Glycan-Mediated Technology for Obtaining Homogeneous Site-Specific Conjugated Antibody–Drug Conjugates: Synthesis and Analytical Characterization by Using Complementary Middle-up LC/HRMS Analysis

Conventional antibody–drug conjugate (ADC) manufacturing methods are based on the nonselective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug–antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice. Here, we evaluated the conjugation process by comparing trastuzumab and trastuzumab conjugated with DM1, following the GlyCLICK procedure. Complementary reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS) were used to analyze the protein subunits (ca. 25–100 kDa) obtained after different levels of enzymatic digestion and chemical reduction. Our results demonstrated that the hydrophobic character of the drug molecule allows to rapidly confirm the Fc-drug conjugation at the chromatographic level. Furthermore, the hyphenation to MS detection provided accurate mass information on the ADC subunits and facilitated the DAR determination of 2.0. Therefore, this work illustrates how middle-up analysis using LC/HRMS can provide accurate and complementary information on the critical quality attributes of these novel site-specific ADC products.