Monoclonal Antibody (mAb) Intact Mass and Subunit Analysis Using Shimadzu Q-TOF LCMS-9030

Significance of the Topic

Monoclonal antibodies serve as critical therapeutics in areas such as oncology and autoimmune diseases. Accurate molecular characterization of these large heterogeneous proteins underpins assessment of critical quality attributes in drug development and regulatory approval. Liquid chromatography coupled with mass spectrometry provides a powerful platform for both intact mass and subunit profiling of antibodies, revealing detailed information on glycosylation, disulfide linkage and oxidative modifications.

Objectives and Study Overview

This application note demonstrates a rapid and streamlined workflow for intact mass and subunit analysis of a humanized IgG1k reference material using the Shimadzu Q-TOF LCMS-9030. The goals are to validate mass accuracy against theoretical values, resolve glycoform distributions and illustrate a practical workflow for routine biopharma quality control.

Methodology

Sample Preparation

• Intact mass: Dilution of reference antibody to 0.1 mg/mL in 0.1% formic acid
• Subunit analysis: Denaturation and reduction with TCEP at 95 °C, enzymatic deglycosylation with PNGase F at 50 °C, final dilution to 0.1 mg/mL

Chromatographic Conditions

• Column: Shim pack Scepter C4 2.1 × 150 mm, 1.9 µm
• Mobile phases: 0.1% formic acid in water and acetonitrile
• Flow rate: 0.3 mL/min, gradient elution over 18 min, column temperature 80 °C
• Injection volume: 5 µL

Mass Spectrometry Conditions

• Instrument: Shimadzu LCMS-9030 Q-TOF with heated electrospray ionization at 4.5 kV
• Interface temperatures: DL 200 °C, heat block 400 °C
• Gas flows: nebulizing 3 L/min, heating 10 L/min, drying 10 L/min
• Acquisition: positive MS mode, m/z 300–4500

Used Instrumentation

• Shimadzu LCMS-9030 Q-TOF mass spectrometer
• Shim pack Scepter C4-300 column
• Shimadzu Torast-H Bio Vials
• Protein Metrics data processing software

Main Results and Discussion

Total ion chromatograms confirmed robust separation for both intact and subunit preparations. Deconvolution by Protein Metrics software yielded measured intact masses within ±20 Da of theoretical values for major glycoforms. Subunit analysis resolved light and heavy chains with mass deviations under 8 Da, enabling clear identification of glycoforms and oxidative modifications. These results highlight high mass accuracy and resolution afforded by the LCMS-9030 platform.

Benefits and Practical Applications

• Rapid turnaround for intact mass and subunit profiling supports process development and batch release testing
• High resolution aids in detecting minor variants and post-translational modifications
• Streamlined workflow reduces sample handling time and minimizes potential artifacts

Future Trends and Possibilities

Advances may include automated top-down fragmentation for sequence confirmation, integration with ion mobility for conformer analysis and adoption of AI-driven data interpretation to accelerate decision making. Expansion into high throughput screening and real-time monitoring could further enhance biopharmaceutical characterization.

Conclusion

The combined intact mass and subunit analysis on the Shimadzu LCMS-9030 Q-TOF, paired with Protein Metrics software, delivers accurate and efficient characterization of monoclonal antibodies, supporting critical quality assessment throughout drug development.

References

1. Srzentic S et al Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry Journal of the American Society for Mass Spectrometry 31(9) 1783–1802 2020
2. Robotham AC Kelly JF LC-MS Characterization of Antibody-Based Therapeutics Elsevier 2020