Pharma Technical Development, F. Hoffmann-La Roche, Grenzacherstrasse 124, 4070 Basel, Switzerland.
Anal Chem. 2022 Jun 14;94(23):8136-8145. doi: 10.1021/acs.analchem.1c04450. Epub 2022 May 11.
For the past few years, multidimensional liquid chromatography-mass spectrometry (LC-MS) systems have been commonly used to characterize post-translational modifications (PTMs) of therapeutic antibodies (mAbs). In most cases, this is performed by fractionation of charge variants by ion-exchange chromatography and subsequent online LC-MS peptide mapping analysis. In this study, we developed a multidimensional ultra-performance-liquid-chromatography-mass spectrometry system (mD-UPLC-MS/MS) for PTM characterization and quantification, allowing both rapid analysis and decreased risk of artificial modifications during sample preparation. We implemented UPLC columns for peptide mapping analysis, facilitating the linkage between mD-LC and routine LC-MS workflows. Furthermore, the introduced system incorporates a novel in-parallel trypsin and LysC on-column digestion setup, followed by a combined peptide mapping analysis. This parallel digestion with different enzymes enhances characterization by generating two distinct peptides. Using this approach, a low retentive ethylene oxide adduct of a bispecific antibody was successfully characterized within this study. In summary, our approach allows versatile and rapid analysis of PTMs, enabling efficient characterization of therapeutic molecules.
在过去的几年中，多维液相色谱-质谱（LC-MS）系统已广泛用于治疗性抗体（mAb）的翻译后修饰（PTM）的特征分析。在大多数情况下，这是通过离子交换色谱对电荷变异体进行分级分离，然后进行在线 LC-MS 肽图分析来实现的。在本研究中，我们开发了一种多维超高效液相色谱-质谱系统（mD-UPLC-MS/MS），用于 PTM 的特征分析和定量，允许快速分析，并降低样品制备过程中人为修饰的风险。我们采用 UPLC 柱进行肽图分析，促进了 mD-LC 与常规 LC-MS 工作流程之间的联系。此外，所引入的系统采用了一种新颖的平行胰蛋白酶和 LysC 在线酶切设置，随后进行组合肽图分析。这种使用不同酶的平行消化通过产生两个不同的肽来增强特征分析。使用这种方法，在本研究中成功地对双特异性抗体的低保留性环氧乙烷加合物进行了特征分析。总之，我们的方法允许灵活快速地分析 PTM，从而有效地对治疗性分子进行特征分析。
