Sorensen Matthew, Harmes David C, Stoll Dwight R, Staples Gregory O, Fekete Szabolcs, Guillarme Davy, Beck Alain
a Gustavus Adolphus College , Department of Chemistry , St. Peter , MN , USA.
b Agilent Technologies , Santa Clara , CA , USA.
MAbs. 2016 Oct;8(7):1224-1234. doi: 10.1080/19420862.2016.1203497. Epub 2016 Jun 30.
As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.
随着生物类似药蛋白质疗法的研究、开发和生产不断增加，人们对持续开发一系列互补分析方法有着浓厚兴趣，这些方法可用于相对于各自的参考（即原研）分子高效且有效地表征生物类似药候选材料。液相分离技术如液相色谱和毛细管电泳是强大的工具，能够提供关于参考材料和生物类似药材料之间异同的定性和定量信息，特别是与质谱联用时。然而，这些蛋白质材料固有的复杂性对即使是最现代的一维（1D）分离方法也构成挑战。二维（2D）分离相对于一维方法具有许多潜在优势，包括增加峰容量、有助于未知物鉴定的二维峰模式，以及改善某些分离方法与质谱的兼容性。在本研究中，我们展示了使用涉及第一维和第二维阳离子交换（CEX）和反相（RP）分离的全二维液相色谱分离，以中观方法比较3对参考/生物类似单克隆抗体（西妥昔单抗、曲妥珠单抗和英夫利昔单抗），它们涵盖了一系列相似性/差异性。第二维RP分离与飞行时间质谱联用，这使得能够直接鉴定用IdeS酶消化单克隆抗体得到的色谱图中的特征，或先用IdeS消化然后用二硫苏糖醇还原后的特征。在单个样品中检测到多达23个化学性质独特的单克隆抗体片段。我们的结果表明，这些丰富的数据集能够轻松评估参考材料和生物类似药材料之间的相似程度。
