Molecular Profiling Research Center for Drug Discovery and Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan.
Research and Development Department, Japan Biological Informatics Consortium, 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan.
J Med Chem. 2020 May 28;63(10):5360-5366. doi: 10.1021/acs.jmedchem.0c00231. Epub 2020 May 6.
Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a N direct detection nuclear magnetic resonance (NMR) technique, the N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the N direct detection.
对三级结构的非侵入性评估是生物制剂的研究、开发和应用的基础。然而，目前几乎没有方法可用于评估像治疗性单克隆抗体（mAb；150 kDa）这样的大分子量生物制剂。在这里，我们新开发了一种 N 直接检测核磁共振（NMR）技术，即 N 直接检测 CRINEPT，它允许观察非氘代蛋白的主链酰胺共振，该蛋白的分子量为 150 kDa。该技术不仅大大扩展了适用于溶液 NMR 研究的蛋白质范围，而且还允许在广泛的温度和溶剂条件下对完整的 mAb 进行非侵入性结构分析。作为原理的证明，我们成功地在 4°C 的配方溶液中获得了完整 mAb 的 N 检测 CRINEPT 谱。该技术能够区分异构半乳糖化状态，显示出 N 直接检测的高分辨率的优势。
