Analysis of Cysteine-Linked Antibody Drug Conjugates

Importance of the Topic

Quantification of the drug-to-antibody ratio (DAR) is a critical quality attribute for antibody-drug conjugates (ADCs), as it directly influences safety, efficacy and pharmacokinetics. Cysteine-linked ADCs, generated by reduction of interchain disulfides, yield a defined set of species with 0, 2, 4, 6 or 8 drugs per antibody. Hydrophobic interaction chromatography (HIC) is the reference method to resolve these species under mild, nondenaturing conditions and to accurately determine DAR.

Objectives and Study Overview

This application note presents the determination of DAR for brentuximab vedotin (Adcetris) using HIC on the Agilent 1260 Infinity II Bio-inert LC. The primary goal was to demonstrate baseline separation of ADC species, calculate average DAR and assess method precision for retention time and peak area.

Methodology

HIC separations were performed at neutral pH with a ternary gradient: mobile phase A (2 M ammonium sulfate in 100 mM sodium phosphate, pH 7), mobile phase B (100 mM sodium phosphate, pH 7) and mobile phase C (isopropanol). The gradient transitioned from high to low salt to elute species with increasing hydrophobicity. A non-denaturing approach preserved intact ADC structure despite disrupted disulfide bridges.

Used Instrumentation

• Agilent 1260 Infinity II Bio-inert LC: G5654A pump, G5668A multisampler with cooler, G7116A multicolumn thermostat with bio-inert heat exchanger, G7115A diode array detector with bio-inert flow cell
• Generic HIC column (stationary phase with hydrophobic surface)
• Chemicals: sodium phosphate monobasic/dibasic, ammonium sulfate, isopropanol; ultrapure water from Milli-Q system
• Software: Agilent OpenLAB CDS Version 2.1

Main Results and Discussion

Trastuzumab confirmed excellent peak shape under the chosen gradient. Brentuximab vedotin produced five well-resolved peaks corresponding to species with 0, 2, 4, 6 and 8 drugs attached (D0–D8). Integration of peak areas yielded an average DAR of ~4, consistent with literature. Precision assessment over seven consecutive injections showed retention time RSDs below 0.085 % (first peak 0.106 %) and area RSDs below 0.92 %.

Benefits and Practical Applications

HIC on a fully inert LC system offers robust, non-corrosive operation for high-salt mobile phases, maintaining protein integrity and delivering high precision. This workflow supports routine DAR monitoring in QA/QC and research labs handling biochromatography methods.

Future Trends and Opportunities

Advancements may include novel HIC stationary phases for faster separations, integration with mass spectrometry for simultaneous mass and DAR determination, and automated platforms for high-throughput ADC characterization. Expanding this approach to diverse ADC linkers and payloads will enhance method versatility.

Conclusion

The Agilent 1260 Infinity II Bio-inert LC demonstrated reliable DAR determination for brentuximab vedotin via HIC. The inert flow path prevented corrosion from high-salt buffers, and the method achieved precise, reproducible separation of ADC species, confirming its suitability for routine biochromatography applications.

References

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3. Younes A, et al. Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med. 2010;363(19):1812–1821.
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6. Agilent Technologies. Agilent InfinityLab Bio-inert LC Solution: True Bio-Inertness for Efficient Biomolecule Analysis. Brochure 5991-7361EN; 2017.
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