Conjugate, clean-up, & characterize ADCs on Unagi & Stunner

Importance of the topic

Antibody-drug conjugates (ADCs) represent a critical class of targeted therapeutics that combine monoclonal antibodies, linkers, and cytotoxic payloads. Effective ADC development depends on precise control of the drug-antibody ratio (DAR), removal of reaction by-products, and monitoring of aggregation. Streamlined workflows for ADC purification and rapid characterization accelerate optimization and ensure product quality in research, QA/QC, and industrial settings.

Objectives and overview of the study

This study demonstrates an integrated bench-scale workflow for ADC conjugation, automated clean-up, and characterization. A human monoclonal antibody was conjugated with three different fluorescent payloads—FITC, Alexa Fluor™ 350 NHS ester, and AF350 maleimide—at two molar ratios (2:1 and 10:1). The performance of automated buffer exchange and desalting on the Unagi platform was assessed, followed by simultaneous UV/Vis and dynamic light scattering (DLS) analysis on the Stunner to quantify concentration, DAR, size, and aggregation profiles.

Methodology and instrumentation

Monoclonal antibody was prepared at 10 mg/mL in borate, bicarbonate, or PBS buffers and reduced where required. Conjugations were performed with dye-to-protein ratios of 2:1 and 10:1 at room temperature. Post-reaction mixtures were processed on the Unagi system using a 10 kDa MWCO filter with controlled pressure (60 psi), mixing (700 rpm), and automated volume and dilution cycles. Final samples were analyzed on the Stunner using UV/Vis absorbance deconvolution and DLS (five 1-second acquisitions) to determine protein concentration, DAR, hydrodynamic diameter, and polydispersity index (PDI).

Použitá instrumentace

• Unagi automated benchtop ultrafiltration/diafiltration system
• Stunner plate-based UV/Vis and DLS analyzer
• 0.1 µm syringe filters and standard lab centrifuge
• Stunner software modules: Store Analyte, ADC + Sizing

Main results and discussion

Automated buffer exchange on Unagi achieved >90% recovery and concentration accuracy within ±5% of the 10 mg/mL target across three reaction buffers. DLS before and after exchange showed monodisperse mAb populations (diameters around 10–12 nm, PDI <0.1), confirming minimal aggregation. Stunner’s spectral deconvolution accurately separated antibody and dye contributions, enabling precise DAR calculation. Measured DARs correlated with input molar ratios and reaction chemistry: AF350 NHS ester displayed higher conjugation efficiency than maleimide or FITC. Unagi removed >96% of unreacted dye, as evidenced by reduced post-exchange optical signals. Conjugated ADCs showed slight size increases consistent with payload attachment; PDI values remained low (<0.1 for most samples), indicating preserved sample homogeneity.

Benefits and practical applications

• Hands-off automation reduces manual steps and sample dilution.
• High-throughput processing (up to 96 samples on Stunner, 8 on Unagi).
• Low sample volumes (2 µL for Stunner analysis) conserve precious materials.
• Integrated UV/Vis and DLS analysis accelerates DAR and aggregation assessment.
• Scalable workflow supports early-stage screening and process optimization.

Future trends and opportunities

Expanding automated clean-up and analytics to a broader range of linkers and payloads will enhance ADC screening libraries. Integration of inline real-time monitoring and machine-learning based spectral models could further streamline process development. Adapting similar workflows to other bioconjugates, such as antibody–oligonucleotide or protein–polymer conjugates, will broaden applications in drug delivery and diagnostics.

Conclusion

The combined use of Unagi for precise buffer exchange and Stunner for rapid UV/Vis-DLS characterization provides a robust, high-throughput workflow for ADC development. This strategy ensures efficient conjugation, thorough removal of impurities, and reliable measurement of critical quality attributes, thereby accelerating ADC process development and ensuring sample integrity.

Reference

• Chen Y. Drug-to-Antibody Ratio (DAR) by UV/Vis Spectroscopy. In: Ducry L, editor. Antibody-Drug Conjugates. Totowa, NJ: Humana Press; 2013:267–273.
• Cockrell GM, Blanco LP, Fadden SI, et al. Photoinduced Aggregation of a Model Antibody–Drug Conjugate. Molecular Pharmaceutics. 2015;12(6):1784–1797.