Mapping Monoclonal Antibody Structure by 2D 13C NMR at Natural Abundance
Mapping Monoclonal Antibody Structure by 2D 13C NMR at Natural Abundance
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DOI：
10.1021/ac504804m
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发表时间：
2015-04-07
影响因子：
7.4
通讯作者：
Marino, John P.
中科院分区：
文献类型：
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作者：
Arbogast, Luke W.;Brinson, Robert G.;Marino, John P.
单抗代表着一类重要且迅速增长的生物治疗药物。MAb的正确折叠对药物疗效至关重要，而错误折叠会引发不想要的免疫反应或其他非靶标反应，从而影响安全性。因此，需要稳健的方法来精确测量单抗结构，以进行药物质量评估和可比性。到目前为止，该领域的看法是，由于分子的大小(约150 kDa)和复杂性，以及方法的不敏感性，核磁共振不能实际应用于mAbs。在大肠杆菌和中国仓鼠卵巢(CHO)细胞表达平台上，已经证明了将核磁共振方法应用于稳定的同位素标记的、被蛋白酶切割的单抗结构域(Fab和Fc)的可行性；然而，在分析药物产品时，同位素标记通常是不可用的。在这里，我们讨论了基于核磁共振的mAb结构作图的可行性问题，首次展示了2DC-13NMR甲基指纹方法在天然同位素丰度下完整mAb结构作图中的应用。此外，我们还证明了蛋白酶裂解的Fc和Fab片段的2D C-13核磁共振谱可以提供准确的结构报告，这些结构可以直接映射到完整的mAb上。通过结合使用快速采集和非均匀采样技术，我们证明了这些FAB和Fc指纹图谱可以在短短30分钟内快速获取。
Monoclonal antibodies (mAbs) represent an important and rapidly growing class of biotherapeutics. Correct folding of a mAb is critical for drug efficacy, while misfolding can impact safety by eliciting unwanted immune or other off-target responses. Robust methods are therefore needed for the precise measurement of mAb structure for drug quality assessment and comparability. To date, the perception in the field has been that NMR could not be applied practically to mAbs due to the size (similar to 150 kDa) and complexity of these molecules, as well as the insensitivity of the method. The feasibility of applying NMR methods to stable isotope-labeled, protease-cleaved, mAb domains (Fab and Fc) has been demonstrated from both E. coli and Chinese hamster ovaries (CHO) cell expression platforms; however, isotopic labeling is not typically available when analyzing drug products. Here, we address the issue of feasibility of NMR-based mapping of mAb structure by demonstrating for the first time the application of a 2D C-13 NMR methyl fingerprint method for structural mapping of an intact mAb at natural isotopic abundance. Further, we show that 2D C-13 NMR spectra of protease-cleaved Fc and Fab fragments can provide accurate reporters on the domain structures that can be mapped directly to the intact mAb. Through combined use of rapid acquisition and nonuniform sampling techniques, we show that these Fab and Fc fingerprint spectra can be rapidly acquired in as short as approximately 30 min.