Use of a stable-isotope-labeled reporter peptide and antioxidants for reliable quantification of methionine oxidation in a monoclonal antibody by liquid chromatography/mass spectrometry.
Use of a stable-isotope-labeled reporter peptide and antioxidants for reliable quantification of methionine oxidation in a monoclonal antibody by liquid chromatography/mass spectrometry.
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使用稳定同位素标记的报告肽和抗氧化剂通过液相色谱/质谱法可靠地定量单克隆抗体中的蛋氨酸氧化。
DOI：
10.1002/rcm.7646
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Routhier,Eric
中科院分区：
文献类型：
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作者：
Kang,Pilsoo;Mezhebovsky,Tanya;Chen,Wei;McCardle,JamesA;Zhang,Sheng;Sullivan,EricP;Sass,PhilipM;Routhier,Eric
原理通过液相色谱/质谱 (LC/MS) 对治疗性蛋白质中的蛋氨酸氧化进行准确定量具有挑战性，因为在肽图分析工作流程中的样品制备和分析过程中可能会引入人为因素。在本研究中，开发了一种优化肽图分析程序的系统方法，以实现单克隆抗体中内源蛋氨酸氧化的可靠定量。方法该方法基于使用稳定同位素标记的报告肽，其序列与含有最容易氧化的蛋氨酸残基的 IgG1 单克隆抗体的胰蛋白酶肽相同。该方法应用于评估各种脱盐程序，并在 nanoLC/MS、microLC/MS 和 UPLC/MS 上进行测试，以对氧化敏感的模型单克隆抗体 IgG1 进行肽图谱分析。结果使用参考稳定同位素标记肽，对使用 LC/MS 检测发生蛋白质氧化的肽图谱程序中的几个步骤进行了识别和优化。因此，验证了目标单克隆抗体中蛋氨酸氧化的可靠定量。结论利用参考稳定同位素标记报告肽的方法适用于单克隆抗体氧化分析，并且一旦鉴定出蛋白质序列中易于氧化的蛋氨酸，就可以扩展到其他生物治疗药物。版权所有 © 2016 约翰·威利父子有限公司
RationaleAccurate quantification of methionine oxidation in therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS) is challenging due to the potential artifacts introduced during sample preparation and analysis in the peptide mapping workflow. In this study, a systematic approach for optimization of the peptide mapping procedure to achieve reliable quantification of endogenous methionine oxidation in monoclonal antibodies was developed.MethodsThe approach is based on usage of a stable‐isotope‐labeled reporter peptide, identical in sequence to the tryptic peptide of an IgG1 monoclonal antibody containing the methionine residue most prone to oxidation. This approach was applied to evaluating various desalting procedures, and tested on nanoLC/MS, microLC/MS and UPLC/MS for the peptide mapping analysis of a model monoclonal antibody IgG1 sensitive to oxidation.ResultsSeveral steps in the peptide mapping procedure with LC/MS detection at which protein oxidation occurred were identified and optimized using the reference stable‐isotope‐labeled peptide. Thus, reliable quantification of methionine oxidation in the target monoclonal antibody was validated.ConclusionsThe methodology which utilizes the reference stable‐isotope‐labeled reporter peptide is applicable to monoclonal antibody oxidation analysis and could be extended to other biotherapeutics once oxidation‐prone methionine(s) in the protein sequence are identified. Copyright © 2016 John Wiley & Sons, Ltd.