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

Significance of the Topic

The rapid growth of monoclonal antibodies (mAbs) and other protein biotherapeutics in pharmaceuticals demands sensitive and high-throughput analytical workflows. Characterization of intact mAbs—including mass, heterogeneity and post-translational modifications—is critical for understanding function, ensuring safety and supporting regulatory compliance.

Objectives and Study Overview

This study evaluates a microflow liquid chromatography–mass spectrometry (LC-MS) workflow using an M3 MicroLC coupled to a TripleTOF 6600 mass spectrometer. Goals include assessing sensitivity, accuracy, dynamic range and throughput for qualitative and quantitative intact mAb analysis and comparing on-column desalting to traditional offline methods.

Methodology

Three IgG1 mAbs (standard, adalimumab, trastuzumab) were serially diluted (0.02–1000 ng) in 0.1% formic acid. On-column desalting was performed during the first 3 min of a 10 min microflow gradient (15 µl/min) on a Waters BEH C4 column. MS acquisition comprised a 3 min salt-wash period (ion spray voltage off) followed by 7 min of intact protein analysis (ion spray voltage 5500 V) over m/z 1000–5000. Data processing used BioPharmaView™ for deconvolution and PTM mapping, and MultiQuant™ for quantitation.

Used Instrumentation

• SCIEX M3 MicroLC system
• SCIEX TripleTOF 6600 mass spectrometer with DuoSpray source
• Waters ACQUITY UPLC M-Class Protein BEH C4 column (300 Å, 1.7 µm, 300 µm × 50 mm)
• BioPharmaView™ v1.4 and MultiQuant™ v3.0.2 software

Key Results and Discussion

• On-column desalting eliminated offline cleanup, reducing sample prep by ~30 min and minimizing loss.
• Retention time reproducibility (stdev ~0.01 min), peak height and area were highly consistent across replicates.
• Quantitation sensitivity reached 0.1 ng on column with linear response over 3–4 orders of magnitude (0.1–1000 ng).
• Successful identification of glycoforms (G0F, G1F, G2F) and PTMs (deamidation, oxidation, pyroglutamate formation) at low-ng levels.
• High throughput capability (~140 samples/day).

Benefits and Practical Applications

This workflow minimizes solvent, enzyme and operation costs while delivering robust qualitative and quantitative data. It is well suited for routine characterization of mAbs in research, quality control and biopharmaceutical development settings.

Future Trends and Opportunities

• Automation of on-line desalting and integration with high-resolution MS/MS for detailed PTM profiling.
• Extension to antibody–drug conjugates (ADCs) and emerging non-IgG biologics.
• Adoption of microfluidic platforms and advanced data analytics for ultra-high throughput.
• Application in low-abundance biomarker and single-cell proteomic studies.

Conclusion

The combination of the M3 MicroLC and TripleTOF 6600 offers a powerful, sensitive and streamlined solution for intact mAb analysis at sub-nanogram levels. On-column desalting enhances throughput and recovery, making this workflow ideal for modern biotherapeutic analytics.

References

• Johansen E, Shin B-H, Hunter C. Simultaneous Quantitative and Qualitative Analysis of Therapeutic Monoclonal Antibodies Using a TripleTOF® System. SCIEX Technical Note; Doc No. 7460213-01.
• Johansen E, Albanese J, Hunter C. Analysis of Intact and Reduced Therapeutic Monoclonal Antibodies Using the TripleTOF 5600 System. SCIEX Technical Note; Doc No. 4220211-01.
• Wagner MH. Linear Programming Techniques for Regression Analysis. Journal of the American Statistical Association. 1959;54(285):207-212.
• Albanese J, Hunter CL, Meitei NS. Profiling the Distribution of N-Glycosylation in Therapeutic Antibodies Using the QTRAP® 6500 System. SCIEX Technical Note; Doc No. RUO-MKT-02-2590-A.