Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis
Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis
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DOI：
10.1016/j.jpba.2017.03.027
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发表时间：
2017-06-05
影响因子：
3.4
通讯作者：
Otsuka, Koji
中科院分区：
文献类型：
--
作者：
Kubota, Kei;Kobayashi, Naoki;Otsuka, Koji
本文介绍了一种新的、全面的鉴定单抗-A(mAb-A)片段峰的方法，该方法用十二烷基硫酸钠-毛细管凝胶电泳法(SDS-CGE)检测。该片段迁移到接近内标物(10 kDa标记物)的SDS-CGE，在25℃条件下6个月增加约0.5%。通常，由于很难从毛细管中收集分析量的馏分，因此在十二烷基硫酸钠-毛细管电泳法中观察到的碎片鉴定具有挑战性。本研究采用凝胶内酶切多肽图谱和反相液相色谱质谱(RPLC-MS)对该片段的结构进行了鉴定。此外，还引入了一种新的Gelless 8100分级系统来收集该片段，并首次将该片段用于单抗的结构分析。这三种分析方法的结果具有可比性，证明该片段是重链HCl-104的一部分。该片段含有对抗原结合有重要意义的互补决定区(CDR)，因此会影响mAb-A的疗效。此外，在没有10 kDa标记的情况下，用十二烷基硫酸钠-毛细管电泳法可以明确片段数量的增加，实验表明，片段在5℃下每年增加0.2%。这三种分析方法的结合成功地鉴定了十二烷基硫酸钠-毛细管电泳法检测到的杂质峰，为确保生物治疗药物的质量和稳定性提供了关键信息。(C)2017爱思唯尔B.V.保留所有权利。
This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE). The fragment migrated close to the internal standard (10 kDa marker) of SDS-cGE and increased about 0.5% under a 25 degrees C condition for 6 months. Generally, identification of fragments observed in SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase liquid chromatography mass spectrometry (RPLC MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10 kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5 degrees C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS-cGE, providing information critical to assuring the quality and stability of the biotherapeutics. (C) 2017 Elsevier B.V. All rights reserved.