Monitoring Product Quality Attributes of Biotherapeutics at the Peptide Level Using the Agilent InfinityLab LC/MSD XT System

Importance of the Topic

In biopharmaceutical development and quality control, precise monitoring of product quality attributes at the peptide level is essential to ensure safety, efficacy, and batch consistency. Peptide mapping by liquid chromatography–mass spectrometry provides detailed insight into sequence integrity, post-translational modifications, and glycosylation patterns. Single quadrupole LC/MS systems, valued for their robustness, simplicity, and cost efficiency, offer an attractive solution for routine analysis in regulated environments.

Objectives and Study Overview

This application note describes a generic peptide mapping workflow using the Agilent InfinityLab LC/MSD XT single quadrupole system coupled with an Agilent 1290 Infinity II LC and OpenLab ChemStation software. The study demonstrates simultaneous monitoring of multiple product quality attributes of a recombinant monoclonal antibody (NISTmAb), including complementary-determining region peptides, deamidated and oxidized peptides, and glycopeptides. It serves as a proof of concept for compliance-ready, single-method analysis in biopharmaceutical QC labs.

Methodology and Instrumentation

Sample Preparation

• NISTmAb reference material was stressed for deamidation (37 °C, pH 8.7, six days) and oxidation (0.002% H2O2, overnight).
• Samples were denatured, reduced, alkylated, digested with trypsin, and desalted using the Agilent AssayMAP Bravo platform.

Used Instrumentation

• Agilent 1290 Infinity II LC with RRHD 300Å StableBond C18 column (2.1×150 mm, 1.8 µm), flow rate 0.25 mL/min, column temperature 50 °C, gradient 1–90% acetonitrile with 0.1% formic acid.
• Agilent InfinityLab LC/MSD XT system with Jet Stream source operating in positive scan mode (m/z 360–1400), drying gas 11 L/min at 325 °C, sheath gas 10 L/min at 325 °C, nebulizer pressure 35 psi, cycle time 0.62 s.
• Agilent OpenLab ChemStation software (version C.01.09) for data acquisition, extracted ion chromatogram (EIC) workflows, and intelligent reporting.

Main Results and Discussion

Performance evaluation with 2.5 µg NISTmAb digest demonstrated high sensitivity and fast scan capabilities of the single quadrupole system. Automated EIC extraction was applied to 15 predefined peptides to enable:

• Confirmation of CDR peptide identities and retention times for product identity verification.
• Quantification of deamidated (H387) and oxidized (H87) peptide forms before and after stress induction, showing expected increases post-stress.
• Relative abundance profiling of key glycopeptides (G0F, G1F, G2F) consistent with high-resolution LC/MS/MS data.
• Streamlined data processing and report generation supporting traceable and compliant workflows.

Benefits and Practical Applications

• A cost-effective, robust single quadrupole LC/MS solution for routine peptide mapping.
• Generic method enabling simultaneous monitoring of multiple quality attributes in one run, reducing analysis time and complexity.
• Compliance-ready data management with OpenLab ECM features such as audit trail, electronic signatures, and secure archival.
• Scalability for high-throughput QC laboratories requiring regulatory compliance.

Future Trends and Opportunities

Adoption of single quadrupole LC/MS workflows for broader biotherapeutic modalities, including fusion proteins and antibody–drug conjugates, is expected. Integration with advanced data analytics and machine learning may further enhance attribute tracking. Development of targeted quantitation approaches using SIM or MRM on SQ platforms could improve sensitivity for low-level modifications. Cloud-based data management and real-time monitoring will facilitate process analytical technology and continuous manufacturing initiatives.

Conclusion

The Agilent InfinityLab LC/MSD XT system combined with Agilent 1290 Infinity II LC and OpenLab ChemStation provides a simple, generic, and compliance-ready workflow for peptide level monitoring of multiple product quality attributes. Automated EIC extraction and intelligent reporting enable high-throughput, traceable analysis of CDR regions, post-translational modifications, and glycopeptides in monoclonal antibodies, delivering a cost-effective solution for routine quality control in biopharmaceutical laboratories.

References

1. OpenLab CDS ChemStation Edition with Content Management Systems Users Guide Agilent Technologies M8301-90089 Rev B
2. OpenLab CDS ChemStation Edition C.01.09 Guide for Administrators Agilent Technologies M8305-90019 Rev B
3. Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform Agilent Technologies 5991-4872EN
4. Dong Q et al The NISTmAb Tryptic Peptide Spectral Library for Monoclonal Antibody Characterization MAbs 2018 10(3) 354–369
5. Dong Q et al In-Depth Characterization and Spectral Library Building of Glycopeptides in the Tryptic Digest of a Monoclonal Antibody Using 1D and 2D LC–MS/MS J Proteome Res 2016 15(5) 1472–1486
6. Quantitation of Chemical-Induced Deamidation and Oxidation on Monoclonal Antibodies Using Agilent 6545XT AdvanceBio LC/Q-TOF and MassHunter BioConfirm Software Agilent Technologies 5994-0406EN
7. Haberger M et al Assessment of Chemical Modifications of Sites in the CDRs of Recombinant Antibodies Susceptibility versus Functionality of Critical Quality Attributes MAbs 2014 6(2) 327–339