Goyon Alexandre, D'Atri Valentina, Bobaly Balazs, Wagner-Rousset Elsa, Beck Alain, Fekete Szabolcs, Guillarme Davy
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Centre Médical Universitaire (CMU) - Rue Michel-Servet 1, 1206 Geneva, Switzerland.
Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160 Saint-Julien-en-Genevois, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Jul 15;1058:73-84. doi: 10.1016/j.jchromb.2017.05.010. Epub 2017 May 11.
Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC, HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods.
生物制药产品中与大小、电荷和疏水性相关的变体必须进行深入表征，以确保批次一致性，并评估免疫原性和安全效应。尺寸排阻色谱法（SEC）和离子交换色谱法（IEX）分别被视为分析高分子量物质（HMWS）和电荷相关变体的金标准。疏水相互作用色谱法（HIC）已重新受到关注，用于监测半胱氨酸连接的抗体药物偶联物（ADC）中小分子药物的分布。这三种色谱技术，即SEC、HIC和IEX，是历史悠久、非变性且稳健的方法，广泛用于生物制药蛋白质的表征。尽管单克隆抗体（mAb）的结构、等电点（pI）和疏水性范围很广，但使用新一代色谱柱和适当的流动相条件，可以应用通用方案来分离其与大小、电荷和疏水性相关的变体。本手稿中描述了简单的方案，并给出了十种不同的美国食品药品监督管理局（FDA）和欧洲药品管理局（EMA）批准的治疗性mAb产品的代表性色谱图，以说明SEC、IEX和HIC方法的性能。
