Monoclonal Antibody Charge Heterogeneity Analysis by CZE and CZE/MS

Significance of the Topic

The analysis of charge heterogeneity in monoclonal antibodies (mAbs) is a critical quality attribute in biopharmaceutical development and manufacturing. Minor variations in post-translational modifications can alter mAb properties such as stability, efficacy, and immunogenicity. Accurate monitoring ensures batch consistency, regulatory compliance, and therapeutic safety.

Objectives and Study Overview

This application note evaluates capillary zone electrophoresis (CZE) and CZE coupled with mass spectrometry (CZE/MS) for profiling charge variants of innovator and biosimilar rituximab using an Agilent 7100 CE system. The goals are to assess method reproducibility, resolution, and variant characterization.

Methodology and Used Instrumentation

The study employed the following setup:

• Agilent 7100 Capillary Electrophoresis System with UV detection at 214 nm
• Fused silica and PVA-coated capillaries (50 µm id, 56–65 cm length)
• Buffer systems: 400 mM e-amino-caproic acid and acetic acid pH 5.7 with HPMC and TETA for CZE-UV; 2 % acetic acid for CZE-MS
• Injection: 5 s at 50 mbar; Separation voltage: 30 kV; Temperature: 20 °C
• Agilent 6530 Accurate-Mass Q-TOF MS with electrospray ionization, sheath liquid of 0.5 % acetic acid in 50 % methanol

Main Results and Discussion

CZE separated rituximab charge variants within 25 minutes, resolving main, early (basic), and late (acidic) migrating peaks. Migration time and peak area RSDs were below 0.6 % and 1.2 %, respectively, indicating high precision. Quantitative analysis revealed that the biosimilar exhibited a higher proportion of basic variants compared to the innovator product. CZE/MS deconvolution of deglycosylated biosimilar rituximab confirmed a series of mass peaks differing by approximately 128 Da, corresponding to C-terminal lysine truncation variants.

Benefits and Practical Applications

CZE offers a rapid, high-resolution alternative to isoelectric focusing for routine charge heterogeneity profiling of mAbs. Coupling with MS provides detailed molecular characterization of basic variants, supporting quality control and biosimilar comparability studies.

Future Trends and Opportunities

Advancements may include automated integration of CZE-MS workflows, novel capillary coatings for enhanced resolution, and real-time process analytical technology (PAT) implementations. Combining high-throughput separation with advanced data analytics and AI-driven peak identification could further streamline mAb development and manufacturing.

Conclusion

Agilent 7100 CZE and CZE/MS methods demonstrated robust, reproducible separation and characterization of rituximab charge variants. The approaches provide essential tools for ensuring biotherapeutic quality and biosimilar comparability.

Reference

1. He, Y et al. High-Resolution Capillary Zone Electrophoresis for Monoclonal Antibody Charge Variant Analysis. Analytical Chemistry, 2010.
2. He, Y et al. Comparative Study of CZE and IEF for mAb Heterogeneity. Journal of Separation Science, 2011.
3. Picometrics. Laser-Induced Fluorescence Detection for Capillary Electrophoresis.
4. Agilent Technologies. Characterization of Monoclonal Antibodies Using CE-ESI-MS, Publication 5991-5212EN, 2015.