Ultimate Resolution HPLC-Based Size Exclusion Chromatography for the Analysis of Small Proteins and Peptides Using 3.5 μm Ethylene Bridged Hybrid (BEH) Particles

Význam tématu

Size-exclusion chromatography (SEC) is critical for the characterization of protein and peptide size distributions in research, quality control, and biopharmaceutical development. The development of BEH-based 3.5 μm, 125 Å SEC columns addresses the need for high-resolution separations of small proteins and peptides on conventional HPLC systems. Enhanced column stability and reproducibility further support validated analytical methods in routine workflows.

Cíle a přehled studie / článku

This application note presents:

• The development of 3.5 μm ethylene bridged hybrid (BEH) SEC columns with 125 Å pore size optimized for HPLC.
• Comparisons with traditional 5 μm silica-based SEC columns.
• Evaluation of resolution, reproducibility, column lifetime, and method transferability to UPLC.

Použitá metodika a instrumentace

Experimental conditions:

• Instrumentation: Waters Alliance HPLC, ACQUITY UPLC H-Class Bio System, ACQUITY UPLC; TUV detectors.
• Columns: XBridge Protein BEH SEC (125 Å, 3.5 μm, 7.8×300 mm), ACQUITY UPLC Protein BEH SEC (125 Å, 1.7 μm, 4.6×300 mm), Comparator silica-diol SEC (125 Å, 5 μm).
• Mobile phase: phosphate buffer (25 mM NaH2PO4, 150 mM NaCl, pH 7.2), optional organic modifiers (ACN, TFA).
• Flow rate: 0.84 mL/min (HPLC), wavelength detection at 280 or 214 nm.
• Samples: Protein and peptide standards (1 mg/mL) run at 10 °C, 10 μL injections.

Hlavní výsledky a diskuse

• Resolution improvement: 3.5 μm BEH columns yielded narrower peaks and higher sensitivity for myoglobin and peptide mixtures compared to 5 μm silica-based columns.
• Enhanced throughput: Two 300 mm BEH columns in series at double flow rate achieved greater resolution without extending run time.
• Reduced secondary interactions: BEH surface exhibited lower silanol-mediated binding for basic peptides (ubiquitin, aprotinin).
• Reproducibility: Batch-to-batch consistency confirmed with <1% RSD in retention times across three production lots.
• Column stability: After >600 injections, BEH columns maintained resolution between monomer and dimer peaks, outperforming silica-based columns at pH 7.2.
• Method transfer: SEC methods scaled from HPLC to UPLC by adjusting column length and linear velocity demonstrated comparable performance, though UPLC delivered faster analysis and lower solvent consumption.
• Pore-size calibration: 125 Å columns cover ~<1–80 kDa, 200 Å ~10–450 kDa, and 450 Å ~50 kDa–1.3 MDa molecular weight ranges.

Přínosy a praktické využití metody

• High-resolution size determination of small proteins and peptides on standard HPLC.
• Robust column lifetime suitable for routine QC environments.
• Seamless transfer to UPLC platforms for increased throughput.
• Improved method confidence due to outstanding column-to-column reproducibility.

Budoucí trendy a možnosti využití

The ongoing evolution of SEC includes:

• Integration of sub-2 μm BEH technology in new HPLC and UPLC formats.
• Expansion of pore-size options for broader molecular weight profiling.
• Coupling SEC with multi-dimensional chromatographic and mass spectrometric techniques.
• Applying advanced data analytics and AI for automated peak assignment and quality control.

Závěr

The 125 Å, 3.5 μm BEH SEC columns combine enhanced resolution, chemical stability, and reproducibility, enabling reliable size-based separations of small proteins and peptides on HPLC. Their compatibility with UPLC methods further supports flexible, high-throughput workflows in research and quality control.

Reference

1. Hong P., Koza S., Fountain K.J. Advances in Size-Exclusion Chromatography for the Analysis of Small Proteins and Peptides: Evaluation of Calibration Curves for Molecular Weight Estimation. Waters Corporation Application Note, 2012.
2. Koza S., Serpa S., Yang H., Bouvier E., Fountain K.J. Advanced HPLC Size-Exclusion Chromatography for the Analysis of Macromolecular Proteins Using 3.5 μm Ethylene Bridged Hybrid (BEH) Particles. Waters Corporation Application Note, 2015.
3. Koza S., Fountain K.J. Successful Transfer of Size Exclusion Separations between HPLC and UPLC. Waters Corporation Application Note, 2014.