Reliable Quantification of Conjugated Peptides from T-DM1 In Vivo by Two-Step Enrichment and Internal Standards from Derivatized Analytes.

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.