Development of fluorescence detection analytical ultracentrifugation

Research Project

Project/Area Number	15K14457
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Structural biochemistry
Research Institution	Osaka University
Principal Investigator	Susumu Uchiyama 大阪大学, 工学研究科, 准教授 (90335381)
Project Period (FY)	2015-04-01 – 2017-03-31
Project Status	Completed (Fiscal Year 2016)
Budget Amount *help	¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000) Fiscal Year 2016: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000) Fiscal Year 2015: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Keywords	抗体 / 複合体 / 蛋白質医薬 / 免疫原性 / 超遠心分析 / 質量分析 / 免疫複合体 / TNF / 抗体医薬 / バイオ医薬品 / 定量評価 / 蛍光ラベル化 / 蛍光超遠心
Outline of Final Research Achievements	We characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum. Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcgRIIa- and FcgRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab.