Charge Variant Analysis of Infliximab on the Alliance, ACQUITY Arc Bio, and ACQUITY UPLC H-Class Bio Liquid Chromatography Systems Using a Quaternary pH Gradient

Significance of the Topic

The separation of monoclonal antibody charge variants is critical in biopharmaceutical development and quality control. Ion exchange chromatography using pH gradients provides high-resolution profiling of therapeutic antibodies such as infliximab.

Objectives and Study Overview

This study compares three liquid chromatography platforms—the Alliance HPLC, ACQUITY Arc Bio, and ACQUITY UPLC H-Class Bio systems—in delivering a quaternary pH gradient for reproducible lysine charge variant analysis of infliximab. Reproducibility of retention times and relative quantitation was assessed across five injections on each system.

Methodology and Instrumentation

A quaternary gradient was generated from four stock solutions (acid, base, salt, and water) using Auto-Blend Plus on the ACQUITY systems and manually configured on the Alliance. Key conditions:

• Column: Protein-Pak Hi Res SP, 4.6×100 mm, 7 µm
• Flow rate: 0.5 mL/min
• Gradient: pH 5.5 to 7.2 with salt ramp (40 to 500 mM)
• Injection: 3 µL of 4 mg/mL infliximab
• Detection: TUV at 280 nm
• Column temperature: 25 °C; sample temperature: 4 °C

Main Findings and Discussion

All systems produced consistent chromatograms over five injections. The ACQUITY Arc Bio system exhibited the lowest retention time standard deviations for the Lys+0, Lys+1, and Lys+2 peaks, while the UPLC H-Class Bio showed the highest. Relative peak areas for lysine variants were comparable across platforms, demonstrating robust method transfer.

Benefits and Practical Applications of the Method

The quaternary pH gradient IEX approach offers:

• Reliable method transfer between LC platforms
• High reproducibility for quality control assays
• Simplified gradient preparation via Auto-Blend Plus

Future Trends and Opportunities

Extensions of this work may include integration with mass spectrometry for detailed variant identification, automation for high-throughput screening, and application to other therapeutic proteins. Software-driven gradient control and data analytics will further enhance method precision.

Conclusion

The quaternary pH gradient IEX method demonstrates excellent reproducibility across three chromatography systems, with the ACQUITY Arc Bio delivering superior retention precision. This workflow supports robust charge variant analysis in biopharmaceutical R&D and QC.

Used Instrumentation

• Alliance HPLC System
• ACQUITY UPLC H-Class Bio System
• ACQUITY Arc Bio System
• Protein-Pak Hi Res SP, 4.6×100 mm, 7 µm
• Empower Software with Auto-Blend Plus
• TUV Detector at 280 nm