Advanced HPLC Size-Exclusion Chromatography for the Analysis of Macromolecular Proteins Using 3.5 μm Ethylene Bridged Hybrid (BEH) Particles

Significance of the topic

Size-exclusion chromatography (SEC) is a cornerstone technique in analytical chemistry for the characterization of macromolecular proteins. It plays a critical role in biopharmaceutical development, quality control of therapeutic proteins, and research into protein aggregation. Reliable SEC methods enable accurate estimation of molecular weight distribution, aggregation state, and sample purity, which are essential parameters in drug development, regulatory submission, and formulation studies.

Study objectives and overview

This study evaluates novel HPLC-compatible size-exclusion columns packed with 3.5 µm ethylene bridged hybrid (BEH) particles having pore sizes of 200 Å and 450 Å. The goals were to compare performance against conventional silica-based SEC columns, assess column-to-column reproducibility and long-term stability, and determine improvements in resolution and sample throughput. Special focus was placed on separating protein standards and monoclonal IgG aggregates under isocratic conditions.

Experimental methodology and instrumentation

Protein standards (10 kDa to 2 MDa) and intact monoclonal antibodies were prepared at ~1 mg/mL in 25 mM sodium phosphate, 150 mM sodium chloride, pH 7.2. Separations were carried out on Waters Alliance HPLC and ACQUITY UPLC H-Class Bio Systems equipped with TUV detectors (280 nm and 214 nm), a column heater, and Auto•Blend Plus™ solvent preparation.

• Columns evaluated: 3.5 µm BEH SEC, 200 Å and 450 Å, 7.8 × 150 mm and 7.8 × 300 mm
• Comparators: silica-DIOL SEC, 5 µm, 250 Å and 8 µm, 450 Å (7.8 × 300 mm)
• Flow rate: 0.84 mL/min (up to 2.0 mL/min for throughput tests)
• Injection volume: 10 µL; sample temperature: 10 °C; column temperature: ambient

Main results and discussion

Compared with silica-based SEC columns, BEH 3.5 µm columns delivered:

• ~40 % higher resolution between IgG monomer (150 kDa) and dimer (300 kDa) on 200 Å particles
• ~75 % resolution improvement for IgG aggregates on 450 Å particles
• Sharper peaks and increased signal sensitivity across the separation range

Doubling the flow rate on 3.5 µm BEH columns reduced run times by 50 % with only a 25 % loss in resolution, demonstrating high mechanical stability. Column-to-column and batch-to-batch reproducibility showed retention time RSDs of 0.04–0.08 min (average ~0.06 min) across six columns. Over 600 injections under pH 7.2 conditions produced minimal performance drift.

Calibration curves defined the linear molecular weight range as ~10 kDa to 450 kDa for the 200 Å column and ~50 kDa to 1.3 MDa for the 450 Å column; partial resolution extended to ~1.8 MDa aggregates.

Benefits and practical applications

The BEH-based SEC columns offer:

• Enhanced resolution for therapeutic proteins, aggregates, and conjugates
• High throughput capability without major loss of separation quality
• Robust lifetime and reproducible performance for validated QC methods
• Compatibility with existing HPLC systems and seamless scaling to UPLC formats

Future trends and opportunities

Anticipated developments include coupling BEH SEC with mass spectrometry for intact protein analysis, expansion of denaturing SEC applications, integration into automated high-throughput workflows, and application of machine-learning algorithms for chromatogram interpretation. Advances in particle chemistry may further extend the molecular weight range and improve resistance to harsh mobile phase conditions.

Conclusion

HPLC-compatible BEH SEC columns with 3.5 µm particles and 200 Å or 450 Å pores deliver superior resolution, throughput, and reproducibility compared to traditional silica-based SEC. Their mechanical strength and stable performance make them well suited for routine biopharmaceutical quality control and research applications, with straightforward scalability to UPLC.

References

• Fountain KJ et al. Analysis of Biomolecules by Size-Exclusion UltraPerformance Liquid Chromatography. Waters Corp. Application Note, 2010; WA64226.
• Hong P, Koza S, Fountain KJ. Advances in Size-Exclusion Chromatography for Peptides and Proteins. Waters Corp. Application Note, 2012; 720004412EN.
• Koza S, Lauber M, Fountain KJ. Analysis of Multimeric Monoclonal Antibody Aggregates. Waters Corp. Application Note, 2013; 720004713EN.
• Koza S, Fountain KJ. Successful Transfer of Size-Exclusion Separations between HPLC and UPLC. Waters Corp. Application Note, 2014; 720005193EN.