Extended Analysis of mAbs Using Ion- Exchange Chromatography on the Alliance™ iS Bio HPLC System

Importance of the Topic

Monoclonal antibodies (mAbs) are cornerstone biologics in therapeutics, demanding precise control of charge variants to ensure consistent efficacy and safety. Ion‐exchange chromatography (IEX) is a non‐denaturing technique widely used for profiling lysine‐related charge heterogeneity. Robust multi‐day performance of IEX methods is critical for process development, quality control, and long‐term stability studies of mAbs.

Objectives and Overview

This study evaluates the long‐term robustness of the Alliance iS Bio HPLC System over 30 consecutive days using a salt‐gradient cation exchange (CEX) method. A three‐protein IEX cation test standard and expired infliximab were analyzed daily. Retention times, total area, and relative peak areas were monitored to assess system reproducibility and hardware durability under high‐salt conditions.

Methodology and Used Instrumentation

A quaternary blending pump delivered four mobile phase lines: 100 mM MES buffer concentrate (acidic and basic), 1 M NaCl, and water. A BioResolve SCX mAb column (4.6×100 mm, 3 µm) was maintained at 30 °C. Samples were thermostatted at 6 °C and detected at 280 nm. Daily sequences included test standard replicates, infliximab injections, and blank runs. Empower 3 CDS managed data acquisition. Key instrumentation:

• Alliance iS Bio HPLC System with corrosion‐resistant titanium, MP35N tubing, and MaxPeak HPS surfaces
• BioResolve SCX mAb column, 4.6×100 mm, 3 µm
• Empower 3.8.0.1 chromatography software
• Sample vials: Maximum Recovery (p/n 186005662CV) and Total Recovery (p/n 186005663CV)

Main Results and Discussion

The Alliance iS Bio system demonstrated exceptional stability:

• Test standard retention time RSD ≤0.24%, total area RSD ≤5.72%, relative area RSD ≤4.06% over 30 days
• Infliximab lysine‐variant peaks showed retention time RSD ≤1.28% and relative area RSD ≤5.93% for low‐abundance acidic/basic variants
• Quaternary pump delivered precise salt gradients up to 500 mM NaCl with negligible clogging or hardware degradation
• Sample manager maintained 6 °C over extended holds, preventing peptide degradation

Benefits and Practical Applications

• Reliable multi‐day IEX performance under high ionic strength without precipitation or flow‐path fouling
• Flexible gradient preparation through quaternary blending, reducing the need for premixed solvents
• Enhanced sample integrity via cooled autosampler for biomolecules
• Applicable to other biologics requiring charge variant monitoring across discovery, manufacturing, and stability phases

Future Trends and Potential Applications

Advances may include coupling IEX to mass spectrometry for deeper structural insights, integration with automated sample preparation workflows, and expansion to high‐throughput screening of biosimilar candidates. Emerging surface technologies and alternative non‐ferrous materials will further extend system longevity under challenging buffer conditions.

Conclusion

The Alliance iS Bio HPLC System sustained consistent CEX performance over 30 days of continuous high‐salt operation. Corrosion‐resistant materials, precise quaternary blending, and robust temperature control ensured minimal variability in retention times and peak areas. This platform offers a reliable solution for rigorous mAb charge variant analysis in research and quality environments.

References

1. Fekete S, Beck A, Fekete J, Guillarme D. Method Development for the Separation of Monoclonal Antibody Charge Variants in Cation Exchange Chromatography, Part I: Salt Gradient Approach. J Pharm Biomed Anal. 2015;102:33–44.
2. Khawli LA et al. Charge Variants in IgG1. mAbs. 2010;2(6):613–624.
3. Goyon A et al. Determination of Isoelectric Points and Relative Charge Variants of 23 Therapeutic Monoclonal Antibodies. J Chromatogr B. 2017;1065–1066:119–128.
4. Grönberg A. Chapter 18 - Ion Exchange Chromatography. In: Biopharmaceutical Processing. Elsevier; 2018:379–399.
5. Spanov B et al. Revealing Charge Heterogeneity of Stressed Trastuzumab at the Subunit Level. Anal Bioanal Chem. 2023;415(8):1505–1513.