High-throughput and Sensitive Size Exclusion Chromatography (SEC) of Biologics Using Agilent AdvanceBio SEC Columns

Significance of the topic

Monoclonal antibodies and antibody–drug conjugates are prone to aggregation during production and storage, posing risks to safety and efficacy in biopharmaceutical applications. Size exclusion chromatography (SEC) is the standard technique for monitoring these aggregates, but conventional methods are time-consuming and may introduce artifacts. High-throughput, sensitive, and reproducible approaches are critical for quality control, stability testing, and process development.

Objectives and study overview

This study evaluates the performance of Agilent AdvanceBio SEC 300 Å columns with 2.7 µm particles in shortened formats (7.8 × 150 mm and 4.6 × 150 mm) for rapid, high-resolution separation and quantitation of monoclonal antibodies and ADCs. Key aims include reducing analysis time below four minutes, achieving low limits of detection and quantitation, and enabling reliable aggregate detection under forced-stress conditions.

Methodology

Samples of innovator and biosimilar rituximab, Herceptin, and an antibody–drug conjugate were prepared in aqueous mobile phase (150 mM sodium phosphate, pH 7.0). Calibration standards spanned 1 kDa to 670 kDa. Limit of detection (LOD) and limit of quantitation (LOQ) were determined based on signal-to-noise ratios of 3 and 10, respectively. Forced-stress aggregates were generated by iterative pH shifts and heating at 60 °C. Chromatographic parameters were optimized for each column, including injection volumes of 5 µL (7.8 mm) and 2 µL (4.6 mm) and flow rates of 1.0 mL/min and 0.35 mL/min, respectively.

Used Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary LC Pump (G5611A)
• Agilent 1260 Infinity Bio-inert High Performance Autosampler (G5667A)
• Agilent 1200 Infinity Series Thermostat (G1330B)
• Agilent 1260 Infinity Thermostatted Column Compartment (G1316C, option 19)
• Agilent 1260 Infinity Diode Array Detector with 60 mm Max-Light flow cell (G4212B option 33)
• AdvanceBio SEC 300 Å columns, 7.8 × 150 mm and 4.6 × 150 mm, packed with 2.7 µm particles

Main results and discussion

Both column formats achieved baseline separation of monomer, aggregates, and fragments in under four minutes. The 4.6 × 150 mm column provided superior resolution and sensitivity. Retention time and peak area relative standard deviations were below 0.02% and 0.21%, demonstrating excellent precision. LOD/LOQ for Herceptin and ADC improved from 78/156 ng on the 7.8 mm column to 31.2/62.4 ng on the 4.6 mm column. Linearity was confirmed from 15.6 to 2000 µg/mL with R2 greater than 0.999. Forced-stress studies showed clear separation of aggregates and degradants without the need for organic modifiers.

Benefits and practical applications

• High-throughput analysis with run times under four minutes
• Enhanced sensitivity and low detection limits for QC and stability testing
• Robust precision and reproducibility for routine use
• Aqueous mobile phase avoids potential protein damage and column degradation
• Applicability to process monitoring, lot release, and formulation development

Future trends and opportunities

Developments may include integration of UHPLC-based SEC with mass spectrometry for molecular characterization, further column miniaturization, advanced stationary phase coatings to minimize secondary interactions, and real-time analysis workflows. Automation and data analytics, including AI-driven interpretation of chromatographic profiles, are expected to enhance throughput and decision support in biopharmaceutical research and manufacturing.

Conclusion

Agilent AdvanceBio SEC columns in shortened formats provide rapid, sensitive, and reproducible size-based separations of monoclonal antibodies and ADCs. Their performance supports high-throughput aggregate analysis without organic modifiers, offering a robust platform for quality control, process development, and stability studies in the biopharmaceutical industry.

References

1. Fekete S, Beck A, Veuthey J-L, Guillarme D. Theory and practice of size exclusion chromatography for the analysis of protein aggregates. J Pharm Biomed Anal. 2014;101:161–173.
2. Kükrer B, Filipe V, van Duijn E, Kasper PT, Vreeken RJ, Heck AJR, Jiskoot W. Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography. Pharm Res. 2010;27:2197–2204.
3. Coffey A. Fast, high-resolution size exclusion chromatography of aggregates in biotherapeutics. Agilent Technol; 2015. Application note 5991-6458EN.