Separation of Trypsin Digested Monoclonal Antibod

Significance of the Topic

Peptide mapping using liquid chromatography–mass spectrometry (LC-MS) is essential for detailed characterization of monoclonal antibodies (mAbs). It enables identification of sequence variants, post-translational modifications and confirmation of glycosylation patterns. High-resolution separation and accurate mass detection are critical for ensuring product quality and comparability in biopharmaceutical development.

Objectives and Study Overview

This study demonstrates the separation performance of a Shim-pack Arata Peptide C18 column in combination with a Nexera X2 UHPLC system and LCMS-9030 mass spectrometer. The aim is to achieve comprehensive peptide coverage of trypsin-digested NIST mAb Reference Material 8671 and to evaluate chromatographic resolution, reproducibility and sensitivity.

Methodology and Used Instrumentation

UHPLC Conditions:

• Column: Shim-pack Arata Peptide C18, 100 × 2.0 mm, 2.2 µm particle size
• Column temperature: 50 °C
• Mobile phases: A—0.1% formic acid in water; B—0.1% formic acid in acetonitrile
• Gradient: 1% B (0–0.5 min) to 60% B (112 min), 95% B (112.05–117 min), re-equilibration to 1% B (117.01–120 min)
• Flow rate: 0.2 mL/min; Injection volume: 2 µL

Mass Spectrometry Conditions:

• Instrument: Shimadzu LCMS-9030 with heated electrospray ionization (ESI) in positive mode
• Interface voltage: 4.5 kV; DL temperature: 250 °C; Heat block: 400 °C; Interface temperature: 300 °C
• Scan range: m/z 350–2000; Nebulizer gas: 2 L/min; Drying and heating gas: 10 L/min each

Main Results and Discussion

The total ion chromatogram (TIC) displayed well-resolved peptide peaks across an 85-minute gradient. Key observations include:

• High peak capacity enabling separation of closely eluting peptides derived from the heavy and light chains.
• Consistent retention times across replicate injections, indicating robust column performance.
• Sensitivity sufficient to detect low-abundance peptides and potential modifications.

The combination of a shallow gradient and optimized temperature control contributed to enhanced resolution of hydrophobic peptides.

Benefits and Practical Applications

This peptide mapping workflow offers several advantages:

• Comprehensive sequence coverage for identity confirmation and variant detection.
• Reproducible performance for lot-to-lot comparison in quality control.
• Scalability for high-throughput environments due to stable baseline and column longevity.

Applications include biosimilar development, stability studies and in-process monitoring during mAb production.

Future Trends and Opportunities for Application

Advancements likely to impact peptide mapping include:

• Faster gradients and sub-2 µm particle columns to reduce analysis time.
• Integration of ion mobility spectrometry for enhanced separation of isobaric peptides.
• Automated data processing pipelines and machine learning for PTM identification and quantitation.
• Miniaturized and microflow LC-MS setups to decrease solvent consumption and improve sensitivity.

Conclusion

The described LC-MS method using Shim-pack Arata Peptide C18 and Nexera X2/LCMS-9030 provides high-resolution, reproducible peptide mapping of trypsin-digested monoclonal antibodies. It supports critical quality attributes assessment and can be adapted for advanced biopharmaceutical applications.