Evaluation of the Aggregation States of Monoclonal Antibodies by Diverse and Complementary Methods
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概要
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Therapeutic monoclonal antibodies (MAbs) with high specificity and fewer adverse effects are becoming widely used for the treatment of various diseases. MAbs need to be stored and administered at high concentrations in solution, the conditions under which MAbs may aggregate. As aggregated MAbs compromise their safety and efficacy, aggregation should be prevented; thus, it is important to analyze the aggregation states of MAbs in detail. We obtained 2 MAbs against dinitrophenol (DNP) that exhibited different aggregation properties: anti-DNP1 exhibited a much higher aggregate content (dimer or trimer) than anti-DNP2 when analyzed by size-exclusion chromatography (SEC). As anti-DNP1 had a longer complementarity-determining region 3 (CDR3) light chain than anti-DNP2 by 2 amino acid residues, we hypothesized that the increased aggregation of DNP1 was due to these extra residues; therefore, we prepared mutant antibodies with shorter CDR3s to compare their aggregation properties. Anti-DNP1-ΔEI, with the same CDR3 length as anti-DNP2, exhibited no aggregates as expected. Anti-DNP1-ΔI, with 1 additional residue, exhibited a smaller peak than wild-type (WT) in SEC, whereas this mutant exhibited stronger thioflavin T fluorescence than WT, which is indicative of amyloid formation. In addition, the anti-DNP1-ΔI solution (but not others) became opalescent at 4°C and exhibited large visible particles, which are undetectable by SEC. The fragment antigen-binding region of this mutant was found to have lower thermal stability than the others by differential scanning calorimetry. These data suggest that diverse analytical methods should be applied to evaluate MAb aggregation, in addition to the commonly used SEC.
著者
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Wakamatsu Kaori
Department Of Biochemical And Chemical Engineering Gunma University
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Ishikawa Tomoyoshi
Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd.
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Yoshino Tetsuya
Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd.
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Ishihara Takashi
Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd.
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Takeuchi Yoshimi
Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd.
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Yoshikawa Hitomi
Antibody Research Laboratories, Kyowa Hakko Kirin Co., Ltd.
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Yoshida Hideaki
Antibody Research Laboratories, Kyowa Hakko Kirin Co., Ltd.
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Yoshida Hitoshi
Innovative Drug Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd.
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Wakamatsu Kaori
Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University
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