Division of Complex Drug Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, United States.
Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, St. Louis, Missouri 63110, United States.
Mol Pharm. 2021 Jan 4;18(1):441-450. doi: 10.1021/acs.molpharmaceut.0c01083. Epub 2020 Dec 11.
The N-glycosylation pattern of Asn-297 may have impacts on monoclonal antibody (mAb) drug plasma clearance, antibody-dependent cell mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Notably, the changes in the relative abundance of certain minor glycans, like the afucosylation, high-mannose, or galactosylation are known to change mAb properties and functions. Here, a middle-down NMR spectroscopy based analytical procedure was applied to assess the composition and structure of glycans on adalimumab and trastuzumab without glycan cleavage from the mAbs. The anomeric 2D H-C spectra showed distinct patterns that could be used to profile and differentiate mAb glycan compositions. Specifically, the anomeric C1/H1 resonances from N-acetylglucosamine (GlcNAc2 and -5) and mannose (Man4) were identified as characteristic peaks for key glycan anomeric linkages and branching states. They were also utilized for measuring the relative abundance of minor glycans of total afucosylation (aFuc%), high mannose (HM%), and branch specific galactosylation (Gal% and Gal%). The obtained total aFuc% value of 11-12% was similar between the two mAbs; however, trastuzumab had significantly lower level of high mannose and a higher level of galactosylation than adalimumab. Overall, the 2D-NMR measurements provided functionally relevant mAb glycan composition and structure information. The method was deemed fit-for-purpose for assessment of these mAb quality attributes and involved fewer chemical preparation steps than the classical approaches that cleave glycans prior to making measurements.
天冬酰胺 297 的 N-糖基化模式可能对单克隆抗体（mAb）药物血浆清除率、抗体依赖性细胞介导的细胞毒性（ADCC）和补体依赖性细胞毒性（CDC）产生影响。值得注意的是，某些次要聚糖（如去岩藻糖基化、高甘露糖或半乳糖基化）的相对丰度的变化已知会改变 mAb 的性质和功能。在此，应用基于中下位 NMR 光谱的分析程序来评估阿达木单抗和曲妥珠单抗的聚糖组成和结构，而无需从 mAb 中裂解聚糖。糖基化 mAb 2D-NMR 图谱。具体而言，N-乙酰葡萄糖胺（GlcNAc2 和 -5）和甘露糖（Man4）的糖醛酸 2D H-C 谱显示出独特的图谱，可用于对 mAb 聚糖组成进行分类和区分。这些特征峰还可用于测量总去岩藻糖基化（aFuc%）、高甘露糖（HM%）和分支特异性半乳糖基化（Gal% 和 Gal%）的次要聚糖的相对丰度。这两种 mAb 的总 aFuc%值为 11-12%，相似；然而，与阿达木单抗相比，曲妥珠单抗的高甘露糖水平显著降低，半乳糖基化水平更高。总体而言，2D-NMR 测量提供了与功能相关的 mAb 聚糖组成和结构信息。该方法被认为适合评估这些 mAb 的质量属性，并且与在进行测量之前裂解聚糖的经典方法相比，涉及的化学制备步骤更少。
