Impact of LC System Dispersion on the Size-Exclusion Chromatography Analysis of Monoclonal IgG Antibody Aggregates and Fragments: Selecting the Optimal Column for Your Method

Importance of the topic

Native SEC is widely used for assessing non-covalent protein aggregation and fragmentation in biotherapeutic monoclonal antibodies, ensuring product purity, safety, and efficacy. Control of LC system dispersion is crucial for reliable resolution and quantitation of high molecular weight species (HMWS) and low molecular weight species (LMWS).

Objectives and study overview

This study systematically evaluates the impact of LC system extra-column dispersion on size-exclusion chromatography separations of IgG monoclonal antibody aggregates (HMWS) and fragments (LMWS). It compares columns of varying particle sizes (1.7, 2.5, 3.5 µm), internal diameters (4.6, 7.8 mm), and lengths (150, 300 mm) on Waters ACQUITY UPLC H-Class Bio and ACQUITY Arc Bio systems. Detection limits, resolution, quantitative reliability, and method transferability are assessed, leading to optimized column recommendations.

Methodology and instrumentation

• Samples: infliximab and rituximab at ~21 mg/mL; trastuzumab at 2 mg/mL.
• LC systems: ACQUITY UPLC H-Class Bio, ACQUITY Arc Bio.
• Columns: BEH200 SEC Protein (1.7 µm, 2.5 µm, 3.5 µm; 4.6×150, 4.6×300, 7.8×150, 7.8×300 mm).
• Mobile phase: 20–25 mM sodium phosphate, 350–400 mM NaCl, pH 6.8–7.2.
• Flow rates: 0.35 mL/min (4.6 mm I.D.), 1.0 mL/min (7.8 mm I.D.), scaled to column dimensions.
• Detection: UV absorbance at 280 nm (5 mm or 10 mm flow cell).
• Data: Empower 3.
• Extra-column dispersion measured by caffeine injections and peak width at 4.4% height (5σec volumes from ~9 to 57 µL).

Main results and discussion

• Extra-column dispersion equally degrades pre- and post-column band broadening in SEC; minimizing 5σec is essential for optimal resolution.
• HMWS separation is robust on 1.7 µm (4.6×300 mm) and 3.5 µm (7.8×300 mm) columns; larger column I.D. and particle sizes show less sensitivity to dispersion.
• LMWS1 separation is most consistent on 1.7 µm (4.6×300 mm) columns when 5σec ≤ ~25 µL; 2.5 µm (7.8×300 mm) columns offer balanced performance at moderate dispersion; 3.5 µm (7.8×300 mm) columns require ≥40 µL dispersion but maintain quantitation.
• Method adaptations include tandem columns and flow rate adjustments to mitigate dispersion on higher-dispersion systems.

Benefits and practical applications

• Offers practical guidance for selecting SEC columns aligned with LC system dispersion capabilities.
• Ensures reliable detection and quantitation of mAb aggregates and fragments in QA/QC workflows.
• Facilitates method transferability and robustness across UHPLC and HPLC platforms.

Future trends and applications

• Emerging 2.5 µm particle size columns for improved resolution-pressure trade-offs.
• Integration of low-dispersion UHPLC and microflow systems for high-throughput SEC.
• Coupling SEC with mass spectrometry for detailed protein characterization.
• Extension to other protein therapeutics, bioconjugates, and complex biologics.

Conclusion

Control of LC system dispersion is critical for high-resolution SEC analysis of mAb aggregates and fragments. Selecting appropriate particle size, column length, and internal diameter in accordance with system dispersion enables reliable separation and quantitation. 1.7 µm (4.6×300 mm) columns excel on low-dispersion systems; 2.5–3.5 µm (7.8×300 mm) formats perform consistently on moderate to high-dispersion platforms.

References

• Hong P., Koza S., Bouvier E.S. Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates. J. Liq. Chromatogr. Relat. Technol. 2012;35:2923-2950.
• Cordoba A.J., Shyong B.-J., Breen D., Harris R.J. Non-Enzymatic Hinge Region Fragmentation of Antibodies in Solution. J. Chromatogr. B 2005;818:115-121.
• Goyon A., Guillarme D., Fekete S. The Importance of System Band Broadening in Modern Size Exclusion Chromatography. J. Pharm. Biomed. Anal. 2017;135:50-60.
• Smith M.A., Easton M., Everett P., Lewis G., Payne M., Riveros-Moreno V., et al. Specific Cleavage of Immunoglobulin G by Copper Ions. Int. J. Pept. Protein Res. 1996;48:48-55.