Successful Transfer of Size-Exclusion Separations Between HPLC and UPLC

Importance of the Topic

Size‐exclusion chromatography (SEC) is a cornerstone technique in protein and peptide analysis, used to assess molecular weight distribution, detect aggregates, and support biopharmaceutical quality control. With growing demand for higher throughput and sensitivity, laboratories seek scalable methods across varied instrumentation, from ultra-performance liquid chromatography (UPLC) to traditional high-performance liquid chromatography (HPLC), without compromising separation performance.

Objectives and Study Overview

This study demonstrates robust transfer of SEC methods between UPLC and HPLC platforms using matched ethylene-bridged hybrid (BEH) particle chemistries. Key goals include:

• Comparing chromatographic performance of 1.7 μm and 2.5 μm UPLC BEH columns with 3.5 μm HPLC BEH columns.
• Establishing scaling relationships for column dimensions, flow rates, and injection volumes.
• Validating method transfer across a broad molecular weight range (112 Da to >1 MDa) for standard proteins and monoclonal antibodies.

Methodology and Instrumentation

Two UPLC formats (200 Å, 1.7 μm; 450 Å, 2.5 μm; 4.6×300 mm) and two HPLC formats (200 Å or 450 Å, 3.5 μm; 7.8×300 mm segments in series to achieve target lengths) were evaluated. Key parameters were scaled according to theoretical relationships:

1. Column length ∝ particle diameter ratio (L_HPLC=L_UPLC×d_p,HPLC/d_p,UPLC).
2. Flow rate ∝ (linear velocity×column I.D.²×particle diameter).
3. Injection volume ∝ column volume (I.D.²×length).

All separations used phosphate‐buffered saline (25 mM sodium phosphate, 150 mM NaCl, pH 7.2) at ambient column temperature, UV detection at 280 or 214 nm, and Waters Empower 3 software.

Instrumentation:

• ACQUITY UPLC H-Class Bio System with TUV detector
• Waters Alliance HPLC System with TUV detector
• ACQUITY UPLC BEH Protein SEC Columns (200 Å/1.7 μm, 450 Å/2.5 μm)
• XBridge BEH Protein SEC Columns (200 Å & 450 Å, 3.5 μm)
• BEH SEC Protein Standard Mixtures and mAb standards

Main Results and Discussion

Comparisons between matched UPLC and HPLC formats revealed equivalent selectivity and resolution across the protein mass range. Illustration highlights:

• 200 Å series: Resolutions for IgG vs BSA were ~30% higher on 7.8 mm I.D. HPLC columns due to reduced extra‐column dispersion.
• Scaled transfers showed overlapping profiles for standard mixes and infliximab monomer/aggregate peaks, with UPLC run times 4–5× shorter.
• 450 Å series: Similar transfer success for large complexes (IgM pentamers/dipentamers), with UPLC achieving ~2× faster analysis.

Benefits and Practical Applications

Method transfer between UPLC and HPLC offers:

• Seamless scalability for labs with mixed instrumentation.
• Streamlined cross-site method deployment.
• Enhanced throughput on UPLC with fallback to HPLC for characterization requiring larger column volumes or lower back-pressure.

Future Trends and Potential Applications

Emerging directions include integration of sub-2 μm SEC for ultra-fast screening, coupling with multi-angle light scattering for absolute molar mass determination, and expanded use in QC environments. Further optimization of buffer systems may reduce subtle surface interactions and extend method universality across novel particle chemistries.

Conclusion

The study confirms that matching BEH particle chemistry and applying systematic scaling rules enables reliable transfer of SEC methods between UPLC and HPLC platforms. This approach preserves chromatographic selectivity while leveraging instrument-specific strengths in throughput or capacity.

Reference

1. Fountain K.J. et al. Analysis of Biomolecules by Size-Exclusion UPLC. Waters Corp. Application Note, 2012.
2. Hong P. et al. Advances in SEC for Small Proteins: Calibration Curves. Waters Corp. Application Note, 2010.
3. Gritti F., Guiochon G. Extra-column Band-broadening in Modern UHPLC. J. Chromatogr. A, 2010;1217(49):7677–7689.
4. Ricker R.D., Sandoval L.A. Fast, Reproducible SEC of Biological Macromolecules. J. Chromatogr. A, 1996;743(1):43–50.