Fast Characterization of Fc-Containing Proteins by Middle-Down Mass Spectrometry Following IdeS Digestion

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• 作者：Tao Liu ; Huaizu Guo ; Lei Zhu ; Yingxin Zheng ; Jin Xu ; Qingcheng Guo…
• 关键词：IdeS ; Middle ; down mass spectrometry ; Antibody ; Fc fusion protein ; Antibody–drug conjugate (ADC) ; Glycosylation ; O ; Acetylated sialic acid
• 刊名：Chromatographia
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：79
• 期：21-22
• 页码：1491-1505
• 全文大小：6,520 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Organic Chemistry
  Pharmacy
  Biochemistry
  Plant Sciences
  Measurement Science and Instrumentation
  
• 出版者：Vieweg Verlag
• ISSN：1612-1112
• 卷排序：79

文摘

The rapid growth of biotherapeutics (monoclonal antibodies and antibody derivatives) demands improved techniques for their quality control and batch-to-batch study. Traditionally, the top-down and bottom-up mass spectrometric approaches may be good options, but they have their share of drawbacks. The middle-down technology has unique superiorities for fast characterization of very large proteins. In this article, we report a systematic approach to characterize Fc-containing proteins (antibody, Fc fusion protein, and antibody–drug conjugate) by middle-down mass spectrometry following IdeS proteolytic digestion. We characterized the main protein modifications including glycosylation, glycation, and other modifications with a relatively small proportion, and results were consistent with free glycan profiling analysis. Meanwhile, the O-acetylated sialic acid modification of cetuximab was reported for the first time; its potential roles in biotherapeutics need further study. The middle-down technology following IdeS proteolytic digestion could be used not only for analyzing the modifications of Fc-containing proteins at the subunit level but also for characterizing the potential modifications at early development stages.