Advances in Size Exclusion Chromatography for the Analysis of Macromolecular Proteins

Importance of the topic

Size‐exclusion chromatography (SEC) is a vital tool for characterizing the size and aggregation state of proteins and other biological macromolecules. Advances in column packing and ultraperformance liquid chromatography (UPLC) hardware have enabled higher resolution, faster analysis, and enhanced reproducibility, meeting the growing demand for reliable quality control and biopharmaceutical development workflows.

Objectives and Study Overview

This study evaluates a new 2.5 µm, 450 Å bridged‐ethane hybrid (BEH) SEC column for UPLC (BEH450 SEC) aimed at:

• Assessing column‐to‐column reproducibility and long‐term stability under repeated injections.
• Comparing separation efficiency against conventional 8 µm SEC media.
• Exploring complementary use with a 1.7 µm, 200 Å BEH SEC column to extend the molecular weight range.

Methodology

Proteins spanning 112 Da to 1.8 MDa were analyzed under isocratic conditions (25 mM phosphate, 250 mM NaCl, pH 6.8) at 10 °C. Injection volumes and flow rates were scaled to column geometry. Retention times, peak shapes, and resolution (USP) were monitored over >800 injections. Comparisons between columns used equivalent system settings and mobile phase.

Instrumentation Used

• Waters ACQUITY UPLC H‐Class Bio System with 30 cm column heater
• ACQUITY UPLC BEH450 SEC columns (4.6 × 150 mm and 4.6 × 300 mm, 2.5 µm, 450 Å)
• ACQUITY UPLC BEH200 SEC columns (4.6 × 150 mm and 4.6 × 300 mm, 1.7 µm, 200 Å)
• ACQUITY UPLC TUV detector (5 mm titanium flow cell, 214/280 nm)
• Wyatt miniDAWN TREOS light scattering detector
• Chromatography data software: Empower Pro, UNIFI, Wyatt Astra

Main Results and Discussion

Column‐to‐column reproducibility across five BEH450 SEC columns from three lots showed retention time RSD ≤0.02 min over eight standards. Stability testing (>800 injections) demonstrated consistent thyroglobulin retention and stable uracil peak asymmetry, confirming mechanical and chemical integrity.

Comparative studies revealed that the 2.5 µm BEH450 SEC column delivered narrower peaks and nearly double the resolution (Rs = 2.49) for apoferritin monomer/dimer versus an 8 µm HPLC SEC packing (Rs = 1.42).

Combining BEH200 and BEH450 SEC columns in series extended the pseudo‐linear separation range from small proteins (17–150 kDa) up to large assemblies (up to ~2 MDa). While each column excels in its optimal mass range, the dual‐column setup offers broad coverage with moderate compromise in peak resolution.

Benefits and Practical Applications

• Exceptional batch‐to‐batch reproducibility and long‐term column stability support robust QC assays.
• Enhanced resolution for high‐molecular‐weight species and aggregates aids detailed impurity profiling.
• Flexible method development using BEH200, BEH450, or both in series enables customization to sample complexity.

Future Trends and Potential Applications

Emerging trends include integration of multi‐detector setups (MALS, viscometry) for deeper structural insights, further miniaturization of columns for high‐throughput screening, and application of advanced packing chemistries to target ultra‐large assemblies such as virus‐based therapeutics and nanoparticles.

Conclusion

The BEH450 SEC column combined with UPLC technology delivers high resolution, robust reproducibility, and expanded molecular weight coverage for macromolecular protein analysis. Its compatibility with BEH200 SEC columns in series offers a versatile platform for comprehensive size‐based separations in research and quality control.

Reference

1. Porath J, Flodin P. Gel filtration: a method for desalting and group separation. Nature. 1959;183:1657–1659.
2. Fountain KJ, Hong P, Serpa S, Bouvier ESP, Morrison D. Analysis of biomolecules by size‐exclusion UPLC. Waters Application Note WA64226. 2010.
3. Hong P, Koza S, Fountain KJ. Advances in SEC for small proteins and peptides: calibration curves for MW estimation. Waters Application Note 720004412EN. 2012.
4. Hong P, Fountain KJ. Method development for SEC of mAbs and aggregates. Waters Application Note 720004076EN. 2011.