Analyzing Monoclonal Antibody (mAb) Fragments and Dimers Using Size Exclusion Chromatography (SEC)

Significance of the topic

Size variants of biotherapeutics including low molecular weight fragments and high molecular weight aggregates directly impact drug safety and efficacy. Robust characterization via size exclusion chromatography (SEC) is essential to control critical quality attributes and mitigate immunogenic responses, ensuring consistent pharmacokinetics and therapeutic potency.

Study objectives and overview

This guide details the application of Agilent AdvanceBio SEC 200 Å 1.9 µm columns for analyzing monoclonal antibody fragments and dimers. It aims to present optimized chromatographic conditions, demonstrate column stability, and provide best practices for operation, maintenance, and consumable selection.

Methodology and Instrumentation

Size-based separation was performed on an Agilent 1260 Infinity II Bio-inert LC System using AdvanceBio SEC columns packed with proprietary 1.9 µm monodisperse silica. Typical conditions included a 4.6 × 300 mm 200 Å column, 150 mM sodium phosphate buffer (pH 7.0), 0.35 mL/min flow rate, ambient temperature, and UV detection at 280 nm. Sample preparation involved stressed mAb mixtures and a protein/uridine mix for performance testing.

Key results and discussion

Column durability tests showed less than 2% loss in plate count after 400 injections, confirming mechanical robustness. Separation of stressed mAb demonstrated clear resolution of aggregates, monomer, and fragments within 12 minutes. Optimization strategies, such as adjusting ionic strength (100–150 mM salts), pH (2.0–8.5), temperature (10–30 °C), and organic modifiers (5–10% ethanol or 5% DMSO), improved peak shape and protein solubility. Operational guidance covered flow rate alignment with column dimensions, pressure limits (≤ 620 bar), cleaning protocols, and proper storage conditions.

Benefits and practical applications

AdvanceBio SEC columns deliver high-resolution, fast separations with minimal secondary interactions, compatible with UHPLC, HPLC, and native MS workflows. The inert hydrophilic surface minimizes nonspecific binding, while PEEK-lined options protect sensitive analytes. Extended column lifetime and reliable quantification make them suitable for QA/QC, biopharmaceutical development, and high-throughput aggregate analysis.

Future Trends and Opportunities

Emerging trends include integration of SEC with multi-angle light scattering and mass spectrometry for comprehensive molecular characterization, development of ultra-low dispersion systems for increased peak capacity, and automation for high-throughput screening. Advances in stationary phase design and mobile phase versatility will further enhance sensitivity and robustness in biotherapeutics analysis.

Conclusion

Agilent AdvanceBio SEC 200 Å 1.9 µm columns represent a robust solution for mAb fragment and aggregate analysis, offering exceptional resolution, stability, and operational flexibility. Adherence to optimized protocols ensures reliable quality monitoring throughout biotherapeutic development.

Reference

1. Fast Separations for Aggregates and Fragments with AdvanceBio SEC columns – 5994-0873EN
2. Size Exclusion Chromatography Analysis of a Monoclonal Antibody and Antibody Drug Conjugate – 5994-0827EN
3. Agilent AdvanceBio SEC 1.9 µm Column user guide – 5994-0739EN
4. Elevate Your mAb Aggregate Analysis – 5994-2709EN
5. Analysis of Antibody Fragment-Drug Conjugates Using an Agilent AdvanceBio SEC 120 Å 1.9 µm PEEK-Lined Column – 5994-3045EN
6. Fast, High-Resolution Size Exclusion Chromatography of Aggregates in Biotherapeutics – 5991-6458EN