pH gradient Analysis of Infliximab charge variant

Importance of the Topic

The characterization of therapeutic antibodies by charge variant analysis is critical for ensuring product consistency, safety and efficacy. Subtle differences in post-translational modifications such as deamidation or glycation can produce acidic or basic species, impacting biological activity, stability and regulatory compliance. A robust, high-resolution method for infliximab charge profiling supports quality control in biopharmaceutical manufacturing.

Objectives and Study Overview

This application note evaluates a pH-gradient ion-exchange chromatography method on a Shim-pack Bio IEX SP-NP column for separating infliximab charge variants. The study compares the performance of the Shim-pack column to a competitor’s stationary phase, focusing on resolution, peak shape and throughput under a defined pH gradient.

Methodology and Instrumentation

System configuration and analytical conditions:

• Instrument: Nexera™ lite inert high-performance liquid chromatograph
• Column: Shim-pack Bio IEX SP-NP, 100 mm × 4.6 mm I.D., 5 µm particles
• Mobile phases: A (30 mmol/L MES-HEPES-sodium acetate, pH 5), B (same buffer at pH 10)
• Gradient: 20 % B at 0 min → 100 % B over 11–13.5 min → 20 % B to 18.5 min
• Flow rate: 1.0 mL/min; Column temp.: 30 °C; Injection: 10 µL; Detection: UV 280 nm

Main Results and Discussion

The Shim-pack Bio IEX SP-NP column delivered sharper peaks and improved separation of acidic, main and basic infliximab variants compared to the competitor’s column. Baseline resolution was achieved within an 18.5-minute run, demonstrating superior selectivity of the stationary phase. Peak capacities were higher, enabling reliable quantitation of low-abundance species.

Benefits and Practical Applications

• Enhanced resolution accelerates method development and shortens analysis time.
• pH-gradient elution provides broad applicability to other mAbs with varying isoelectric points.
• Reproducible performance supports routine quality control and stability studies.

Future Trends and Potential Applications

Advances in mixed-mode and monolithic stationary phases may further reduce analysis time and increase robustness. Integration with mass spectrometry detection can yield detailed structural insights into charge variants. Automated high-throughput platforms will support bioprocess monitoring and comparability studies in biosimilar development.

Conclusion

The pH-gradient IEX method on Shim-pack Bio IEX SP-NP provides an efficient, high-resolution approach for infliximab charge variant analysis. Its superior selectivity and reproducibility make it a valuable tool for biopharmaceutical quality control.

Reference

Shimadzu Corporation. pH Gradient Analysis of Infliximab Charge Variants on Shim-pack Bio IEX SP-NP. Application Note ERAS-1000-0603. First Edition: Sep. 2025.