Improving SEC-MALS Data Quality with Ethylene Bridged Hybrid HPLC Size-Exclusion Columns

Significance of the Topic

Protein aggregation assessment is crucial in biotherapeutic development because it affects drug efficacy and safety by modulating immunogenic responses. Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) enables direct measurement of molecular weight and aggregate levels in protein samples.

Objectives and Study Overview

This study compares the performance of an ethylene bridged hybrid (BEH) organosilica-based SEC column against a standard diol-bonded silica SEC column in SEC-MALS experiments. Key goals include evaluating conditioning time, baseline noise, chromatographic efficiency, and molecular weight accuracy for both protein standards and a monoclonal antibody sample.

Methodology and Instrumentation

Experiments were conducted on a Waters ACQUITY UPLC H-Class Bio system equipped with a TUV detector (280 nm) and a Wyatt miniDAWN TREOS MALS detector. Columns tested were XBridge Protein BEH SEC (200 Å, 3.5 µm) and a silica-based SEC column (250 Å, 5 µm). The mobile phase was phosphate buffered saline (20 mM phosphate, 5.4 mM KCl, 274 mM NaCl, pH 7.4). A standard mixture included thyroglobulin, IgG, BSA, myoglobin, and uracil. Adalimumab was analyzed at 1 mg/mL.

Main Results and Discussion

• The BEH column required shorter conditioning and exhibited dramatically lower baseline disturbance and noise in the MALS signal compared to the silica column.
• Injection-related pressure transient peaks were reduced to ~0.1 mV initially and ~0.02 mV after repeats on BEH, versus ~1–4 mV on silica.
• Baseline noise on the BEH column was fourfold lower, improving sensitivity for low molecular weight species.
• Chromatographic efficiency increased by 41% on the BEH column, yielding higher resolution and plate counts for monomer and aggregate peaks.
• Signal-to-noise for myoglobin (17 kDa) improved from 9 to 25, supporting more accurate molecular weight assignments.
• Adalimumab analysis showed enhanced separation of fragments and aggregates, enabling reliable dimer mass assignment (270 kDa ±5%) and accurate monomer mass (140 kDa ±0.4%).

Benefits and Practical Applications

• Enhanced detection and quantitation of low-abundance species and small proteins.
• Improved resolution across a wide molecular weight range (up to ~660 kDa).
• Reduced method setup time due to faster column conditioning.
• Lower MALS baseline noise for reliable molecular weight determination.

Future Trends and Applications

Advancements in hybrid column materials and detector integration are expected to further minimize artefacts and noise. Applications may extend to high-throughput biotherapeutic screening, detailed aggregation profiling, and routine biosimilarity assessments.

Conclusion

The XBridge BEH SEC column demonstrates superior SEC-MALS performance over a conventional silica SEC column by offering faster conditioning, lower baseline noise, and higher chromatographic efficiency, leading to improved molecular weight accuracy and resolution of protein species.

Used Instrumentation

• Waters ACQUITY UPLC H-Class Bio system with TUV detector (280 nm)
• Waters XBridge Protein BEH SEC column (200 Å, 3.5 µm)
• Silica-based diol-bonded SEC column (250 Å, 5 µm)
• Wyatt miniDAWN TREOS MALS detector
• Empower 3 and Wyatt Astra 7 software packages

References

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