Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies
Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies
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DOI：
10.1021/acs.analchem.8b00912
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发表时间：
2018-08-07
影响因子：
7.4
通讯作者：
Cianferani, Sarah
中科院分区：
文献类型：
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作者：
Hemandez-Alba, Oscar;Wagner-Rousset, Elsa;Cianferani, Sarah
尽管大多数FDA和EMA批准的治疗性单克隆抗体（mab）是IgGl，但进入市场的基于igg4的单克隆抗体的数量正在增加。IgG4与其他单克隆抗体同型的不同之处在于，它可以形成半单克隆抗体，并通过称为单克隆抗体臂交换（FAE）的过程重组为双特异性（bsAbs）分子。在此，我们报告了天然质谱（MS）、离子迁移率（IM）和碰撞诱导展开（CIU）实验对IgG4亚家族成员（野生型（wt）、铰链稳定型（hs， S228P突变）以及由此产生的bsAb IgG4s）结构表征的互互性。原生MS允许确认/无效FAE的发生作为这些不同类型IgG4的功能。虽然IM-MS无法区分等截面IgG4物种，但CIU实验根据每个IgG4的特定展开途径提供了独特的特定结构特征。IgG4格式的常见CIU特征包括观察到三个构象状态和两个跃迁。此外，CIU实验表明，S228P突变稳定了hsIgG4的气相构象，这与更刚性铰链区域相关的稳定性增加相一致。在bsAb结构表征方面，CIU模式似乎比IM-MS更能提供信息，揭示了bsAb在前母体wt-IgG4s之间的特征，强调了bsAb CIU谱保留了它们起源的记忆。总之，我们的研究结果表明，CIU模式可以作为mAb特定的结构特征，并且成熟到可以包含在基于ms的分析工作流程中，用于在早期开发阶段对mAb格式进行构象/结构表征，并用于多属性监测。
Although the majority of FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are IgGl, the number of IgG4-based formats reaching the market is increasing. IgG4 differs from other mAb isotypes by its specificity to form half mAbs that recombine into bispecific (bsAbs) molecules, through a process termed fab-arm exchange (FAE). We report here the complementarity of native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) experiments for the structural characterization of members of the IgG4 subfamily (wild-type (wt), hinge-stabilized (hs, S228P mutation), and the resulting bsAb IgG4s). Native MS allows confirming/invalidating the occurrence of FAE as a function of these different types of IgG4. While IM-MS was unable to distinguish iso-cross-section IgG4 species, CIU experiments provide unique specific structural signatures of each individual IgG4 based on their specific unfolding pathways. Common CIU features of IgG4 formats include the observation of three conformational states and two transitions. In addition, CIU experiments demonstrated that S228P mutation stabilizes gas phase conformations of hsIgG4, in agreement with increased stability related to more rigid hinge regions. CIU patterns also appear to be more informative than IM-MS for bsAb structural characterization, unfolding signature of the bsAb being intermediate to the ones of the former parent wt-IgG4s, highlighting that bsAb CIU profiles keep the memory of their origins. Altogether, our results demonstrate that CIU patterns can serve as mAb specific structural signatures and are mature to be included in MS-based analytical workflows for conformational/structural characterization of mAb formats in early development phases and for multiple attribute monitoring.