Developing Robust and Efficient IEX Methods for Charge Variant Analysis of Biotherapeutics Using ACQUIT Y UPLC H-Class System and Auto•Blend Plus

Significance of the Topic

Charge variant analysis is a cornerstone of biotherapeutic development and quality control. Subtle modifications such as deamidation, N-terminal cyclization, glycan sialylation and C-terminal lysine clipping can alter protein charge distribution, affecting binding affinity, biological activity, immunogenicity and shelf life. Reliable characterization of these variants ensures product safety and efficacy throughout development and manufacturing.

Study Objectives and Overview

This application note describes the efficient development of ion-exchange chromatography (IEX) methods for charge variant profiling of a chimeric monoclonal antibody (infliximab) using Waters ACQUITY UPLC H-Class System equipped with Auto•Blend Plus Technology. The goals were to automate buffer preparation, streamline method optimization and demonstrate high reproducibility across varying pH, ionic strength and sample loads.

Methods and Instrumentation

The following instrumentation and software were employed:

• ACQUITY UPLC H-Class System with quaternary solvent management and Auto•Blend Plus Technology
• ACQUITY UPLC Tunable UV Detector (TUV) at 280 nm
• Protein-Pak Hi Res SP strong cation-exchange column (4.6 × 100 mm, 7 µm)
• UNIFI Scientific Information System v1.6 for data acquisition and analysis

Auto•Blend Plus blended concentrated buffer stocks (MES monohydrate, MES sodium salt, NaCl and water) to target pH and ionic strength gradients without manual preparation. Method parameters (pH 5.1–7.1, 20–65 mM NaCl gradient, 0.5 mL/min, 25 °C) were optimized systematically.

Results and Discussion

Auto•Blend Plus achieved precise buffer pH with %RSD ≤ 1.4 across three independent preparations. Retention times remained constant across a 9-fold increase in protein load (20–180 µg), demonstrating linearity and quantitation fidelity. Over 40 consecutive injections spanning three days, individual variant peak areas showed %RSD < 12% and total area %RSD < 9%, meeting FDA bioanalytical validation guidelines. Automated blending reduced buffer preparation error and development time compared to discrete manual mixing.

Benefits and Practical Applications

• Increased productivity through end-to-end method automation
• Reproducible separations across instruments, analysts and labs
• Elimination of manual buffer mixing and associated errors
• Rapid qualification and transfer of IEX methods for QA/QC and regulatory submission

Future Trends and Opportunities

Integration of intelligent software with UPLC platforms will further accelerate biotherapeutic characterization. Future developments may include real-time gradient optimization, AI-driven method scouting and extended applicability to multi-attribute monitoring by coupling to MS detection. Expanded libraries of buffer systems and calibration models will support diverse molecule formats and automated high-throughput workflows.

Conclusion

The combination of Auto•Blend Plus Technology with the ACQUITY UPLC H-Class System offers a robust, automated solution for charge variant analysis of biotherapeutics. It delivers consistent buffer blending, high reproducibility under varied conditions and streamlined method transfer, meeting the stringent demands of biopharmaceutical development and quality control.

References

1. Du Y, Walsh A, Ehrick R, Wang J, et al. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. mAbs. 2012;4(5):578–585.
2. Guidance for Industry: Bioanalytical Method Validation. U.S. FDA, May 2001.