Structural Fingerprints of an Intact Monoclonal Antibody Acquired under Formulated Storage Conditions via 15N Direct Detection Nuclear Magnetic Resonance
Structural Fingerprints of an Intact Monoclonal Antibody Acquired under Formulated Storage Conditions via 15N Direct Detection Nuclear Magnetic Resonance
复制标题
在配制储存条件下通过 15N 直接检测核磁共振获得完整单克隆抗体的结构指纹
DOI：
10.1021/acs.jmedchem.0c00231
复制
发表时间：
2020
影响因子：
7.3
通讯作者：
Shimada Ichio
中科院分区：
文献类型：
--
作者：
Tokunaga Yuji;Takeuchi Koh;Okude Junya;Ori Kazutomo;Torizawa Takuya;Shimada Ichio
三级结构的非侵入性评价是生物制品研究、开发和使用的基础。然而，目前很少有方法可用于评估大分子量(MW)生物制品，如治疗性单抗(mAbs；150 kDa)。在这里，我们新开发了一种15N直接检测核磁共振(NMR)技术，即15N直接检测CRINEPT，它可以观察到分子量为150 kDa的非氚蛋白质的主链酰胺共振。该技术不仅大大扩展了适用于溶液核磁共振研究的蛋白质范围，而且还允许在广泛的温度和溶剂条件下对完整的mAbs进行非侵入性的结构分析。作为原理的证明，我们成功地获得了完整单抗在4°C的配方溶液中的15N检测CRINEPT谱。该技术能够区分非均相半乳糖化状态，证明了15N直接检测的高分辨率的好处。
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 a15N direct detection nuclear magnetic resonance (NMR) technique, the15N 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 the15N-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 the15N direct detection.