title: Effects of subclass change on the structural stability of chimeric, humanized, and human antibodies under thermal stress.
abstract: To address how changes in the subclass of antibody molecules affect their thermodynamic stability, we prepared three types of four monoclonal antibody molecules (chimeric, humanized, and human) and analyzed their structural stability under thermal stress by using size-exclusion chromatography, differential scanning calorimetry (DSC), circular dichroism (CD), and differential scanning fluoroscopy (DSF) with SYPRO Orange as a dye probe. All four molecules showed the same trend in change of structural stability; the order of the total amount of aggregates was IgG1 < IgG2 < IgG4. We thus successfully cross-validated the effects of subclass change on the structural stability of antibodies under thermal stress by using four methods. The T(h) values obtained with DSF were well correlated with the onset temperatures obtained with DSC and CD, suggesting that structural perturbation of the CH2 region could be monitored by using DSF. Our results suggested that variable domains dominated changes in structural stability and that the physicochemical properties of the constant regions of IgG were not altered, regardless of the variable regions fused.
authors: Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Company Limited, 100-1 Hagiwara-machi, Takasaki, Gunma, 370-0013, Japan; Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan; Department of Medical Genome Sciences, Graduate School of Frontier Sciences, , The University of Tokyo, Kashiwa, 277-8562, Japan.
authors: Takahiko
authors: T
authors: Ito
authors: Kouhei
authors: K
authors: Tsumoto
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000276:immunology; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003412:Cricetulus; D005456:Fluorescent Dyes; D005471:Fluoroscopy; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology*; D015203:Reproducibility of Results; D013312:Stress, Physiological; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins
keywords: antibody; circular dichroism; differential scanning calorimetry; differential scanning fluoroscopy; subclass change; thermodynamic stability
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