIs
Publication statusPublished - Dec 1 2018

All Science Journal Classification (ASJC) codes

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    Kiyoshi, M., Caaveiro, J. M. M., Tada, M., Tamura, H., Tanaka, T., Terao, Y., Morante, K., Harazono, A., Hashii, N., Shibata, H., Kuroda, D., Nagatoishi, S., Oe, S., Ide, T., Tsumoto, K., & Ishii-Watabe, A. (2018). Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column. Scientific Reports, 8(1), [3955]. https://doi.org/10.1038/s41598-018-22199-8