Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain
Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain
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DOI：
10.1016/j.ab.2006.10.012
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发表时间：
2007-01-01
影响因子：
2.9
通讯作者：
Rouse, Jason C.
中科院分区：
文献类型：
--
作者：
Johnson, Keith A.;Paisley-Flango, Karl;Rouse, Jason C.
对于重组单克隆抗体 (mAb) 样品，通过弱阳离子交换 HPLC (WCX) 观察到独特的延迟洗脱“基本峰”（相对于“主峰”）。收集、脱盐并通过高分辨率 MS 使用自上而下的表征方法对峰组分进行分析，该方法提供了完整 mAb 电荷亚型的准确质量和结构异质性的全面概况。二硫键还原和半胱氨酸烷基化后，通过反相 (RP) HPLC/MS 分析主峰和基本峰中的各个轻 (L) 和重 (H) 链亚基。检测到三种 mAb 亚型，其修饰位于 H 链。使用 RP-HPLC/MS 肽图谱和精确质量测量进行自下而上的表征，鉴定出三种不同的 H 链 C 端肽，以甘氨酸、赖氨酸或 α-酰胺化脯氨酸结尾。组合分析表明，主要的 WCX 峰 mAb 同工型包含两条未修饰的 L 链和两条以甘氨酸终止的 H 链。在基本峰中共洗脱的每种 mAb 同工型均由两个未修饰的 L 链亚基和一条以甘氨酸结尾的 H 链组成，但一个同工型的第二个 H 链以赖氨酸终止，而另一种同工型的第二个 H 链以赖氨酸终止。异构体的 WCX 洗脱位置与其各自的净电荷一致。据我们所知，以前没有报道过单克隆抗体中 C 端α-酰胺化的发生。 (c) 2006 Elsevier Inc. 保留所有权利。
A unique, late-eluting "basic peak" (relative to the "main peak") was observed by weak cation exchange-HPLC (WCX) for a recombinant monoclonal antibody (mAb) sample. Peak fractions were collected, desalted, and analyzed by high-resolution MS using a topdown characterization approach that provided accurate masses of intact mAb charge isoforms and a comprehensive profile of the structural heterogeneity. The individual light (L) and heavy (H) chain subunits from the main and basic peaks were analyzed by reversedphase (RP) HPLC/MS after disulfide bond reduction and cysteine alkylation. Three mAb isoforms were detected, and their modifications were localized to H chain. Bottom-up characterization using RP-HPLC/MS peptide mapping and accurate mass measurements identified three distinct H chain C-terminal peptides ending in glycine, lysine, or alpha-amidated proline. The combined analyses showed that the main WCX peak mAb isoform contained two unmodified L chains and two H chains terminating in glycine. Each mAb isoform that coeluted in the basic peak consisted of two unmodified L chain subunits and a single H chain ending in glycine, but the second H chain terminated in lysine for one isoform and alpha-amidated proline for another isoform. The WCX elution positions of the isoforms were consistent with their respective net charge. To the best of our knowledge, the occurrence of C-terminal a-amidation in mAbs has not been reported previously. (c) 2006 Elsevier Inc. All rights reserved.