Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry
Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry
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DOI：
10.1021/ac4036857
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发表时间：
2014-03-18
影响因子：
7.4
通讯作者：
Tsybin, Yury O.
中科院分区：
文献类型：
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作者：
Fornelli, Luca;Ayoub, Daniel;Tsybin, Yury O.
批准的生物治疗药物的快速增长，例如，单克隆抗体或免疫球蛋白G（IgG）需要改进的质量控制技术。传统上，已采用基于蛋白水解的自下而上质谱法（MS）。然而，已知自下而上MS所需的长时间、多步骤样品制备方案可能会在原始样品中引入伪影。因此，最好采用自上而下的管理系统方法。然而，完整单克隆IG Gs的自上而下MS的当前性能只能达到接近30%的序列覆盖率，并且对于IgG抗原结合至关重要的互补决定区的测序不完整。在这里，我们描述了一种基于使用化脓性链球菌的免疫球蛋白G降解酶（IdeS）的中-下MS方案，该酶能够在仅30分钟内消化IgG。通过与电子转移解离（ETD）联用的反相液相色谱（LC）在线分析获得的类似于25 kDa的蛋白水解片段。启用混合Orbitrap傅里叶变换质谱仪（Orbitrap Elite FTMS）。在优化ETD和产物离子转移参数后，结果显示，在单个LC运行中达到所选IgG片段的高达50%的序列覆盖率，并且当对通过不同LC MS运行获得的数据进行平均时，达到高达70%的序列覆盖率。重要的是，我们证明了这种中间向下的方法在鉴定氧化蛋氨酸残基中的潜力。所描述的方法显示了用于分析IgG混合物的特定潜力。
The rapid growth of approved biotherapeutics, e.g., monoclonal antibodies or immunoglobulins G (IgGs), demands improved techniques for their quality control. Traditionally, proteolysis-based bottom-up mass spectrometry (MS) has been employed. However, the long, multistep sample preparation protocols required for bottom-up MS are known to potentially introduce artifacts in the original sample. For this reason, a top-down MS approach would be preferable. The current performance of top-down MS of intact monoclonal Ig Gs, though, enables reaching only up to similar to 30% sequence coverage, with incomplete sequencing of the complementarity determining regions which are fundamental for IgG's antigen binding. Here, we describe a middle-down MS protocol based on the use of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS), which is capable of digesting IgGs in only 30 min. After chemical reduction, the obtained similar to 25 kDa proteolytic fragments were analyzed by reversed phase liquid chromatography (LC) coupled online with an electron transfer dissociation (ETD)-enabled hybrid Orbitrap Fourier transform mass spectrometer (Orbitrap Elite FTMS). Upon optimization of ETD and product ion transfer parameters, results show that up to similar to 50% sequence coverage for selected IgG fragments is reached in a single LC run and up to similar to 70% when data obtained by distinct LC MS runs are averaged. Importantly, we demonstrate the potential of this middle-down approach in the identification of oxidized methionine residues. The described approach shows a particular potential for the analysis of IgG mixtures.