Advantages of Agilent AdvanceBio SEC Columns for Biopharmaceutical Analysis

Significance of the Topic

Size exclusion chromatography (SEC) is a critical method for characterizing molecular weight distributions and assessing biomolecular size variants in biopharmaceutical development and quality control.

Study Objectives and Overview

This Technical Note compares Agilent AdvanceBio SEC columns with competing products using a common phosphate buffer mobile phase to separate protein standards, monoclonal antibodies (rituximab, trastuzumab), and an antibody–drug conjugate (ADC). The aim is to evaluate column performance in terms of resolution, sensitivity, and robustness.

Materials and Methods

• Columns: Agilent AdvanceBio SEC 300Å (2.7 µm) in 7.8 × 300 mm and 4.6 × 300 mm formats; competitor columns with pore sizes from 200 Å to 450 Å and particle sizes 1.7 µm to 5 µm.
• Mobile phase: 150 mM phosphate buffer at pH 7.0.
• Flow rates: 0.8 mL/min for 7.8 mm ID; 0.4 mL/min for 4.6 mm ID columns.
• Injection volume: 5 µL; column temperature ambient; detection UV at 220 nm.
• Samples: protein molecular weight markers (thyroglobulin aggregate, thyroglobulin monomer, IgG, BSA, myoglobin, uracil), monoclonal antibodies, and ADC.

Used Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary LC system.
• Agilent 1290 Infinity Binary LC with G1315D DAD and bio-inert flow cell (1200 bar).

Main Results and Discussion

• Resolution and Peak Shape: AdvanceBio SEC showed sharper, better-resolved peaks across all molecular weight markers compared to competitor columns, with resolution factors (Rs) exceeding 2.5 in many cases.
• Calibration Linearity: AdvanceBio SEC produced more linear calibration curves (R2 > 0.98) over a wide molecular weight range (112 Da to 662 kDa), indicating consistent size-based separation.
• Pore Volume: The 7.8 × 300 mm AdvanceBio SEC column exhibited the highest pore volume (7.11 mL), contributing to improved peak capacity.
• Monoclonal Antibodies: Trastuzumab and rituximab separations achieved higher Rs values and clear aggregate resolution on AdvanceBio SEC versus other vendors.
• Antibody–Drug Conjugate Analysis: Use of an aqueous phosphate buffer with AdvanceBio SEC eliminated nonspecific interactions, delivering symmetric peaks (Rs ~1.75) and reliable aggregate quantitation without organic modifiers.
• 4.6 × 300 mm Format: The smaller ID AdvanceBio SEC column maintained high resolution at lower pressures (~176 bar) and offered comparable performance to sub-2 µm columns operating at elevated pressures.

Benefits and Practical Applications

• Enhanced resolution and sensitivity enable accurate quantitation of aggregates and fragments.
• Aqueous mobile phase supports sample integrity for hydrophobic ADCs.
• Lower operating pressure extends column lifespan and maintains instrument compatibility.
• Improved reproducibility and data quality streamline biopharmaceutical development and QC workflows.

Future Trends and Opportunities

• Serial coupling of SEC columns may further increase peak capacity for complex mixtures.
• Integration with high-throughput and multi-detector systems to deepen biomolecular characterization.
• Expansion of applications in ADC formulation screening, stability studies, and regulatory compliance testing.

Conclusion

Agilent AdvanceBio SEC columns demonstrate superior performance in size-based separations for biopharmaceutical applications, combining optimal pore structure, hydrophilic coating, and robust design to deliver high-resolution, reproducible, and reliable data for proteins, monoclonal antibodies, and ADCs.