Qi Meiling, Xiong Ying, Chen Meiling, Zhang Cui, Zhu Chenyue, Chen Yi, Wang Chenxi, Ye Xinyuan, Jiang Lili, Li Sen, Cheng Zhongzhe, Jiang Hongliang, Du Zhifeng
Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, China.
Medical Sub-center of Analytical and Testing Center, Huazhong University of Science and Technology, Wuhan 430030, China.
Anal Chem. 2025 Jul 1;97(25):13522-13531. doi: 10.1021/acs.analchem.5c01960. Epub 2025 Jun 17.
Antibody-drug conjugates (ADCs) are the conjugation of antibody with cytotoxic payloads. Conjugated peptides that originate from ADCs are commonly used for pharmacokinetics (PK) study. However, a reliable and sensitive method for the quantitative analysis of conjugated peptides in vivo remains elusive, especially for randomly conjugated ADCs. In this study, a strategy that was based on a liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify conjugated peptides from trastuzumab emtansine (T-DM1) in vivo. To correct variations introduced during sample processing and LC-MS detection, a dimethyl labeling strategy was developed to generate one-to-one structural analogues for each conjugated peptide from T-DM1, serving as internal standards (ISs). For sample preparation, protein A/G bead enrichment from the protein level and high-pH reverse-phase fractionation from the peptide level were utilized to enrich conjugated peptides from plasma, leading to the detection of more conjugated peptides. The established method was then validated and applied to quantify conjugated peptides in plasma from rats administered with T-DM1, resulting in the detection of 17 distinct conjugated peptides. Notably, differences in stability across various conjugation sites were observed for the first time, leading to different PK profiles depending on the analytes used. This method is applicable to ADCs and peptide-drug conjugates, considering that they have complex structures and the stable isotope-labeled internal standard (SIL-IS) of conjugated peptide is unavailable.
抗体药物偶联物（ADCs）是抗体与细胞毒性载荷的缀合物。源自ADCs的缀合肽通常用于药代动力学（PK）研究。然而，一种可靠且灵敏的体内缀合肽定量分析方法仍然难以捉摸，尤其是对于随机缀合的ADCs。在本研究中，开发了一种基于液相色谱-质谱（LC-MS）方法的策略，用于定量分析体内曲妥珠单抗（T-DM1）中的缀合肽。为了校正样品处理和LC-MS检测过程中引入的变化，开发了一种二甲基标记策略，为T-DM1中的每个缀合肽生成一对一的结构类似物，作为内标（ISs）。对于样品制备，利用蛋白A/G磁珠从蛋白质水平进行富集，以及从肽水平进行高pH反相分级分离，从血浆中富集缀合肽，从而检测到更多的缀合肽。然后对建立的方法进行验证，并应用于定量分析给予T-DM1的大鼠血浆中的缀合肽，结果检测到17种不同的缀合肽。值得注意的是，首次观察到不同缀合位点的稳定性差异，导致根据所使用的分析物不同而有不同的PK谱。考虑到ADCs和肽-药物缀合物结构复杂且无法获得缀合肽的稳定同位素标记内标（SIL-IS），该方法适用于它们。
