单克隆抗体药物的高级结构与其安全性和有效性密切相关，是单抗类药物的关键质量属性。高场液相核磁共振波谱技术作为蛋白质精细高级结构的主要表征手段，目前在单抗类药物上的应用却十分有限，其中一个重要原因是单克隆抗体的核磁共振波谱信号指认难以获得。针对这个问题，本项目拟开发一种借助质谱技术的新方法对单抗的核磁共振波谱信号进行指认的方法，并且在此基础上进一步开发利用核磁共振对单克隆抗体药物高级结构表征以及质控的方法。目前我们已成功地对自然丰度的蛋清溶菌酶和胃蛋白酶的部分核磁共振波谱信号进行了指认，验证了该方法的可行性。本项目的成功实施将有效提高液相核磁共振波谱技术在单克隆抗体药物及其他蛋白质药物中的应用。

The higher order structure (HOS) is one of the critical quality attributes (CQA) of therapeutic monoclonal antibodies (mAbs) because it is related to the safety, efficacy and pharmacokinetic properties of mAbs. As one of the major techniques in structural biology, nuclear magnetic resonance (NMR) offers unique advantage of high resolution to study HOS of proteins. However, its application on mAbs is mitigated by the difficulty in NMR resonance assignment as the isotopically labeled mAbs are not always available. Here we propose an unique NMR assignment strategy for mAbs using the natural-abundance samples. The strategy involves selective modification of the target amino acid and quantification of the modification rate by both NMR and MS. The NMR assignment of target amino acid is made by correlating the quantitative results obtained from both NMR and MS. Eventually we will establish an NMR platform to study the HOS of mAbs with site-specific information. The feasibility of this proposal has been demonstrated on tnatural-abundance proteins such as hen egg white lysozyme and porcine pepsin. We believe the success of this program will open a new avenue to structural characterization and quality control of therapeutic mAbs and other biotherapeutic molecules.