Characterization of Antibody-Drug Conjugate Critical Quality Attributes Using the Agilent Cary 3500 UV-Vis Multizone Temperature Capability

Meaning of the topic

Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of small-molecule drugs, making them essential in targeted cancer therapy. Monitoring critical quality attributes such as drug-to-antibody ratio (DAR) and aggregation is vital to ensure safety, efficacy, and regulatory compliance.

Objectives and study overview

This application note demonstrates how the Agilent Cary 3500 UV-Vis multicell spectrophotometer with a multizone temperature feature can be used to determine DAR and aggregation index of ADCs accurately and efficiently. The study evaluates mixing ratios, temperature effects, and time-dependent aggregation behavior using Herceptin and its DM1 conjugate.

Applied methodology and instrumentation

Samples of Herceptin and its DM1-conjugated analog were prepared at 0.5 mg/mL for mixing experiments and at 25 mg/mL for temperature and aggregation studies, followed by 40× dilution. The Cary 3500 UV-Vis multicell spectrophotometer was fitted with eight Peltier-controlled cuvettes, enabling four simultaneous temperature conditions (60, 70, 80, 90 °C). Spectral scans were performed from 200 to 400 nm (1 nm interval, 1 nm bandwidth), with 0.020 s averaging time and 3000 nm/min scan rate. Data acquisition and custom calculations were handled by Cary UV Workstation software with a multizone add-on.
Equations:

• Average DAR = (ε252 mAb – R × ε280 mAb) / (R × ε280 DM1 – ε252 DM1), where R = A252/A280
• Aggregation index (%) = (OD350 / (OD280 – OD350)) × 100

Main results and discussion

• Mixing experiments produced DAR values proportional to ADC content: 0 % ADC → DAR 0; 25 % → 0.7; 50 % → 1.6; 75 % → 2.4; 100 % → 3.5.
• Temperature elevation from 60 to 90 °C caused a slight decrease in measured DAR, attributed to partial aggregation.
• Aggregation index at 90 °C increased over time, confirming time- and temperature-dependent aggregation kinetics.

Benefits and practical applications of the method

• Simultaneous multi-temperature control increases throughput and minimizes variability.
• Custom equation feature in the software streamlines calculation of DAR and aggregation index.
• Accurate temperature regulation ensures reproducible results.
• Compatibility with 21 CFR Part 11 in Agilent OpenLab software supports regulated laboratory environments.

Future trends and opportunities

• Integration with automation and high-throughput workflows for accelerated ADC development.
• Expansion to other bioconjugates and protein–drug modalities.
• Coupling with real-time monitoring and data analytics, including AI-driven spectral deconvolution.
• Miniaturization and microfluidic interfacing for reduced sample volume and higher screening capacity.

Conclusion

The Agilent Cary 3500 UV-Vis multicell spectrophotometer with multizone temperature capability presents a simple, reliable, and high-throughput solution for ADC quality control. Its ability to assess DAR and aggregation index under multiple conditions simultaneously supports robust biopharmaceutical development and regulatory compliance.

References

1. Wag A et al. Challenges and New Frontiers in Analytical Characterization of Antibody-Drug Conjugates. MAbs. 2018;10(2):222–243.
2. Li W et al. Antibody Aggregation: Insights from Sequence and Structure. Antibodies (Basel). 2016;5(3):19.
3. Chen Y. Drug-to-Antibody Ratio (DAR) by UV/Vis spectroscopy. Methods Mol Biol. 2013;1045:267–273.
4. Abedi M et al. Novel Trastuzumab-DM1 Conjugate: Synthesis and Bio-Evaluation. J Cell Physiol. 2019;234(10):18206–18213.
5. Padmanaban A, Menon S. Characterization of mAb Aggregation. Agilent Technologies Application Note. 2020.