Monoclonal Antibody Workflows on the Shimadzu Q-TOF LCMS-9030 Using the Protein Metrics Software Suite

Significance of the Topic

Monoclonal antibodies (mAbs) are prominent biotherapeutics used in diagnostics and treatment of numerous diseases. Thorough molecular characterization of mAbs ensures batch consistency, efficacy, and safety during development and production. High-resolution liquid chromatography–mass spectrometry (LC-MS) is a powerful analytical tool to dissect the structural complexity of intact antibodies, their subunits, and post-translational modifications such as glycosylation.

Study Objectives and Overview

This application note demonstrates a comprehensive workflow for mAb analysis using the Shimadzu Q-TOF LCMS-9030 combined with Protein Metrics software. The recombinant human IgG1κ NIST mAb reference standard serves as a model system to illustrate:

• Intact mass analysis and deconvolution of major glycoforms.
• Subunit (heavy and light chain) profiling after reduction.
• Peptide mapping with multiple proteases for sequence confirmation.
• Assessment of N-glycan removal by PNGase F deglycosylation.

Methodology and Instrumentation

The workflow comprises three tiers:

1. Intact and reduced subunit analysis: NIST mAb was diluted to 1 mg/mL, reduced with DTT, and optionally deglycosylated with PNGase F. Separation was achieved on a Restek Ultra C4 column, followed by MS acquisition (m/z 1 000–4 000 for intact, 800–4 000 for subunits).
2. Proteolytic digestion: Heavy and light chains were alkylated, then digested with trypsin, Glu-C, and Lys-C at 1:25 enzyme-to-substrate ratio. Peptides were desalted and separated on a Restek Raptor ARC-18 column, with data-dependent acquisition in the m/z 100–2 000 range.
3. Data analysis: Protein Metrics Intact Mass and PTM Workflows were employed to deconvolute mass spectra, assign glycoforms, and confirm peptide sequences against theoretical in silico digests.

Key Results and Discussion

Intact mAb analysis yielded chromatograms and deconvoluted spectra consistent with theoretical masses for major glycoforms, with mass errors below 25 ppm. Reduced subunit profiling separated light and heavy chains clearly, confirming expected masses. Peptide mapping with combined proteases achieved complete sequence coverage for both chains. PNGase F treatment effectively removed N-glycans, evidenced by mass shifts in intact and subunit spectra.

Benefits and Practical Applications

This integrated LC-MS approach provides:

• Rapid verification of mAb identity and glycoform distribution.
• Accurate subunit mass measurement to detect truncations or modifications.
• Comprehensive peptide mapping to confirm sequence integrity and locate PTMs.
• Flexible workflows supporting intact, subunit, and peptide-level analyses in a single platform.

Future Trends and Potential Use Cases

Advancements in high-resolution mass spectrometry and software algorithms will streamline biotherapeutic characterization further. Potential directions include automation of sample preparation, deeper glycan structural elucidation, high-throughput screening in QA/QC environments, and application to novel antibody formats such as bispecifics and antibody–drug conjugates.

Conclusion

The Shimadzu Q-TOF LCMS-9030 paired with Protein Metrics software offers a robust, end-to-end solution for detailed monoclonal antibody characterization. Its high sensitivity, mass accuracy, and adaptable workflows enable researchers and quality laboratories to ensure product consistency and accelerate biotherapeutic development.

Instrumentation Used

• Shimadzu UHPLC: LC-30AD binary pumps, DGU-20A5R degasser, SIL-30ACMP autosampler, CTO-20AC oven, CBM-20A controller.
• Shimadzu Q-TOF LCMS-9030 mass spectrometer with LabSolutions Ver. 5.95.
• Columns: Restek Ultra C4 for intact/subunit; Restek Raptor ARC-18 for peptide mapping.

Reference

• Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm. 1999;185(2):129–188.
• Chen G. Characterization of Protein Therapeutics using Mass Spectrometry. Springer, New York; 2013.
• Varki A, Cummings RD, Esko JD, et al. Essentials of Glycobiology. 2nd ed. Cold Spring Harbor Laboratory Press; 2009.