High-Performance Vion IMS QTof for Monoclonal Antibody Intact and Subunit Mass Analysis

Importance of the Topic

Monoclonal antibodies (mAbs) are key biotherapeutics whose structural heterogeneity arises from post-translational modifications and manufacturing changes. Regulatory guidelines require detailed characterization of intact mAbs and their subunits to ensure consistency, safety, and efficacy of these complex molecules.

Objectives and Study Overview

This study evaluates the performance of the Waters Vion IMS QTof Mass Spectrometer combined with the UNIFI Scientific Information System for high-resolution analysis of intact mAbs and their subunits. Key aims include demonstrating sensitivity, mass accuracy, reproducibility, and robust glycoform quantitation.

Methodology and Instrumentation

Sample Preparation and LC Conditions:

• Intact mAb standards (Waters and NIST) diluted to 0.01–1.0 mg/mL in 25 mM ammonium bicarbonate and injected at 1–1000 ng on column.
• Subunit analysis: Trastuzumab digested with IdeS, reduced with DTT, quenched, and injected at 50 ng on column.
• Chromatography: ACQUITY UPLC H-Class Bio with Protein BEH C4 column (2.1×50 mm, 1.7 µm) at 80 °C; gradient of 0.1% FA in water/ACN.

Mass Spectrometry and Data Processing:

• Vion IMS QTof operated in ESI+ across m/z 500–4000; automatic tuning and calibration.
• Parameters: cone voltage 80–140 V, capillary 2.75 kV, source/desolvation temps up to 600 °C.
• UNIFI software enabled workflow-driven acquisition, automated deconvolution, and reporting.

Results and Discussion

Intact mAb Analysis:

• Detectable down to 1 ng with clear charge envelopes and deconvoluted spectra.
• Five major glycoforms in NIST mAb identified with mass errors under 10 ppm and relative abundance %RSD below 1.5% across 11 injections.

Subunit Analysis:

• Baseline separation of scFc, LC, and Fd subunits at 50 ng on column.
• High signal-to-noise; mass accuracy <10 ppm for all subunits and glycoforms.

Benefits and Practical Applications

• Minimal tuning and rapid setup for intact protein workflows.
• High sensitivity supports low-level detection of mAbs.
• Accurate mass measurements and reliable glycoform quantitation enhance QC and biopharma R&D.
• Automated workflows reduce turnaround time and maintain data integrity.

Future Trends and Potential Applications

Continued advances may push detection limits below 0.1 ng and expand to other biotherapeutic modalities. Integration of ion mobility separation, AI-driven data analysis, and high-throughput automation will further streamline comprehensive protein characterization.

Conclusion

The Vion IMS QTof system with UNIFI software provides a user-friendly, high-performance platform for intact and subunit mAb analysis, delivering exceptional sensitivity, mass accuracy, and reproducible glycosylation profiling ideal for biopharmaceutical development and quality control.

Used Instrumentation

• Vion IMS QTof Mass Spectrometer with ADC2 (QuanTof2) detector
• ACQUITY UPLC H-Class Bio System
• ACQUITY UPLC Tunable Ultraviolet (TUV) Detector
• BEH Protein C4 Column (1.7 µm, 2.1×50 mm)
• UNIFI Scientific Information System v1.8.2