Agilent MassHunter BioConfirm software - Enabling Routine Characterization of Proteins

Importance of the Topic

Rapid and reliable characterization of protein therapeutics is critical in biopharmaceutical research and quality control. Detailed information on molecular weight, post-translational modifications, glycoforms and drug-to-antibody ratios supports decision making throughout development, ensuring safety, efficacy and regulatory compliance.

Objectives and Study Overview

This study introduces the MassHunter BioConfirm platform as an integrated solution for routine intact protein analysis, peptide mapping, released glycan profiling and antibody–drug conjugate (ADC) DAR calculation. It highlights workflow automation from sample preparation through data reporting, aiming to streamline biopharma analytics and improve throughput.

Methodology and Instrumentation

MassHunter BioConfirm workflows combine advanced sample handling, high-resolution separation, accurate mass detection and automated data processing:

• Sample Preparation: Automated pipetting with AssayMAP Bravo cartridges for reproducible protein digests and glycan release.
• Separation: AdvanceBio analytical columns coupled to Agilent 1290 Infinity II UHPLC for high-efficiency chromatographic resolution.
• Detection: 6545XT AdvanceBio LC/Q-TOF mass spectrometer delivering accurate mass measurements for intact proteins, peptides and glycans.
• Data Processing: MassHunter BioConfirm software for deconvolution, sequence confirmation, glycan database searching and one-click DAR calculation; Walkup environment for queue management and PDF report generation.

Key Results and Discussion

The platform demonstrated robust performance across multiple workflows:

• Intact Protein Analysis: Automatic deconvolution of mass spectra to determine molecular weight, label glycoforms on spectra and quantify relative abundances via histograms.
• Peptide Mapping: MS/MS-driven identification of peptides, generating sequence coverage maps for different proteases and enabling fast disulfide bond mapping in monoclonal antibodies.
• Released Glycan Profiling: Annotated mass spectra of labeled glycans, supported by a curated database and customizable entries for comprehensive glycan characterization.
• ADC DAR Calculation: Automated determination of drug-to-antibody ratios for intact and reduced ADCs with minimal manual input, producing standardized reports.

Benefits and Practical Applications

BioConfirm streamlines biopharmaceutical analyses by reducing setup time, minimizing manual data review and enhancing reproducibility. Its intuitive interface allows non-experts to obtain expert-level results, making it suitable for process development, quality control and comparability studies.

Future Trends and Potential Applications

Emerging developments may include integration of machine learning for peak annotation, expansion of glycan libraries with predictive modeling, cloud-based data sharing and remote monitoring. Further automation of DAR prediction and single-cell proteomics workflows could extend the platform’s reach in advanced biopharma research.

Conclusion

Agilent MassHunter BioConfirm provides a comprehensive, automated suite for protein therapeutic characterization, from intact mass analysis to detailed peptide and glycan profiling. By unifying sample prep, instrumental analysis and report generation, it accelerates workflows and supports high-confidence decision making in biopharmaceutical development.