Characterization of Monoclonal Antibodies Using Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry (CE-ESI-MS)

Importance of the Topic

Monoclonal antibodies (mAbs) are critical therapeutic proteins whose structural integrity and heterogeneity directly impact safety and efficacy. Monitoring molecular weight, purity, and post-translational modifications is essential in biopharmaceutical development and quality control. Capillary electrophoresis–mass spectrometry (CE-MS) offers high sensitivity, minimal sample consumption, and accurate mass measurement, making it a promising complementary technique to liquid chromatography–MS (LC-MS).

Objectives and Study Overview

This application note evaluates CE-ESI-MS for characterizing intact and reduced forms of two proprietary IgG1 and IgG2 mAbs. The goals are to demonstrate CE-MS capability in resolving charge variants, detecting glycoforms, and identifying modifications that may escape LC-MS analysis.

Methodology and Instrumentation

The analysis employed an Agilent 7100 CE System coupled to an Agilent 6530 Accurate-Mass Q-TOF via an orthogonal coaxial sheath-liquid electrospray interface. Key CE parameters:

• Separation capillary: 70 cm total length, 50 μm i.d., PVA coated
• Buffer: 50 mM acetic acid, 20 °C, 30 kV
• Sample injection: 3 μg/μL mAb, 10–20 s at 50 mbar

The sheath liquid (0.5 % acetic acid in 50 % methanol) was delivered at 5 μL/min. MS settings included 1 000–6 000 m/z range, drying gas 5 L/min at 250 °C, fragmentor 350 V, and Vcap 3 500 V. Data were acquired with MassHunter Acquisition and processed in BioConfirm using peak modeling deconvolution.

Main Results and Discussion

Intact mAb CE-MS traces revealed broad peaks at 12–18 min. Charge envelopes ranged from 2 400–5 100 m/z, shifted higher than typical LC-MS. Deconvoluted masses identified four major glycoforms for both IgG1 and IgG2, matching LC-MS data.

Reduction with DTT produced light and heavy chains that separated in the electropherogram. Light chains showed single masses (~22 930 Da). Heavy chains exhibited glycoforms consistent with intact analysis. CE-MS uniquely detected a +203 Da mass shift (N-acetyl glucosamine) on IgG1 heavy chain not observed by LC-MS, highlighting the technique’s sensitivity to subtle modifications.

Benefits and Practical Applications

• Low sample volume (1–20 nL) and rapid separation reduce solvent and time requirements.
• Accurate mass measurement and high resolution enable confident glycoform and variant identification.
• CE-MS complements LC-MS by resolving variants suppressed or unresolved in reversed-phase separations.

Future Trends and Applications

Advances in sheath-liquid interfaces and high-resolution MS will further improve CE-MS sensitivity and coverage. Integration with automated sample preparation and bioinformatics pipelines can streamline mAb characterization workflows. Emerging applications include detailed mapping of glycosylation microheterogeneity and monitoring of charge variants in biosimilar comparability studies.

Conclusion

This study demonstrates that CE-ESI-MS is a robust, complementary platform for comprehensive primary characterization of mAbs. It provides accurate mass data, resolves glycoforms and subtle modifications, and enhances analytical depth beyond conventional LC-MS approaches.

References

1. Crank, J. A.; Fraczkiewicz, R.; Mouchahoir, T.; et al. Anal. Chem. 2006, 78 (18), 6583–6594.
2. Beck, A.; Wagner-Rousset, E.; Ayoub, D.; et al. Anal. Chem. 2012, 85 (2), 715–736.
3. Camilleri, P.; Zilch, L. W.; Haselberg, R.; et al. Electrophoresis 2014, 35 (1), 96–108.
4. Agilent Technologies. CE/MS and LC/MS Synergy, 2013, publication 5991-2583EN.
5. Agilent Technologies. Analysis of Monoclonal Antibody Using 1290 Infinity LC and 6530 Q-TOF, 2014, publication 5991-4266EN.