Guide to Size-Exclusion Chromatography (SEC) of mAb Aggregates, Monomers, and Fragments

Importance of Topic

Size-exclusion chromatography (SEC) is a vital analytical tool in biopharmaceutical research and quality control, enabling precise separation and quantitation of monoclonal antibody (mAb) aggregates, monomers, and fragments. Accurate profiling of these species is essential for ensuring the safety, efficacy, and stability of therapeutic proteins.

Study Objectives and Overview

This guide outlines critical considerations for developing robust LC-based SEC methods using bridged ethylene hybrid (BEH) SEC columns. Key goals include selecting appropriate column pore size and particle chemistry, optimizing mobile phase conditions to minimize secondary interactions, managing system dispersion effects, and maximizing column lifetime through proper maintenance.

Methodology and Instrumentation

Recommended workflow and instrumentation:

• Chromatography system: Waters ACQUITY UPLC H-Class Bio with Tunable UV detection at 280 nm
• Columns: ACQUITY UPLC Protein BEH SEC columns (particle sizes 1.7 µm, 2.5 µm, 3.5 µm; pore sizes 125 Å, 200 Å, 450 Å)
• Mobile phases: aqueous buffers (e.g., 20–25 mM sodium phosphate with 0.1–0.35 M NaCl, pH 6.8–7.0)
• Flow rates and temperature: 0.35–0.5 mL/min at 30 °C
• Dispersion measurement: zero-volume union with caffeine injections to ensure total system dispersion <15 µL
• Cleaning protocol: sequential purging with 70% isopropanol, 100% methanol, 10% phosphoric acid, and high-purity water
• Sample preparation: sterile filtration (≤0.2 µm) or centrifugation to remove particulates

Main Results and Discussion

BEH SEC columns with surface-modified diol groups exhibit reduced silanol activity, allowing use of sub-2 µm particles at UPLC pressures for higher resolution and faster separations. Calibration data demonstrate column pore size selection based on sample molecular weight range (125 Å for 1–80 kDa; 200 Å for 10–450 kDa; 450 Å for 100–1500 kDa). Minimizing extra-column dispersion is critical for resolving closely eluting species. Proper column installation, mobile phase filtration, and microbial control prevent column fouling. The use of short guard columns further extends analytical column lifetime beyond 1000 injections.

Benefits and Practical Applications

Optimized BEH SEC methods deliver higher chromatographic efficiency, improved reproducibility, and shorter analysis times compared to traditional silica-based SEC. These advantages support applications in biopharmaceutical R&D, quality control, stability testing, and batch release assays.

Future Trends and Possibilities

• Integration of SEC with mass spectrometry and advanced detectors for detailed structural characterization of protein species
• Development of novel hybrid particle materials and sub-2 µm chemistries for enhanced separation performance
• Automation and high-throughput workflows tailored to process development and manufacturing environments
• Utilization of machine learning and predictive modelling for method optimization and data interpretation

Conclusion

Implementing a well-designed SEC protocol with appropriate BEH SEC column selection, control of system dispersion, rigorous sample and mobile phase preparation, and routine instrument maintenance yields consistent, high-quality analysis of mAb aggregates, monomers, and fragments. These best practices ensure reliable data essential for biotherapeutic development and regulatory compliance.

References

1. Hong P, Koza SM, Bouvier ESP. Size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chrom Rel Tech. 2012;35(20):2923-2950.
2. Bouvier ESP, Koza SM. Advances in size-exclusion separations of proteins and polymers by UHPLC. Trends Analyt Chem. 2014;63:85-94.
3. Rutala WA, Weber DJ; HICPAC. Guideline for Disinfection and Sterilization in Healthcare Facilities. CDC; 2008.
4. Koza SM, Reed CE, Chen W. Impact of LC system dispersion on SEC analysis of proteins. Waters Application Note. 2019;720006337EN.
5. Koza SM, Reed C, Chen W. Column configuration for mAb aggregate analysis. Waters Application Note. 2019;720006336EN.
6. Hong P, Fountain K. Method development for SEC of mAbs and aggregates. Waters Application Note. 2011;720004076EN.