Characterize mAb Charge Variants by Cation-Exchange Chromatography

Importance of Topic

The analysis of monoclonal antibody (mAb) charge variants is critical for ensuring biopharmaceutical safety and efficacy. Charge heterogeneity can arise during production and storage, affecting product performance. Ion-exchange chromatography is a key tool for sensitive detection and quantification of these variants.

Objectives and Study Overview

This application note demonstrates the separation of charge variants of trastuzumab (Herceptin) using a weak cation-exchange column, followed by detailed characterization of collected fractions by reversed-phase liquid chromatography (RPLC) and mass spectrometry (MS). Batch consistency and stress-induced modifications are evaluated.

Methodology and Instrumentation

Experimental workflow and instrumentation:

• Sample preparation: Herceptin diluted to 2.1 mg/mL in 10 mM phosphate buffer pH 7.65; pH stress by incubation at pH 9.0 for 1 day and 3 days.
• CEX separation: Agilent Bio MAb NP5 column (2.1×250 mm, 5 µm) with gradient elution (10 mM phosphate ± 100 mM NaCl) at 200 µL/min, 30 °C, UV detection at 214 nm; time-based fraction collection.
• Reduction and RPLC: Collected fractions treated with 10 mM DTT; analysis on Agilent ZORBAX RRHD 300SB-C8 (2.1×100 mm, 1.8 µm) at 80 °C with 0.1% TFA in water/acetonitrile, 200 µL/min.
• MS detection: Agilent 6540 UHD Q-TOF with JetStream ESI in positive mode; mass range m/z 500–3200, high-resolution mode.

Key Results and Discussion

• High precision: Five replicate CEX injections showed reproducible separation of Herceptin charge variants.
• Batch consistency: Three production lots displayed similar profiles with ~28–30% acidic pre-peaks, ~58–60% main peak, and ~11–14% basic post-peaks.
• Stress-induced variants: pH stress generated additional acidic peaks; 1-day stressed samples revealed two early CEX peaks, while 3-day stressed samples exhibited multiple distinct acidic variants.
• Structural characterization: RPLC-UV/MS of collected peaks identified light-chain deamidation events (+1 Da), with equimolar and fully converted forms indicating single or double deamidations per mAb.

Benefits and Practical Applications

The combined CEX and RPLC-UV/MS workflow enables robust profiling of mAb charge heterogeneity, supporting batch release testing, comparability assessments between biosimilars and originators, and stability studies under stress conditions.

Future Trends and Opportunities

Advances in multidimensional separations, automation, and high-resolution mass spectrometry will enhance the depth and throughput of mAb variant analysis. Integration with AI-driven data processing and top-down/middle-down MS approaches could provide site-specific modification mapping in a single workflow.

Conclusion

The Agilent Bio MAb weak cation-exchange column effectively separates intact mAb charge variants, and subsequent RPLC-UV/MS characterization offers detailed structural insights into post-translational modifications.