The importance of correct UHPLC instrument setup for protein aggregate analysis by size-exclusion chromatography

Importance of the topic

A robust UHPLC setup with low extra-column dispersion is essential for reliable size-exclusion chromatography (SEC) analysis of monoclonal antibody aggregates. Protein aggregation impacts efficacy, safety, and stability of biotherapeutics and must be quantified accurately in research, quality control, and regulatory testing.

Objectives and overview of the article

This study evaluates Thermo Scientific™ Vanquish™ Flex UHPLC system equipped with MAbPac™ SEC-1 columns of 7.8 mm and 4 mm internal diameter for mAb aggregate analysis. The goal was to demonstrate how pre-column dispersion and operational parameters affect peak quality and resolution in SEC applications.

Methodology and instrumentation

The analysis employed:

• Vanquish Flex Quaternary UHPLC system with low-dispersion flow path, split sampler, column compartment, and diode array detector (214 nm).
• MAbPac SEC-1 columns: 7.8 × 300 mm and 4.0 × 300 mm.
• Mobile phase: 0.2 M NaCl in 100 mM phosphate buffer, pH 6.8.
• Flow rates: 1.0 mL/min (7.8 mm i.d.) and 0.3 mL/min (4.0 mm i.d.).
• Injection volume: 1 µL of bevacizumab at 25 mg/mL, with additional tests at 10 µL and varied solvent content (0–20% acetonitrile).

Main results and discussion

The 7.8 mm column at 1.0 mL/min showed negligible impact from reduced tubing inner diameter (100 vs 75 µm), maintaining peak width and resolution. Conversely, the 4.0 mm column at 0.3 mL/min experienced significant peak broadening and loss of resolution when using larger tubing (180 µm). Reducing transfer tubing to 75 µm minimized dispersion. Increasing injection volume from 1 to 10 µL amplified peak broadening and masked minor aggregates. Addition of 5–20% acetonitrile did not improve peak symmetry but caused partial protein unfolding, increasing peak width and reducing height.

Benefits and practical applications

• Optimized UHPLC setup with low-dispersion tubing ensures accurate quantification of monoclonal antibody aggregates.
• Proper choice of column diameter, flow rate, and tubing minimizes peak dispersion and improves resolution of low-abundance species.
• Avoiding organic solvents prevents protein unfolding and preserves native conformations.

Future trends and potential applications

Advances in ultra-low dispersion UHPLC systems, microbore column technology, and integration with mass spectrometry will further enhance the detection of trace aggregates. Automation and real-time data processing will streamline quality control workflows in biopharmaceutical manufacturing.

Conclusion

Maintaining low extra-column dispersion through optimized tubing, minimal injection volumes, and appropriate flow rates is crucial for high-resolution SEC analysis of monoclonal antibodies. The MAbPac SEC-1 column delivers robust performance without the need for organic modifiers.

Instrument used

• Thermo Scientific Vanquish Flex Quaternary UHPLC system (VF-S01-A, VF-P20-A, VF-A10-A, VH-C10-A, VH-D10-A).
• LightPipe Flow Cell, standard 10 mm (P/N 6083.0100).
• MAbPac SEC-1 columns 7.8 × 300 mm (P/N 088460) and 4 × 300 mm (P/N 074696).

Reference

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2. Hong P., Koza S., Bouvier E.S.P. Size exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chromatogr Relat Technol. 2012;35(20):2923–2950.
3. Arakawa T., Philo J.S., Ejima D., Tsumoto K., Arisaka F. Aggregation analysis of therapeutic proteins. BioProcess Int. 2006;4(10):42–43.