Analysis of pharmaceuticals and drug related impurities using Agilent instrumentation

Peptide Mapping of a Monoclonal Antibody Using a Microfluidic-based HPLC-Chip Coupled to an Agilent Accurate-Mass Q-TOF LC/MS

Significance

Peptide mapping provides definitive confirmation of the amino acid sequence of therapeutic monoclonal antibodies (mAbs). High-resolution LC/MS peptide maps can detect sequence variants, post-translational modifications, and degradation products in complex biopharmaceuticals. The integration of microfluidic HPLC-Chip technology with an accurate-mass Q-TOF MS offers high sensitivity and mass accuracy when only nanogram quantities are available.

Study Overview

• A purified mAb was digested with trypsin to generate peptide fragments.
• Peptide mixtures were separated on an Agilent HPLC-Chip with integrated enrichment and analytical columns.
• Eluting peptides were analyzed by an Agilent Accurate-Mass Q-TOF LC/MS.
• Data were processed using Agilent MassHunter and BioConfirm software to assign peptide sequences and confirm modifications.

HPLC-Chip/MS Method

• Trap column: 40 nL enrichment phase; analytical column: 75 µm × 150 mm, C18, 5 µm.
• Gradient: 5→40% acetonitrile in 0.1% formic acid over 40 min at 0.6 µL/min.
• MS settings: positive ESI, capillary voltage 3.8 kV, fragmentor 175 V, m/z 300–1700, acquisition rate 1–2 spectra/s.
• Data-dependent MS/MS for peptide fragmentation and sequence confirmation.

Main Results

• Complete sequence coverage achieved for both heavy and light chains of the mAb using 50 ng of digest per injection.
• Mass accuracy of peptide precursor ions consistently within ±4 ppm provided high confidence in peptide assignments.
• Agilent BioConfirm software automatically matched observed masses and MS/MS spectra to the theoretical digest, revealing 100% coverage for all expected peptides.
• Low carryover and excellent retention time reproducibility (RT RSD ≤0.1%) demonstrated system robustness.

Benefits and Practical Application

• The microfluidic HPLC-Chip design enables low dead volume and high sensitivity with minimal sample consumption (nanogram levels).
• Accurate-mass Q-TOF detection ensures reliable identification of low-abundance peptides and subtle modifications.
• Automated data processing in MassHunter and BioConfirm accelerates method development and routine sequence confirmation.

Conclusions

• The Agilent HPLC-Chip/Q-TOF LC/MS platform provides a robust, sensitive, and accurate solution for mAb peptide mapping in a routine laboratory environment.
• Mass accuracy, seamless chromatography, and advanced data analysis tools enable confident sequence confirmation and modification analysis.