Effects of subclass change on the structural stability of chimeric, humanized, and human antibodies under thermal stress

Authors

  • Takahiko Ito,

    1. Bio Process Research and Development Laboratories, Production Division, Takasaki, Gunma, Japan
    2. Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
    3. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, , The University of Tokyo, Kashiwa, Japan
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  • Kouhei Tsumoto

    Corresponding author
    1. Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan
    2. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, , The University of Tokyo, Kashiwa, Japan
    3. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan
    4. Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan
    • Correspondence to: Kouhei Tsumoto, Institute of Medical Science, The University of Tokyo, 4–6-1 Shirokanedai, Minato-ku, Tokyo 108–8639, Japan. E-mail: tsumoto@ims.u-tokyo.ac.jp

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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 Th 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.

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