Pan Xiao, Zhao Yimeng, Wu Zhijie, Qiu Haibo, Li Ning
Analytical Chemistry, Regeneron Pharmaceuticals Inc, 777 Old Saw Mill River Road, Tarrytown, NY, 10591, United States.
Analytical Chemistry, Regeneron Pharmaceuticals Inc, 777 Old Saw Mill River Road, Tarrytown, NY, 10591, United States.
Anal Chim Acta. 2025 Sep 22;1368:344315. doi: 10.1016/j.aca.2025.344315. Epub 2025 Jun 9.
Antibody-drug conjugates (ADCs) have become a promising treatment for various cancers, by combining the specificity of monoclonal antibodies (mAbs) with the potent cytotoxicity of small-molecule drugs. The intricate structure of ADCs necessitates comprehensive analytical characterization to ensure their safety, efficacy, and quality. Reduced peptide mapping, integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS), is essential for assessing critical quality attributes (CQAs) of ADCs, including linker-payload (LP) integrity, post-translational modifications (PTMs) of the mAb backbone, and the distribution of conjugation sites.
Traditional tryptic peptide mapping protocols optimized for mAbs are often inadequate for ADCs, because of the hydrophobic nature of LPs. These LPs alter the physicochemical properties of conjugated peptides, thus leading to challenges such as suboptimal recovery because of interactions with other hydrophobic peptides and steric hindrance impeding enzymatic digestion. Herein, we explored strategies to enhance the recovery of hydrophobic peptides by incorporating various surfactants and denaturants. The addition of 2.7 M guanidine hydrochloride significantly increased the recovery of hydrophobic peptides. Digestion conditions were optimized to increase the digestion efficiency of the conjugated peptides. A 3-h incubation at 37 °C with a 1:10 (w/w) ratio of Promega sequencing grade trypsin to ADCs yielded minimal missed cleavages and a minimal increase in non-specific cleavages.
The optimized peptide mapping workflow demonstrates high hydrophobic peptide recovery and reproducibility, and provides a robust platform for detailed characterization of ADCs, thereby ensuring their consistent product quality.
抗体药物偶联物（ADCs）通过将单克隆抗体（mAbs）的特异性与小分子药物的强效细胞毒性相结合，已成为治疗多种癌症的一种有前景的方法。ADCs的复杂结构需要全面的分析表征，以确保其安全性、有效性和质量。与液相色谱 - 串联质谱（LC-MS/MS）相结合的还原肽图谱分析对于评估ADCs的关键质量属性（CQAs）至关重要，这些属性包括连接子 - 载荷（LP）完整性、mAb骨架的翻译后修饰（PTMs）以及偶联位点的分布。
由于LP具有疏水性，为mAbs优化的传统胰蛋白酶肽图谱分析方案通常不适用于ADCs。这些LP改变了偶联肽的物理化学性质，从而导致诸如由于与其他疏水肽相互作用而回收率欠佳以及空间位阻阻碍酶切消化等挑战。在此，我们探索了通过加入各种表面活性剂和变性剂来提高疏水肽回收率的策略。添加2.7 M盐酸胍显著提高了疏水肽的回收率。优化了消化条件以提高偶联肽的消化效率。在37°C下以Promega测序级胰蛋白酶与ADCs 1:10（w/w）的比例孵育3小时，产生的漏切最少且非特异性切割增加最少。
优化后的肽图谱分析工作流程显示出高疏水肽回收率和重现性，并为ADCs的详细表征提供了一个强大的平台，从而确保其产品质量的一致性。
