Pfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, 1 Burtt Road, Andover, MA, 01810, USA.
Pfizer, Inc. Pharmaceutical Sciences Small Molecules, Analytical Research and Development, Discovery Park, Ramsgate Road, Sandwich, CT13 9FF, UK.
Pharm Res. 2023 Oct;40(10):2457-2467. doi: 10.1007/s11095-023-03600-2. Epub 2023 Oct 5.
Nuclear magnetic resonance (NMR) spectroscopy provides the sensitivity and specificity to probe the higher order structure (HOS) of monoclonal antibodies (mAbs) for potential changes. This study demonstrates an application of chemometric tools to measure differences in the NMR spectra of mAbs after forced degradation relative to the respective unstressed starting materials.
Samples of adalimumab (Humira, ADL-REF) and trastuzumab (Herceptin, TRA-REF) were incubated in three buffer-pH conditions at 40°C for 4 weeks to compare to a control sample that was left unstressed. Replicate 1D H and 2D H-C HMQC NMR spectra were collected on all samples. Chemometric analyses such as Easy Comparability of HOS (ECHOS), PROtein FIngerprinting by Lineshape Enhancement (PROFILE), and Principal Component Analysis (PCA) were applied to capture and quantitate differences between the spectra.
Visual and statistical inspection of the 2D H-C HMQC spectra of adalimumab and trastuzumab after forced degradation conditions shows no changes in the spectra relative to the unstressed material. Chemometric analysis of the 1D H NMR spectra shows only minor changes in the spectra of adalimumab after forced degradation, but significant differences in trastuzumab.
The chemometric analyses support the lack of statistical differences in the structure of pH-thermal stressed adalimumab, however, it reveals conformational changes or chemical modifications in trastuzumab after forced degradation. Application of chemometrics in comparative NMR studies enables HOS characterization and showcases the sensitivity and specificity in detecting differences in the spectra of mAbs after pH-thermal forced degradation with respect to local and global protein structure.
核磁共振（NMR）光谱提供了灵敏度和特异性，可用于探测单克隆抗体（mAb）的高级结构（HOS）是否发生潜在变化。本研究展示了化学计量学工具在测量 mAb 在强制降解后的 NMR 光谱与相应未受应力起始材料之间的差异方面的应用。
阿达木单抗（Humira，ADL-REF）和曲妥珠单抗（Herceptin，TRA-REF）的样品在 40°C 的三种缓冲液-pH 条件下孵育 4 周，与未受应力的对照样品进行比较。对所有样品都收集了重复的一维 H 和二维 H-C HMQC NMR 光谱。应用 Easy Comparability of HOS（ECHOS）、PROtein FIngerprinting by Lineshape Enhancement（PROFILE）和主成分分析（PCA）等化学计量分析方法来捕捉和量化光谱之间的差异。
对阿达木单抗和曲妥珠单抗在强制降解条件下的二维 H-C HMQC 光谱进行目视和统计检查，发现相对于未受应力的材料，光谱没有变化。对一维 H NMR 光谱的化学计量分析显示，阿达木单抗在强制降解后光谱只有微小变化，但曲妥珠单抗则有明显差异。
化学计量分析支持 pH-热应激阿达木单抗结构无统计学差异，但揭示了强制降解后曲妥珠单抗的构象变化或化学修饰。化学计量学在比较 NMR 研究中的应用支持了 HOS 特征描述，并展示了在 pH-热强制降解后检测 mAb 光谱差异方面的灵敏度和特异性，无论是局部还是全局蛋白质结构。
