Agilent MassHunter DAR Calculator Software - Quick Start Guide

Significance of the Topic

The accurate determination of drug-to-antibody ratio (DAR) in antibody-drug conjugates (ADCs) is critical for therapeutic efficacy, safety, and quality control in biopharmaceutical development. Reliable DAR calculation supports process development, formulation stability studies, and final product release testing.

Objectives and Study Overview

This Quick Start Guide describes the installation, user interface, and core workflows for Agilent MassHunter DAR Calculator Software B.01.01. It outlines manual and fully automated procedures to process deconvoluted LC/MS data from ADCs, generate reports, and integrate DAR calculations into broader MassHunter BioConfirm, Data Acquisition, and Walkup environments.

Methodology and Instrumentation

The DAR Calculator is a standalone application that imports CSV files exported from the MassHunter BioConfirm deconvolution algorithm (Intact or Reduced). Key methodological steps include:

• Loading deconvoluted mass spectra for intact ADCs or separate heavy/light chains
• Defining the unconjugated antibody mass (DAR 0) and drug/linker mass
• Adjusting peak detection parameters, integration limits, and maximum DAR peaks
• Reviewing and editing peak assignments interactively or via a peak list table
• Generating DAR reports in PDF or DOCX formats

Instrumentation required:

• Agilent LC/MS system with Time-of-Flight (TOF) or Q-TOF detector
• MassHunter Workstation software (BioConfirm B.08.00+, Data Acquisition, Walkup)
• Microsoft Excel 2013 or 2016 for reporting

Main Results and Discussion

New features in version B.01.01 enable direct execution of DAR calculations within BioConfirm and automated DAR columns in Data Acquisition and Walkup. The guide demonstrates basic tasks:

• Installing the G4994AA DAR Calculator add-on and configuring Excel integration
• Navigating the user interface: File menu, Ribbon groups, spectrum and peak list panes
• Processing intact ADC CSV files: setting DAR 0 and drug/linker masses, selecting linkage type (Lysine-linked or Cystine-linked)
• Handling reduced ADC data: separate heavy and light chain spectra, combined DAR determination
• Editing integration boundaries, deleting or adding peaks, and customizing global or per-file methods
• Exporting peak tables (CSV/XLS) and spectrum images (BMP, JPEG, PNG, TIFF)
• Implementing automated workflows: creating DAR methods, embedding them in BioConfirm custom workflows, linking in Data Acquisition methods, and configuring Walkup methods/workflows with DAR 0 and drug/linker input columns

The integrated workflow reduces manual intervention, ensures parameter consistency across runs, and supports high-throughput sample analysis.

Benefits and Practical Applications

By centralizing DAR computations in a dedicated tool and embedding them into automated sequences, laboratories can achieve:

• Standardized and reproducible DAR determinations for regulatory compliance
• Rapid turnaround of ADC characterization data to guide process optimization
• Seamless integration with Agilent’s MassHunter ecosystem for end-to-end data handling
• Flexible reporting options to support documentation and archival requirements

Future Trends and Opportunities

Emerging enhancements may include:

• Advanced deconvolution algorithms with improved resolution of overlapping DAR species
• Machine learning-driven peak selection and quality control flags
• Cloud-based data management and collaboration features
• Extension of DAR analysis workflows to novel bioconjugates beyond classical ADC formats

Conclusion

Agilent MassHunter DAR Calculator Software provides an expert solution for accurate DAR analysis of ADCs, offering both interactive and fully automated workflows that integrate seamlessly with BioConfirm, Data Acquisition, and Walkup platforms. Its robust feature set enhances data integrity, throughput, and regulatory readiness in biotherapeutic development.

Reference

Agilent Technologies, MassHunter DAR Calculator Software Quick Start Guide, Revision A, June 2016.