TY - JOUR
T1 - Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column
AU - Kiyoshi, Masato
AU - Caaveiro, Jose M.M.
AU - Tada, Minoru
AU - Tamura, Hiroko
AU - Tanaka, Toru
AU - Terao, Yosuke
AU - Morante, Koldo
AU - Harazono, Akira
AU - Hashii, Noritaka
AU - Shibata, Hiroko
AU - Kuroda, Daisuke
AU - Nagatoishi, Satoru
AU - Oe, Seigo
AU - Ide, Teruhiko
AU - Tsumoto, Kouhei
AU - Ishii-Watabe, Akiko
N1 - Funding Information:
This work was supported in part by the grant of ‘Research on Regulatory Science of Pharmaceuticals and Medical Devices’ from AMED (Japan agency for medical research and development). We also acknowledge funding from Grant-in-Aid for young scientists (B) 16K18973 (to M.K.), JSPS KAKENHI-A grants 25249115 (to K.T.) and by a JSPS KAKENHI-C grant 15K06962 (to J.M.M.C.). Access to beamline BL-5A was granted by the Photon Factory Advisory Committee (Proposals 2016G199).
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-018-22199-8
DO - 10.1038/s41598-018-22199-8
M3 - Article
C2 - 29500371
AN - SCOPUS:85042802181
SN - 2045-2322
VL - 8
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 3955
ER -