Conjugation site analysis of antibody-drug-conjugates (ADCs) by signature ion fingerprinting and normalized area quantitation approach using nano-liquid chromatography coupled to high resolution mass spectrometry.

Trastuzumab-MCC-DM1 (T-DM1) is an antibody-drug conjugate (ADC) that consists of a monoclonal antibody (mAb) trastuzumab non-cleavably linked to a cytotoxic drug DM1. During production, the DM1 agents were conjugated to the lysine residues of the mAb in a non-specific manner, yielding a heterogeneous mixture of ADC molecules that differ with respect to both the number and the conjugation sites of DM1 per mAb molecule. Since drug conjugation sites of ADC can significantly impact properties such as stability and pharmacokinetic behaviors, a rapid and reliable approach for conjugation site analysis of ADCs is highly demanded. Herein, we have employed a signature ion fingerprinting approach to specifically determine lysine residues with DM1 conjugation, and developed a normalized peak area quantitation method to characterize the percentage of DM1-conjugated lysine for each putative site using a T-DM1 biosimilar as a model drug. With this integrative approach, 38 lysine residues were identified with DM1 conjugation among 90 possible sites. More interestingly, we found that the T-DM1 biosimilar exhibited a specific preference of DM1-conjugation for several lysine residues, and such preference was consistent among three production batches. A molecular modeling approach was subsequently utilized to analyze all the conjugation sites, and revealed an intriguing correlation of the conjugated residue's microenvironment with the conjugation level. In summary, our study introduced an approach that is widely applicable to ADCs of interest for conjugation site analysis. Moreover, it suggests the necessity of performing conjugation site analysis for product and process characterization and also for routine use in lot release and stability testing of manufactured ADCs.