Kiyoshi Masato, Oyama Taiji, Shibata Hiroko, Suzuki Satoko, Higuchi Yuji, Tsumoto Kouhei, Ishii-Watabe Akiko
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan.
JASCO Corporation, 2967-5 Ishikawamachi, Hachioji, Tokyo 192-8537, Japan.
Anal Chem. 2025 Jun 24;97(24):12578-12586. doi: 10.1021/acs.analchem.5c00718. Epub 2025 Jun 10.
The higher-order structure (HOS) of therapeutic monoclonal antibodies is one of the important quality characteristics in terms of efficacy and safety. However, the HOS of therapeutic monoclonal antibodies has been assessed using low- to medium-resolution spectroscopy. Furthermore, because the analysis of the obtained spectra has been performed by visual evaluation in many cases, their specificity, structural similarity, and statistical significance have not been thoroughly discussed. Therefore, there is an increasing need for advanced, statistically comparable, and highly specific analytical methods for the HOS of therapeutic monoclonal antibodies. Herein, we describe an analytical method for the HOS of therapeutic monoclonal antibodies using a powerful combination of near-UV circular dichroism (CD) and statistical analysis. The obtained near-UV CD spectra of 14 therapeutic monoclonal antibodies were employed for principal component analysis. Moreover, the spectral data were converted into Euclidean distances to perform equivalence tests and significance tests. The results clearly demonstrated that each antibody had a unique near-UV CD spectrum, like a structural fingerprint. All antibodies were judged to be not equivalent to other antibodies and were also judged to be significantly different from other antibodies. Moreover, the equivalence tests were performed on several lots of antibodies, and each lot of the antibodies were judged to be equivalent. We believe that our methods are useful for identity testing and also for comparative analysis of HOS of therapeutic monoclonal antibodies.
治疗性单克隆抗体的高阶结构（HOS）是影响疗效和安全性的重要质量特征之一。然而，治疗性单克隆抗体的高阶结构一直是通过低至中分辨率光谱进行评估的。此外，由于在许多情况下对所得光谱的分析是通过目视评估进行的，其特异性、结构相似性和统计意义尚未得到充分讨论。因此，对于治疗性单克隆抗体高阶结构的先进、具有统计可比性且高度特异的分析方法的需求日益增加。在此，我们描述了一种使用近紫外圆二色性（CD）和统计分析的强大组合来分析治疗性单克隆抗体高阶结构的方法。对14种治疗性单克隆抗体获得的近紫外CD光谱进行主成分分析。此外，将光谱数据转换为欧几里得距离以进行等效性检验和显著性检验。结果清楚地表明，每种抗体都有独特的近紫外CD光谱，就像结构指纹一样。所有抗体均被判定与其他抗体不等效，并且也被判定与其他抗体有显著差异。此外，对多个批次的抗体进行了等效性检验，并且判定每批抗体都是等效的。我们认为我们的方法对于鉴别测试以及治疗性单克隆抗体高阶结构的比较分析很有用。
