title: Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments.
abstract: A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). 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. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. 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 FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.
authors.affiliation: a Graduate School of Engineering, Osaka University , Yamadaoka, Suita , Osaka , Japan.|b U-Medico Inc. , Yamadaoka, Suita , Osaka , Japan.
authors.forename: Elena
authors.initials: E
authors.lastname: Krayukhina
authors.affiliation: a Graduate School of Engineering, Osaka University , Yamadaoka, Suita , Osaka , Japan.|b U-Medico Inc. , Yamadaoka, Suita , Osaka , Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Noda
authors.affiliation: c Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences , Higashiyama, Myodaiji, Okazaki , Aichi , Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Ishii
authors.affiliation: a Graduate School of Engineering, Osaka University , Yamadaoka, Suita , Osaka , Japan.|b U-Medico Inc. , Yamadaoka, Suita , Osaka , Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Maruno
authors.affiliation: a Graduate School of Engineering, Osaka University , Yamadaoka, Suita , Osaka , Japan.
authors.forename: Hirotsugu
authors.initials: H
authors.lastname: Wakabayashi
authors.affiliation: d Division of Biological Chemistry and Biologicals , National Institute of Health Sciences , Kamiyoga, Setagaya-ku , Tokyo , Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Tada
authors.affiliation: d Division of Biological Chemistry and Biologicals , National Institute of Health Sciences , Kamiyoga, Setagaya-ku , Tokyo , Japan.
authors.forename: Takuo
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: d Division of Biological Chemistry and Biologicals , National Institute of Health Sciences , Kamiyoga, Setagaya-ku , Tokyo , Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishii-Watabe
authors.affiliation: e Sysmex Corporation , Murotani, Nishi-ku, Kobe-shi , Hyogo , Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kato
authors.affiliation: a Graduate School of Engineering, Osaka University , Yamadaoka, Suita , Osaka , Japan.|b U-Medico Inc. , Yamadaoka, Suita , Osaka , Japan.|c Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences , Higashiyama, Myodaiji, Okazaki , Aichi , Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
mesh_terms: D000068879:Adalimumab / Q000032:analysis* / Q000737:chemistry; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D000069285:Infliximab / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000737:chemistry; D014461:Ultracentrifugation
chemical_list: D000936:Antigen-Antibody Complex; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D000069285:Infliximab; D000068879:Adalimumab
keywords: Adalimumab; FcγR cell-reporter assay; TNF; analytical ultracentrifugation with fluorescence detection; etanercept; immune complex; immunogenicity; infliximab; native mass spectrometry; size distribution