Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation
Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation
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DOI：
10.1021/acs.analchem.6b00302
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发表时间：
2016-04-05
影响因子：
7.4
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
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作者：
Cotham, Victoria C.;Brodbelt, Jennifer S.
单抗是一类发展迅速的治疗性糖蛋白，具有生物相容性、高度的抗原特异性和靶向性免疫刺激等优点，具有广泛的临床应用价值。这些治疗特性在很大程度上取决于免疫球蛋白G(Ig G)结构的组成，无论是初级序列还是翻译后修饰(PTM)；然而，细胞培养中的大规模生产往往会导致不同种类的混合物，这可能会深刻影响临床安全性和有效性。这就对分析方法提出了很高的要求，这些方法能够全面地表征单抗的结构，以确保其严格的质量控制。在这里，我们报告了使用定向中向下193 nm紫外光解离(UVPD)来提供治疗性单抗亚基(类似于25 kDa)的详细初级序列分析和PTM位点定位，该亚基是由化脓性链球菌重组免疫球蛋白G降解酶(IDES)消化后经化学还原而产生的。在一次LC-MS/MS实验中，除了明确的糖基化位点定位和抗原结合互补决定区(CDR)的广泛覆盖外，导致了大约60%的总覆盖。结合UVPD和ETD数据提供了更深的测序和更全面的免疫球蛋白亚基特征。总体而言，这种有针对性的UVPD方法代表了一种有前景的新策略，用于全面描述基于抗体的治疗方法。
Monoclonal antibodies (mAbs) are a rapidly advancing class of therapeutic glycoproteins that possess wide clinical utility owing to their biocompatibility, high antigen specificity, and targeted immune stimulation. These therapeutic properties depend greatly on the composition of the immunoglobulin G (IgG) structure, both in terms of primary sequence and post-translational modifications (PTMs); however, large-scale production in cell culture often results in heterogeneous mixtures that can profoundly affect clinical safety and efficacy. This places a high demand on analytical methods that afford comprehensive structural characterization of mAbs to ensure their stringent quality control. Here we report the use of targeted middle-down 193 nm ultraviolet photo dissociation (UVPD) to provide detailed primary sequence analysis and PTM site localization of therapeutic monoclonal antibody subunits (similar to 25 kDa) generated upon digestion with recombinant immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) followed by chemical reduction. resulted in approximately 60% overall coverage of the IgG sequence, in addition to unambiguous glycosylation site localization and extensive coverage of the antigen-binding complementarity determining regions (CDRs) in a single LC-MS/MS experiment. Combining UVPD and ETD data afforded even deeper sequencing and greater overall characterization of IgG subunits. Overall, this targeted UVPD approach represents a promising new strategy for the comprehensive characterization of antibody-based therapeutics.