Novel Native Reversed-Phase Liquid Chromatography (nRPLC)/MS for Antibody–Drug Conjugates (ADCs) Characterization and Drug–Antibody Ratio (DAR) Assessment

Characterization of drug–antibody ratio (DAR) species in antibody–drug conjugates (ADCs) is crucial for assessing the developability/manufacturability and downstream development of drug candidates. Although hydrophobic interaction chromatography (HIC) is the gold standard for DAR analysis, elucidating DAR species within each HIC peak has historically been challenging. This is due to the nonvolatility and high ionic strength of conventional buffer systems, which necessitate labor-intensive offline fractionation, followed by MS analysis. To address these challenges, an innovative alternative strategy has been developed that directly couples native reversed-phase liquid chromatography (nRPLC) to high-resolution Orbitrap MS for online native MS analysis (nRPLC-MS). In collaboration with Phenomenex, two types of columns, each with a different hydrophobicity, were developed, allowing for elution with low concentration of MS-friendly salt and organic buffer. LC and MS parameters were optimized to enhance the detection of intact DAR species under high flow rate conditions. To demonstrate the feasibility of the platform for characterizing different types of ADCs, both interchain-linked (heterogeneous DAR of 0 to 8) and site-specific ADCs were evaluated. The method enables the nondenatured separation and simultaneous characterization of different DAR species, and strong correlation was observed between this approach and analysis by HIC. This integrated strategy allows unbiased characterization of DAR species without postcolumn flow splitting or peak fractionation. Furthermore, comparisons with two commonly used methods (native SEC-MS and RPLC-MS) have shown that superior separation in terms of selectivity and resolution is achieved with the nRPLC method. Notably, unconjugated antibody (DAR0) was successfully retained with a low-ionic-strength salt using this method. Moreover, the method facilitated the chromatographic separation of positional isomers of DAR4 species with different conjugation linkages, which was not achievable with traditional HIC. As a result, this method holds great promise for high-throughput screening and characterization of ADCs across conjugation methods and payload classes.