Simultaneous Analysis of the Drug-to-Antibody Ratio, Free-Drug-Related Impurities, and Purity of Antibody–Drug Conjugates Based on Size Exclusion Chromatography
Woran Song, Lifeng Yin, Jun Ren, Lai Jiang, Zi‐Ao Huang, Yamin Fan, Rong Tao, Tingting Duan, Zijun Su, Yanjing Cao, Jing Song, Ancha Xu
Abstract
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.