Agilent Biopharma Workflow Solutions

Significance of the Topic

Antibody-drug conjugates (ADCs) combine the targeting specificity of monoclonal antibodies with the potency of small-molecule drugs. Accurate determination of the drug-to-antibody ratio (DAR) is essential to balance efficacy, minimize off-target toxicity, and optimize pharmacokinetics during drug discovery and quality control.

Goals and Study Overview

This workflow guide presents a suite of complementary strategies for ADC characterization. Key objectives include quantifying intact ADC distributions, simplifying spectral complexity through chemical processing, and mapping payload attachment sites at the peptide level.

Methodology and Instrumentation

Analytical approaches described:

• Intact ADC analysis by UHPLC coupled to high-resolution TOF/Q-TOF MS for DAR distribution and glycoform profiling.
• Chemical modifications: deglycosylation, reduction to separate light and heavy chains, and sequential reduction/deglycosylation to simplify spectra and determine chain-specific DAR.
• Peptide mapping: denaturation, reduction, alkylation, enzymatic digestion, and cleanup for site-specific payload localization and occupancy assessment.

Instrumentation Used:

• Agilent 1290 Infinity LC System (Binary Pump, TCC, Sampler, FC/ALSTherm).
• Agilent 6230 TOF LC/MS and 6530/6550 iFunnel Q-TOF LC/MS.
• Analytical columns: PLRP-S, Poroshell 300SB-C3/C8, AdvanceBio RP-mAb, AdvancedBio Peptide Map, RRHD Eclipse Plus C18.
• Software: MassHunter Qualitative Analysis, BioConfirm, DAR Calculator, Walkup, and AssayMAP Bravo Protein Sample Prep Workbench.

Main Results and Discussion

Intact LC/MS workflows yield detailed DAR distributions and glycoform masses. Deglycosylation and reduction steps markedly simplify spectra, enabling precise deconvolution and reproducible DAR calculations via integrated software. Peptide mapping protocols unambiguously identify conjugation sites and quantify site occupancy, informing structure–activity relationships.

Benefits and Practical Applications

• Enhanced accuracy in DAR determination for lead candidate selection.
• Reduced spectral complexity through targeted chemical processing.
• Automated sample preparation and data analysis for increased throughput and reproducibility.
• Site-specific payload localization supporting pharmacokinetic and efficacy studies.

Future Trends and Opportunities

Advances in full automation of sample prep, real-time DAR monitoring, AI-driven spectral deconvolution, and multiplexed MS approaches are poised to further accelerate ADC development. Enhanced software interoperability and cloud-based analytics will democratize ADC characterization across diverse laboratory settings.

Conclusion

Combining intact LC/MS analysis, targeted chemical workflows, and peptide-level mapping delivers a robust, multi-dimensional toolkit for ADC DAR characterization. Integrated hardware and software platforms optimize precision, throughput, and accessibility for research and quality control.

References

1. Agilent Technologies. Analysis of Antibody-Drug Conjugate Through Drug Antibody Ratio Characterization Using 1290 Infinity LC and 6550 iFunnel Q-TOF. Application Note, 2016.
2. Agilent Technologies. Drug-to-Antibody Ratio Calculation of ADCs Using Automated Sample Preparation and DAR Calculator Software. Application Note, 2016.
3. Agilent Technologies. DAR Calculation of ADCs Purified from Serum Using Automated Affinity Purification, LC/MS, and DAR Software. Application Note, 2016.
4. Agilent Technologies. Mapping Drug Conjugation Sites of an ADC Using Automated Sample Preparation and LC/MS Analysis. Application Note, 2016.
5. Agilent Technologies. Automation for High Throughput In-Solution Digestion and Peptide Cleanup via AssayMAP Bravo. Application Note, 2016.
6. Agilent Technologies. Integrated Workflow for Automated DAR Calculation Using MassHunter Walkup. Application Note, 2016.