Analysis of Intact Monoclonal Antibodies Using an M3 MicroLC with the TripleTOF® 6600

Importance of the Topic

The rapid growth of therapeutic monoclonal antibodies (mAbs) in biopharmaceutical pipelines demands robust analytical workflows. Intact mass analysis provides critical insights into primary structure, heterogeneity and post-translational modifications that directly influence safety, efficacy and stability of these biologics.

Study Objectives and Overview

This study evaluates a microflow liquid chromatography–mass spectrometry (LC–MS) workflow using the SCIEX M3 MicroLC coupled to a TripleTOF 6600 system. Key aims include demonstrating on-column desalting for streamlined sample preparation, assessing sensitivity and quantitation performance across a wide dynamic range, and characterizing glyco-isoforms and other modifications on intact IgG1 antibodies.

Methodology and Instrumentation

Sample Preparation and On-Column Desalting

• Three IgG1 molecules (commercial standard, trastuzumab, adalimumab) were serially diluted from nanogram to subnanogram levels in 0.1% formic acid.
• On-column desalting was performed directly on the microLC system to minimize sample loss and preparation time, avoiding offline membrane filters.

LC–MS Conditions

• Microflow separation on SCIEX M3 MicroLC with Waters ACQUITY UPLC M-Class Protein BEH C4 column (300 µm × 50 mm, 1.7 µm, 300 Å) at 15 µl/min and 60 °C.
• Gradient: 3 min desalting at 20% organic, followed by 7 min linear ramp to 80% acetonitrile and reequilibration to 5%.
• MS acquisition on TripleTOF 6600 in intact protein mode with DuoSpray source and 25 µm electrode; spray voltage floated to zero during desalting, then set to 5500 V for analysis.

Key Results and Discussion

Reproducibility and Sensitivity

• Retention time variation below 0.01 % and consistent peak areas across replicates.
• Quantitative linear dynamic range covering 0.1–100 ng per injection for multiple charge states with R²≥0.99.

Enhanced Detection

• On-column desalting outperformed offline membrane methods, yielding improved signal-to-noise and reduced sample loss.
• High-resolution mass accuracy enabled characterization of mAb glyco-isoforms down to 2.5 ng, including major variants (G0F, G1F, G2F) and PTMs such as deamidation, oxidation and N-terminal modifications.

Benefits and Practical Applications

• Single-run quantitation and characterization of intact mAbs with minimal sample handling and ~30 min prep time.
• High throughput capability (~140 samples per day) driven by a 10 min cycle time.
• Cost savings in solvents, desalting consumables and enzymes by eliminating offline cleanup.
• Robust quantitation over three orders of magnitude dynamic range suitable for QA/QC and bioanalysis.

Future Trends and Opportunities

Emerging needs in biologics analysis will drive integration of microflow LC-MS with advanced data processing to deepen proteoform profiling. Expanded use cases include high-throughput comparability studies, real-time process monitoring, and regulatory-compliant characterization of next-generation antibody-drug conjugates and biosimilars. Ongoing improvements in microfluidics, source design and software algorithms will further enhance sensitivity and throughput.

Conclusion

The combination of M3 MicroLC with on-column desalting and the TripleTOF 6600 platform delivers a robust, sensitive and high-throughput solution for intact mAb quantitative and qualitative analysis. This workflow simplifies sample preparation, extends dynamic range, and provides detailed molecular information critical for biotherapeutic development and quality control.

Reference

1. Johansen E, Shin B-H, Hunter C. Simultaneous Quantitative and Qualitative Analysis of Proteolytic Digests of Therapeutic Monoclonal Antibodies Using a TripleTOF System. SCIEX Technical Note 7460213-01.
2. Johansen E, Albanese J, Hunter C. Analysis of Intact and Reduced Therapeutic Monoclonal Antibodies using the TripleTOF 5600 System. SCIEX Technical Note 4220211-01.
3. Wagner HM. Linear Programming Techniques for Regression Analysis. Journal of the American Statistical Association, March 1959; p 207-212.
4. Albanese J, Hunter CL, Meitei NS. Profiling the Distribution of N-Glycosylation in Therapeutic Antibodies using the QTRAP 6500 System. SCIEX Technical Note RUO-MKT-02-2590-A.