Method Transfer of a Monoclonal Antibody Ion-Exchange Separation Across Biocompatible Systems

Importance of the Topic

Ion exchange chromatography under biocompatible conditions enables the analysis of monoclonal antibodies in their native form, preserving structural integrity and minimizing interactions with metal surfaces. This approach is vital for detecting charge variants and degradants in biotherapeutics while reducing corrosion and oxidation caused by high salt concentrations and iron ions.

Aims and Study Overview

The primary goal was to develop and transfer a cation exchange separation method for infliximab and its lysine variants from a Waters ACQUITY Arc Bio System to an Agilent 1260 Infinity Bio-inert Quaternary System. This comparison demonstrates method robustness and reproducibility across different biocompatible platforms.

Methodology and Instrumentation

Infliximab at 1 mg/mL in 20 mM sodium phosphate buffer pH 6.8 was separated on a Protein-Pak Hi Res CM column (7 µm, 4.6×100 mm) at 0.5 mL/min and 30 °C. A quaternary gradient of phosphate monobasic, phosphate dibasic, sodium chloride, and water was employed over 60 minutes. Both systems used passive preheating, diode array detection at 280 nm, and comparable sampling rates (1–1.25 Hz). Data were acquired with Empower 3 FR3 and OpenLAB CDS Chemstation, and transfer was facilitated by a data converter.

Main Results and Discussion

The separation resolved the main antibody peak (IgG), single and double lysine variants (IgGK, IgGKK), as well as acidic and basic impurities. Measured dwell volumes were 1.32 mL (Path 1) for the Arc Bio and 1.57 mL for the Agilent system, accounting for observed retention time shifts (average –0.34 min). Relative retention times remained stable with an average difference of 0.02. Quantitative comparison of percent area for major peaks showed differences within 0.20% and repeatability with RSDs below 1.2% on both platforms. Minor degradant groups under 6% area exhibited slightly higher variability but remained within acceptable limits.

Benefits and Practical Applications

• Reliable method transfer between biocompatible LC systems
• Stable quantification of charge variants and degradants
• Reduced system corrosion and contamination risk
• Use of relative retention times for robust method comparison

Future Trends and Applications

Advances in pump flow path design and dwell volume control will further enhance method transfer efficiency. Integration of automated gradient calibration and enhanced data processing algorithms will support faster method development and compliance in QC laboratories. Expansion to other biotherapeutic modalities and high-throughput workflows is anticipated.

Conclusion

The cation exchange method for infliximab was successfully transferred between two biocompatible systems with minimal retention time shifts and excellent repeatability. Relative retention time emerged as a reliable metric for method equivalence, and the approach supports robust charge variant analysis in regulated environments.

References

1. Hong P, Fountain KJ, Wheat TE, Morrison D. IEX Method Development of a Monoclonal Antibody and Its Charge Variants. Application Note 720003836EN, Waters Corporation, 2011.
2. Hong P, McConville PR. Dwell Volume and Extra-Column Volume: What Are They and How Do They Impact Method Transfer. Application Note 720005723EN, Waters Corporation, 2016.