Solutions for Aggregate and Fragment Analyses - SEC COLUMN TECHNOLOGY

Significance of the Topic

Size-exclusion chromatography (SEC) is a cornerstone technique for assessing aggregate and fragment content in biotherapeutic proteins and viral vectors. Reliable aggregate analysis is critical in quality control, process development, and regulatory compliance. Optimizing SEC column technology enhances data quality, accelerates method development, and supports scale-up from discovery to manufacturing.

Objectives and Overview

This application note presents a comprehensive overview of SEC column selection and performance criteria. Key goals include guiding users through pore size, particle size, and column dimension choices based on sample molecular weight and system dispersion. Representative chromatograms illustrate monomer and size variant separation using both UPLC and HPLC platforms.

Methodology and Used Instrumentation

Instrument platforms

• ACQUITY UPLC H-Class Bio System with dispersion <20 µL
• ACQUITY Premier UPLC with Protein SEC columns
• XBridge Premier HPLC for larger particle formats

Mobile phase and conditions

• 2× PBS buffer (20 mM phosphate, 276 mM NaCl, 5.4 mM KCl, pH 7.4)
• Flow rates: 300 µL/min for 1.7 µm UPLC; up to 575 µL/min for 2.5 µm HPLC
• Column temperatures maintained at 35 °C
• Detection at 280 nm

Main Results and Discussion

Column selection guidelines

• Pore sizes: 125 Å for peptides and small proteins (1–80 kDa); 200/250 Å for monoclonal antibodies and analogs (10–450/650 kDa); 450 Å for large assemblies (100–1500 kDa).
• Particle sizes: 1.7 µm for low dispersion UPLC (<20 µL); 2.5 µm for mid-range dispersion (20–35 µL); 2.5 µm for HPLC (>35 µL).
• Column dimensions: longer columns improve resolution for closely sized species; 4.6×300 mm or 7.8×300 mm for <2× molecular weight differences; 4.6×150 mm or 7.8×150 mm for ≥2× differences.

Chromatographic performance
UPLC with 1.7 µm, 250 Å columns achieved baseline resolution of IgG1 monomer, high-molecular-weight species, and low-molecular-weight fragments within ten minutes. HPLC with 2.5 µm, 250 Å columns demonstrated comparable separation in extended run times, supporting flexible platform choices.

Benefits and Practical Applications

• Minimized secondary interactions through carefully tuned stationary phase chemistry.
• pH stability above neutral for method robustness.
• Scalable column chemistries applicable from discovery to manufacturing scales.
• High lot-to-lot reproducibility supported by stringent quality control standards.

Future Trends and Potential Applications

Emerging directions include microflow SEC for reduced sample consumption, integrated multi-detector workflows (e.g., light scattering, mass spectrometry), and AI-driven method optimization. Advances in column packing materials and hardware will further improve resolution and throughput, enabling real-time process monitoring in biomanufacturing.

Conclusion

Optimizing SEC column selection based on molecular weight range, system dispersion, and resolution requirements is essential for accurate aggregate and fragment analysis. Waters Premier and XBridge column families offer scalable, high-performance solutions supporting diverse applications in biotherapeutic development and quality control.

Reference

• Waters SEC Optimization Guide, p/n 720006067EN
• Waters Application Note on SEC Column Recommendations, p/n 720006336EN