Peptide Mapping - Agilent BioHPLC Columns Application Compendium

Importance of the topic

Peptide mapping is a cornerstone analytical technique in biopharmaceutical development and quality control. It provides a precise "fingerprint" of protein therapeutics, enabling confirmation of primary sequence, detection of post-translational modifications, and monitoring of critical quality attributes such as oxidation, deamidation, glycosylation, and charge variants. High-throughput, reproducible peptide maps support comparability studies, biosimilar evaluation, and ongoing process monitoring in regulated environments.

Objectives and overview

This compendium of application notes demonstrates streamlined workflows for peptide mapping of monoclonal antibodies using Agilent technologies. Key goals include:

• Automating sample preparation to reduce hands-on time and variability (AssayMAP Bravo).
• Accelerating separations with superficially porous C18 columns (AdvanceBio Peptide Mapping).
• Coupling high-resolution mass spectrometry (6545XT AdvanceBio LC/Q-TOF) and compliance-ready single-quadrupole detection (InfinityLab LC/MSD XT) to achieve detailed peptide identification and quantification.
• Implementing software tools (MassHunter BioConfirm and OpenLab ChemStation) for automatic data processing, reporting, and regulatory compliance.

Methods and instrumentation

Sample preparation employed robotic in-solution denaturation, reduction/alkylation, digestion (trypsin/Lys-C), and desalting on the Agilent AssayMAP Bravo. Chromatographic separations used:

• Agilent AdvanceBio Peptide Mapping columns (2.7 µm superficially porous C18, 120 Å) on 1290 Infinity II UHPLC: 0–65% ACN over 15 min at 0.4 mL/min, 55–60 °C.
• Agilent ZORBAX 300StableBond C18 (1.8 µm, 300 Å) on 1290 Infinity II LC: 1–35% ACN over 5 min, 35–90% over 2 min at 0.25 mL/min, 50 °C for single-quadrupole methods.

Mass detection was performed on:

• 6545XT AdvanceBio LC/Q-TOF (extended dynamic range, 2 GHz), ESI source, MS and auto MS/MS acquisition for comprehensive mapping.
• InfinityLab LC/MSD XT single quadrupole (m/z 360–1,400), ESI with JetStream, for targeted PQA monitoring.

Main results and discussion

Automated workflows produced complete peptide maps of NISTmAb in under 20 minutes. BioConfirm software achieved >99% sequence coverage with <5 ppm mass errors and annotated PTMs such as methionine oxidation and asparagine deamidation. Reproducibility studies on AdvanceBio columns showed consistent retention times and peak shapes over 200 injections and across lots. The single-quad LC/MSD XT system successfully monitored multiple PQAs—complementarity-determining region (CDR) peptides, oxidized/deamidated forms, and glycopeptides—in a single run using extracted ion chromatograms with tailored OpenLab ChemStation processing methods.

Benefits and practical application

• Automation on the AssayMAP Bravo reduced hands-on time by ~50% and improved reproducibility (<5% CV).
• AdvanceBio Peptide Mapping columns delivered 2–3× faster separations than fully porous media without sacrificing resolution, compatible with legacy and UHPLC instruments.
• 6545XT Q-TOF provided high sensitivity and resolution for full peptide mapping and PTM localization.
• InfinityLab LC/MSD XT offered a cost-effective, robust platform for routine PQA monitoring with simple software integration.
• MassHunter BioConfirm and OpenLab ChemStation enabled end-to-end automation of data analysis, reporting, and compliance controls.

Future trends and possibilities

• Integration of peptide mapping with multi-attribute methods (MAM) for simultaneous analysis of sequence variants, PTMs, and host-cell impurities.
• Increased use of high-throughput robotics and microflow LC to reduce solvent consumption and increase assay density.
• Application of real-time data analytics and machine learning for peak detection, PTM prediction, and proactive quality monitoring.
• Expansion of single-quadrupole LC/MS into regulated QC labs for targeted attribute monitoring, complementing high-resolution methods for detailed characterization.

Conclusion

The combination of automated sample preparation, rapid UHPLC separations, versatile column chemistries, and both high-resolution and single-quad MS detection transforms peptide mapping from a laborious task into a routine, high-throughput operation. Agilent’s integrated hardware and software solutions deliver robust, reproducible, and compliance-ready workflows, facilitating accelerated biotherapeutic development and stringent quality control.

Použitá instrumentace

• Agilent AssayMAP Bravo liquid-handling platform
• Agilent 1290 Infinity II UHPLC system
• Agilent AdvanceBio Peptide Mapping columns
• Agilent ZORBAX 300StableBond C18 columns
• Agilent 6545XT AdvanceBio LC/Q-TOF system
• Agilent InfinityLab LC/MSD XT single-quadrupole mass detector
• Agilent MassHunter BioConfirm B.08 software
• Agilent OpenLab ChemStation C.01.09

Reference

1. Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform. Agilent Technologies, publication 5991-2957EN.
2. Fast and Efficient Peptide Mapping of a Monoclonal Antibody (mAb): UHPLC Performance with Superficially Porous Particles. Agilent Technologies, publication 5991-3585EN.
3. High Resolution and Rapid Peptide Mapping of Monoclonal Antibody Using an Agilent 1290 Infinity UHPLC and an Agilent 6550 iFunnel Q-TOF LC/MS System. Agilent Technologies, publication 5991-3600EN.
4. Quantitation of Chemical-Induced Deamidation and Oxidation on Monoclonal Antibodies Using Agilent 6545XT AdvanceBio LC/Q-TOF and Agilent MassHunter BioConfirm Software. Agilent Technologies, publication 5994-0406EN.
5. The NISTmAb Tryptic Peptide Spectral Library for Monoclonal Antibody Characterization. Dong Q. et al., MAbs 2018, 10(3):354–369.
6. In-Depth Characterization and Spectral Library Building of Glycopeptides in the Tryptic Digest of a Monoclonal Antibody Using 1D and 2D LC–MS/MS. Dong Q. et al., J. Proteome Res. 2016, 15(5):1472–1486.
7. Assessment of Chemical Modifications of Sites in the CDRs of Recombinant Antibodies: Susceptibility versus Functionality of Critical Quality Attributes. Haberger M. et al., MAbs 2014, 6(2):327–339.