Simultaneous Analysis of the Drug-to-Antibody Ratio, Free-Drug-Related Impurities, and Purity of Antibody-Drug Conjugates Based on Size Exclusion Chromatography.

Drug-to-antibody ratio (DAR), free-drug-related impurities (FDRI) content, and purity are critical quality attributes of antibody-drug conjugates (ADCs), which substantially impact product safety and efficacy. However, growing efforts in developing ADCs with higher complexity in a faster timeline impose a great challenge to their analytical support. Herein, for the ADC carries an antibody and linker-payload with distinct UV/vis absorption maxima, we propose a high-throughput and multiattribute ADC analysis strategy based on size exclusion chromatography coupled with UV detection (SEC-UV). Briefly, in addition to the quantitation of aggregates and fragments, SEC excludes FDRI from ADC-related peaks and improves the accuracy of DAR determination via dual-wavelength UV detection. Relative FDRI content can be determined subsequently by comparing the free-drug-related and ADC-related peaks at a wavelength where UV absorbance is exclusively attributed to the linker-payload. Afterward, a quantitative consistency in DAR and FDRI analysis was established between SEC-UV and orthogonal methodologies widely adopted by the pharmaceutical industry. The applicability of the developed SEC-UV method was further extended through feasiblity studies on ADC process-intermediates, bispecific ADC, and photodegraded ADC. Despite lacking characterization of drug load distribution and profiling of individual FDRI species, its efficiency, simplicity, and high throughput make SEC-UV a phase-appropriate strategy for supporting in-process monitoring and early-stage process development of diverse ADC projects.