Imaged Capillary Isoelectric Focusing Coupled to High-Resolution Mass Spectrometry (icIEF-MS) for Cysteine-Linked Antibody-Drug Conjugate (ADC) Heterogeneity Characterization Under Native Condition.

IF 3 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Xiaoxi Zhang, Gang Wu, Min Du, Tao Bo, Tong Chen, Tiemin Huang
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Abstract

Native mass spectrometry (nMS) is a cutting-edge technique that leverages electrospray ionization MS (ESI-MS) to investigate large biomolecules and their complexes in solution. The goal of nMS is to retain the native structural features and interactions of the analytes during the transition to the gas phase, providing insights into their natural conformations. In biopharmaceutical development, nMS serves as a powerful tool for analyzing complex protein heterogeneity, allowing for the examination of non-covalently bonded assemblies in a state that closely resembles their natural folded form. Herein, we present an imaged capillary isoelectric focusing-MS (icIEF-MS) workflow to characterize cysteine-linked antibody-drug conjugate (ADC) under native conditions. Two ADCs were analyzed: a latest generation cysteine-linked ADC polatuzumab vedotin and the first FDA-approved cysteine-linked ADC brentuximab vedotin. This workflow benefits from a recently developed icIEF system that is MS-friendly and capable of directly coupling to a high-sensitivity MS instrument. Results show that the icIEF separation is influenced by both drug payloads and the post-translational modifications (PTMs), which are then promptly identified by MS. Overall, this native icIEF-MS method demonstrates the potential to understand and control the critical quality attributes (CQAs) that are essential for the safe and effective use of ADCs.

成像毛细管等电聚焦与高分辨率质谱联用技术(icIEF-MS)用于原生态条件下半胱氨酸连接抗体-药物共轭物(ADC)的异质性表征。
原生质谱(nMS)是一种尖端技术,它利用电喷雾离子化质谱(ESI-MS)来研究溶液中的大型生物分子及其复合物。nMS 的目标是在向气相过渡的过程中保留分析物的原生结构特征和相互作用,从而深入了解它们的天然构象。在生物制药开发过程中,nMS 是分析复杂蛋白质异质性的有力工具,可以在与其自然折叠形态极为相似的状态下检查非共价键结合的集合体。在此,我们介绍了一种成像毛细管等电聚焦-MS(icIEF-MS)工作流程,用于表征原生条件下半胱氨酸连接的抗体-药物共轭物(ADC)。对两种 ADC 进行了分析:最新一代半胱氨酸连接型 ADC polatuzumab vedotin 和首个获得 FDA 批准的半胱氨酸连接型 ADC brentuximab vedotin。该工作流程得益于最近开发的 icIEF 系统,该系统便于 MS 使用,能够直接与高灵敏度 MS 仪器连接。结果表明,icIEF 分离受药物有效载荷和翻译后修饰 (PTM) 的影响,而翻译后修饰可通过 MS 快速鉴定。总之,这种原生 icIEF-MS 方法展示了了解和控制关键质量属性 (CQAs) 的潜力,而关键质量属性对安全有效地使用 ADCs 至关重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
ELECTROPHORESIS
ELECTROPHORESIS 生物-分析化学
CiteScore
6.30
自引率
13.80%
发文量
244
审稿时长
1.9 months
期刊介绍: ELECTROPHORESIS is an international journal that publishes original manuscripts on all aspects of electrophoresis, and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid-phase micro-extractions, etc.). Topics include new or improved analytical and preparative methods, sample preparation, development of theory, and innovative applications of electrophoretic and liquid phase separations methods in the study of nucleic acids, proteins, carbohydrates natural products, pharmaceuticals, food analysis, environmental species and other compounds of importance to the life sciences. Papers in the areas of microfluidics and proteomics, which are not limited to electrophoresis-based methods, will also be accepted for publication. Contributions focused on hyphenated and omics techniques are also of interest. Proteomics is within the scope, if related to its fundamentals and new technical approaches. Proteomics applications are only considered in particular cases. Papers describing the application of standard electrophoretic methods will not be considered. Papers on nanoanalysis intended for publication in ELECTROPHORESIS should focus on one or more of the following topics: • Nanoscale electrokinetics and phenomena related to electric double layer and/or confinement in nano-sized geometry • Single cell and subcellular analysis • Nanosensors and ultrasensitive detection aspects (e.g., involving quantum dots, "nanoelectrodes" or nanospray MS) • Nanoscale/nanopore DNA sequencing (next generation sequencing) • Micro- and nanoscale sample preparation • Nanoparticles and cells analyses by dielectrophoresis • Separation-based analysis using nanoparticles, nanotubes and nanowires.
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