Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry.
Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry.
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DOI：
10.1080/19420862.2018.1525253
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发表时间：
2019-01
期刊：
影响因子：
5.3
通讯作者：
Ge Y
中科院分区：
文献类型：
--
作者：
Jin Y;Lin Z;Xu Q;Fu C;Zhang Z;Zhang Q;Pritts WA;Ge Y
制药工业对单克隆抗体（mAb）及其衍生物的兴趣刺激了这些有效治疗药物的商业和临床管道的快速增长。mAb复杂的微观异质性要求对关键质量属性评估和质量保证进行深入的结构表征。目前，基于质谱（MS）的方法是mAb分析的金标准，主要采用自下而上的方法，其中免疫球蛋白G（IgG）及其变体被消化成肽以促进分析。IgG和微变体的全面表征在蛋白质组水平上仍然具有挑战性。在这里，我们使用了自上而下和中间向下的MS，使用超高分辨率傅立叶变换MS对人IgG 1进行深入表征。我们的自上而下的MS分析提供了单位质量分辨率的IgG 1蛋白质型的特征指纹图谱。随后，完整IgG 1的串联MS分析能够在完整蛋白水平上对代表性IgG 1蛋白质型进行详细的序列表征。此外，我们使用中-下MS分析来表征主要糖型和微变体。微变体，如低丰度糖型，C-末端甘氨酸剪切，和C-末端脯氨酸酰胺化的特点是键裂解高于44%的亚基水平。结合自上而下和自下而上的分析，实现了76%的键断裂（509/666个氨基酸键断裂）的IgG 1。综上所述，我们证明了自上而下和中间向下MS的组合作为mAb综合表征的强大工具。
The pharmaceutical industry’s interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial and clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis. Comprehensive characterization of IgGs and the micro-variants remains challenging at the proteoform level. Here, we used both top-down and middle-down MS for in-depth characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top-down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we used the middle-down MS analysis to characterize the primary glycoforms and micro-variants. Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal proline amidation were characterized with bond cleavages higher than 44% at the subunit level. By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top-down and middle-down MS as powerful tools in the comprehensive characterization of mAbs.
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