Agilent MassHunter BioConfirm software - Protein Characterization Made Routine and Secure

Importance of the Topic

The accurate and efficient characterization of proteins and their post-translational modifications is essential in biopharmaceutical development and quality control. Streamlined workflows that integrate sample preparation, separation, detection, and data analysis reduce time-to-result, improve data integrity, and support regulatory compliance in GxP environments.

Objectives and Study Overview

This application note presents Agilent MassHunter BioConfirm software as a comprehensive solution for routine intact protein analysis, peptide mapping, and released glycan profiling. It aims to demonstrate how BioConfirm enhances confidence in molecular weight determination, sequence confirmation, PTM identification, and compliance-ready reporting.

Methodology and Instrumentation

BioConfirm employs advanced deconvolution algorithms to assign accurate intact protein masses and identify glycoforms or disulfide bonds. MS/MS–based peptide mapping narrows candidate peptides and visualizes sequence coverage across enzymes or sample conditions. The released glycan workflow integrates chemical tagging, curated databases, and spectral annotation to profile diverse glycan structures.

Key Results and Discussion

• Intact Protein Analysis: Rapid deconvolution produced clear molecular weight assignments, labeled PTMs in spectra, and provided relative quantitation of glycoforms via histograms.
• Peptide Mapping: Sequence coverage maps combined MS-only and MS/MS evidence, enabling quick comparison of digestion conditions and disulfide bond mapping.
• Released Glycan Profiling: Annotated glycan spectra aligned experimental isotope distributions with theoretical patterns, demonstrating sensitivity and specificity across custom and commercial tags.
• Compliance Features: Role-based access, audit trails with checksum-based tamper detection, and 21 CFR Part 11 / Annex 11 controls supported data integrity.
• Reporting: Customizable PDF templates allowed insertion of logos, tables, chromatograms, and spectra, streamlining report generation.

Benefits and Practical Applications

BioConfirm accelerates biopharmaceutical workflows by reducing manual data review, improving quantitative PTM analysis, and ensuring regulatory readiness. Laboratories can standardize methods for intact protein QC, peptide mapping for sequence verification, and glycan profiling for critical quality attributes.

Instrumentation

• AssayMAP Bravo sample preparation platform
• Agilent AdvanceBio liquid chromatography columns
• Agilent 1290 Infinity II Bio LC system
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer

Future Trends and Possibilities

Emerging developments may include artificial intelligence–driven spectral interpretation, expanded in silico glycan libraries, deeper integration of networked workstations with cloud-based data management, real-time process monitoring, and enhanced automation across sample prep and reporting.

Conclusion

Agilent MassHunter BioConfirm software delivers a unified, secure, and compliant platform for protein and glycan analysis. By combining robust algorithms with flexible reporting and regulatory controls, it empowers biopharma laboratories to achieve high confidence in critical quality attribute assessment.

References

Agilent Technologies. BioConfirm Application Note 5991-8466EN, April 2021.