Comparative Orthogonal RP-UHPLC and SEC-UHPLC Methods for Quantitative Analysis and Integrity Assessment of Cetuximab-Based Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are structurally complex biopharmaceuticals whose quantitative characterization is often complicated by payload-related interferences and molecular heterogeneity. In this study, two complementary chromatographic methods based on reverse-phase ultra-high-performance liquid chromatography (RP-UHPLC) and size-exclusion ultra-performance liquid chromatography (SEC-UHPLC) coupled with diode array detection were developed and comparatively evaluated for quantitative and integrity assessment of Cetuximab (CET) in ADC formulations. The RP-UHPLC method, developed on a C8 stationary phase under denaturing conditions, showed linearity over the range 0.05-5 mg/mL (R2 = 0.9979), with LOD and LOQ values of 10 and 40 mu g/mL, respectively. The SEC-UHPLC method, optimized under native conditions on a Yarra SEC-3000 column, exhibited linearity within the 0.5-5 mg/mL range (R2 = 0.9998), with LOD and LOQ values of 17 and 52 mu g/mL, respectively. Both methods showed satisfactory accuracy, precision, and robustness according to a fit-for-purpose validation approach. Application to CET-based ADCs bearing different aminobisphosphonate payloads confirmed reliable quantification. RP-UHPLC enabled sensitive determination of antibody concentration, whereas SEC-UHPLC enabled assessment of monomeric integrity and sample heterogeneity under native conditions. Comparative analysis of aged ADC samples demonstrated that SEC-UHPLC revealed loss of structural integrity and sample heterogeneity that were not detectable by RP-UHPLC or direct UV spectrophotometry. The combined RP-UHPLC/SEC-UHPLC workflow provides a robust, accessible, and complementary platform for routine quantitative analysis and integrity assessment of ADC formulations.