Post-column denaturation-assisted hydrophobic interaction chromatography−mass spectrometry for rapid and in-depth characterization of positional isomers in cysteine-based antibody-drug conjugates

IF 3.1 3区 医学 Q2 CHEMISTRY, ANALYTICAL
Zhengqi Zhang, Anita P. Liu, Hongxia Wang, Hillary A. Schuessler
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引用次数: 0

Abstract

Antibody-drug conjugates (ADCs) represent a significant advancement in targeted cancer therapy, offering the potential to selectively deliver cytotoxic drugs to tumor cells while minimizing systemic toxicity. However, the structural complexity of ADCs, particularly those conjugated through cysteine residues, poses significant analytical challenges. Due to the hydrophobicity of ADCs, Hydrophobic interaction chromatography (HIC) is often the method of choice to analyze the drug-to-antibody ratio (DAR). However, it requires high-concentration salts, which are often incompatible with mass spectrometry (MS) analysis. By employing ammonium acetate as an MS-compatible salt and integrating a 4-way liquid junction cross configuration for simultaneous introduction of the makeup flow and splitting the flow right before coupling to a mass spectrometer, we achieve high-quality separation and sensitive mass spectrometric analysis. This innovative setup allows for simultaneous DAR measurement and positional isomer characterization by switching the makeup flow solvent from water to a denaturation solution. Our method offers a streamlined and effective approach to ADC characterization, facilitating the identification of positional isomers without the need for fractionation or multiple chromatographic steps. The versatility and robustness of this HIC-MS method are demonstrated through the analysis of two ADCs, highlighting its potential for broad application in ADC development and quality control.
柱后变性辅助疏水相互作用色谱-质谱法用于快速深入鉴定半胱氨酸抗体-药物共轭物中的位置异构体。
抗体-药物偶联物(adc)代表了靶向癌症治疗的重大进展,提供了选择性地向肿瘤细胞递送细胞毒性药物的潜力,同时将全身毒性降到最低。然而,adc的结构复杂性,特别是那些通过半胱氨酸残基偶联的adc,提出了重大的分析挑战。由于adc的疏水性,疏水相互作用色谱(HIC)通常是分析药物抗体比(DAR)的首选方法。然而,它需要高浓度的盐,这往往与质谱(MS)分析不相容。通过采用乙酸铵作为ms兼容盐,并集成4路液体结交叉配置,同时引入组成流并在耦合到质谱仪之前分裂流动,我们实现了高质量的分离和敏感的质谱分析。这种创新的装置允许同时进行DAR测量和位置异构体表征,通过将补流溶剂从水切换到变性溶液。我们的方法提供了一种简化有效的ADC表征方法,方便了位置异构体的鉴定,而不需要分离或多个色谱步骤。通过对两个ADC的分析,证明了这种HIC-MS方法的通用性和鲁棒性,突出了它在ADC开发和质量控制方面的广泛应用潜力。
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来源期刊
CiteScore
6.70
自引率
5.90%
发文量
588
审稿时长
37 days
期刊介绍: This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome. Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.
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