ADC drug-antibody ratio (DAR) determined by SEC-MALS

Importance of Topic

Antibody-drug conjugates (ADCs) offer targeted delivery of cytotoxic agents to cancer cells, combining the selectivity of monoclonal antibodies with the potency of small-molecule drugs. The drug-to-antibody ratio (DAR) is a critical parameter influencing therapeutic efficacy, safety, stability and aggregation behavior of ADC products.

Study Objectives and Overview

This application note describes a size-exclusion chromatography coupled with multi-angle light scattering, UV absorption and differential refractive index detection (SEC-MALS-UV-dRI) approach to accurately determine DAR in ADCs. Two model ADCs, sharing the same antibody and drug-linker system but differing in conjugation levels, were analyzed to demonstrate method performance.

Methodology and Instrumentation

The two ADC samples were separated by SEC. Eluted species were detected sequentially by a UV detector, a DAWN multi-angle light-scattering detector and an Optilab differential refractive index detector. Data were processed using ASTRA software’s Protein Conjugate Analysis module, employing empirically determined dn/dc values and UV extinction coefficients to deconvolute protein and drug contributions to molar mass.

Used Instrumentation

• High-performance liquid chromatography system with UV detector
• DAWN multi-angle light scattering (MALS) detector
• Optilab differential refractive index (dRI) detector

Key Results and Discussion

UV chromatograms revealed consistent antibody masses for both ADCs, while drug mass differed significantly. For ADC1, the complex mass averaged 167.8 kDa with a drug mass of 12.6 kDa, yielding a DAR of 10.1. ADC2 showed a complex mass of 163.7 kDa, drug mass of 8.1 kDa and DAR of 6.5. The method reliably resolved drug contributions as low as 3 of total mass, demonstrating sensitivity and reproducibility across elution peaks.

Benefits and Practical Applications

SEC-MALS-UV-dRI provides a label-free, matrix-independent means to quantify DAR without reliance on ionization efficiency or overlapping UV extinction coefficients. This method supports ADC development, process optimization and quality control by offering direct mass measurement and automated analysis.

Future Trends and Potential Applications

Advancements may include sensitivity enhancements enabling analysis of lower DAR species, integration with automation for high-throughput screening, coupling with orthogonal detectors for site-specific conjugation mapping, and application to more heterogeneous ADC formats. Data-driven approaches may further refine dn/dc determination and predictive modeling of ADC behavior.

Conclusion

SEC-MALS combined with UV and dRI detection offers a robust, accurate and straightforward platform for DAR determination in ADCs, fulfilling key needs in therapeutic development and quality assurance.