Separation of Trypsin Digested Monoclonal Antibody

Importance of the Topic

The detailed characterization of monoclonal antibodies by peptide mapping is critical for biopharmaceutical quality control and stability studies. High-resolution separation and accurate mass detection of tryptic digests enable verification of sequence integrity, post-translational modifications, and batch consistency. This approach supports regulatory compliance and ensures therapeutic safety and efficacy.

Objectives and Study Overview

This application note describes the separation and mass spectrometric analysis of trypsin-digested monoclonal antibody reference material (NIST RM 8671). The primary objectives are to demonstrate chromatographic performance of the Shim-pack GISS C18 column and to evaluate the LC-MS method for comprehensive peptide mapping of a complex biopolymer.

Methodology and Instrumentation

The peptide mapping workflow combines reversed-phase ultra-high-performance liquid chromatography (UHPLC) with high-resolution mass spectrometry. Key methodological features include:

• Sample preparation: Trypsin digestion of NIST monoclonal antibody reference material.
• Chromatography: Shim-pack GISS C18 column (150 × 2.1 mm, 1.9 µm) operated at 50 °C with a gradient from 1 % to 60 % acetonitrile over 112 minutes at 0.2 mL/min.
• Mass spectrometry: LCMS-9030 equipped with heated electrospray ionization in positive mode, scanning m/z 350–2000.

Results and Discussion

The total ion chromatogram shows baseline separation of over 50 tryptic peptides with sharp, symmetric peaks spanning a 0–70 minute retention window. High-resolution MS data confirm accurate mass assignments and enable detection of minor modifications such as deamidation and oxidation. Consistent retention times and signal intensities demonstrate column reproducibility and method robustness.

Benefits and Practical Applications

The described LC-MS setup offers:

• Comprehensive sequence coverage for monoclonal antibody mapping.
• High sensitivity for low-abundance variant detection.
• Reproducible chromatography suitable for routine QC laboratories.
• Flexibility to adapt gradient programs for different biopharmaceutical molecules.

Future Trends and Opportunities

Advances in peptide mapping may include faster gradient capabilities, integration of ion mobility separation, automated data processing pipelines, and targeted MS/MS workflows for in-depth characterization of glycoforms and other modifications. Combining these innovations will further streamline biopharmaceutical analysis and accelerate product development.

Conclusion

The Shim-pack GISS C18 column paired with the LCMS-9030 system delivers high-resolution, reproducible peptide mapping of monoclonal antibodies. This method supports rigorous quality control, enabling reliable detection of sequence variants and post-translational modifications in complex biotherapeutic samples.

Used Instrumentation

• UHPLC: Nexera X2 system with Shim-pack GISS C18 (150 × 2.1 mm, 1.9 µm).
• Mobile phases: 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B).
• Gradient: 1 % B (0–0.5 min) → 60 % B (0.5–112 min) → 95 % B (112.05–117 min) → 1 % B (117.01–120 min).
• Flow rate: 0.2 mL/min; column oven: 50 °C; injection: 1 µL.
• Mass spectrometer: Shimadzu LCMS-9030, heated ESI positive mode, DL 250 °C, interface 300 °C, heat block 400 °C, voltage 4.5 kV, scan range m/z 350–2000.