Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

中文翻译：

---

通过阳离子交换色谱-质谱法和自上而下分析表征治疗性蛋白质。


最近，使用水性挥发性缓冲液的阳离子交换色谱（CEX）直接与质谱（MS）结合，并应用于治疗性蛋白质和抗体的完整分析。在我们的研究中，通过 CEX-UV-MS 鉴定了单克隆抗体 (mAb)、双特异性抗体和 Fc 融合蛋白中导致电荷变体的化学修饰。我们还报告了 CEX 柱后添加有机溶剂和酸，然后在升高的温度下混合，这使蛋白质展开，增加了离子强度（灵敏度）并促进了自上而下的分析。受到过氧化氢氧化应激的 mAb 被用作模型系统，它产生了额外的 CEX 峰。在线 CEX-UV-MS 自上而下分析产生含有一个或两个蛋氨酸残基的气相片段。一些蛋氨酸残基的氧化导致较早的（酸性）洗脱峰，一些导致较晚的（碱性）洗脱峰，而其他残基的氧化不会改变 CEX 洗脱。氧化和非氧化碎片离子的丰度还可以估计受胁迫的 mAb 中不同蛋氨酸残基的氧化百分比。 CEX-UV-MS 测量揭示了一种新的完整抗体蛋白质形式，浓度为 5%，作为基本峰洗脱，并包含配对修饰：高甘露糖糖基化和剩余的 C 末端赖氨酸残基 (M5/M5 + K)。这一发现通过肽图谱和柱上二硫键还原以及反相液相色谱 - 对收集的基本峰进行自上而下的 MS 分析得到了证实。总体而言，我们的结果证明了在线方法在提供电荷修饰的位点特异性结构信息方面的实用性，而无需馏分收集和费力的肽图分析。