Makey Devin M, Ruotolo Brandon T, Kennedy Robert T
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, United States.
Anal Chem. 2025 Aug 12;97(31):16742-16750. doi: 10.1021/acs.analchem.5c00741. Epub 2025 Aug 1.
Identification and quantification of critical quality attributes (CQAs) such as sequence variants and post-translational modifications (PTMs) at the residue level are essential for ensuring the safety and efficacy of monoclonal antibody (mAb) therapeutics. Peptide mapping using liquid chromatography coupled to mass spectrometry (LC-MS) allows for the simultaneous monitoring of multiple CQAs, but conventional methods often suffer from low throughput, limiting their utility in applications requiring fast analysis. Here, we present a multidimensional high-throughput peptide mapping workflow that combines fast LC with cyclic ion mobility-mass spectrometry (LC-cIM-MS) for the high-resolution analysis of mAb tryptic digests. A 15-s LC gradient coupled to single-pass cIM-MS achieved a peak capacity of 490 and allowed for a 96-well plate to be analyzed in 37 min, including time for column re-equilibration and autosampler needle washing between each injection. The method yielded a 97% sequence coverage. Repeatability assessments demonstrated robust retention time, arrival time, and peak intensity reproducibility, and a linear dynamic range was observed across nearly 2 orders of magnitude. Mobility-aligned collision-induced dissociation was used to unambiguously localize PTM sites. The fast LC-cIM-MS platform provided site-specific tracking of the oxidation, deamidation, and isomerization kinetics during forced degradation studies. The method offers an approach to assessing CQAs in high-throughput analysis applications such as stability studies, formulation screening, and process monitoring and has the potential to accelerate mAb development and manufacturing. These results also demonstrate the potential of fast multidimensional separations for complex sample analysis.
在残基水平上识别和量化关键质量属性（CQA），如序列变异和翻译后修饰（PTM），对于确保单克隆抗体（mAb）治疗药物的安全性和有效性至关重要。使用液相色谱-质谱联用（LC-MS）的肽图分析能够同时监测多个CQA，但传统方法往往通量较低，限制了它们在需要快速分析的应用中的实用性。在此，我们提出了一种多维高通量肽图分析工作流程，该流程将快速液相色谱与循环离子淌度-质谱联用（LC-cIM-MS）相结合，用于对mAb胰蛋白酶消化产物进行高分辨率分析。一个15秒的液相色谱梯度与单通道cIM-MS联用实现了490的峰容量，并能在37分钟内分析一个96孔板，包括每次进样之间的柱重新平衡和自动进样器针清洗时间。该方法的序列覆盖率达到97%。重复性评估表明保留时间、到达时间和峰强度具有良好的重现性，并且在近2个数量级内观察到线性动态范围。利用淌度对齐的碰撞诱导解离明确地定位PTM位点。快速LC-cIM-MS平台在强制降解研究期间提供了对氧化、脱酰胺和异构化动力学的位点特异性跟踪。该方法为在稳定性研究、制剂筛选和过程监测等高通量分析应用中评估CQA提供了一种途径，并且有潜力加速mAb的开发和生产。这些结果还证明了快速多维分离在复杂样品分析中的潜力。
