Lifetime stability of size exclusion chromatography columns for protein aggregate analysis

Significance of the topic

Monoclonal antibodies are a cornerstone of modern biotherapeutics, offering high specificity and long serum half-lives. Aggregation of these proteins can compromise efficacy, safety, and immunogenicity, making reliable monitoring of aggregate levels a critical quality attribute in biopharmaceutical development and manufacturing.

Study objectives and overview

This study evaluates the long-term stability of Thermo Scientific MAbPac SEC-1 columns (4.0 × 300 mm and 7.8 × 300 mm, 5 µm, 300 Å) for protein aggregate analysis. Using a Vanquish Flex Quaternary UHPLC system, the team performed repeated injections of bevacizumab to assess retention time consistency, peak symmetry, and column efficiency over hundreds to thousands of injections.

Methodology and instrumentation

Sample preparation and mobile phases:

• Bevacizumab (25 mg/mL) diluted 1:10 in phosphate buffer (100 mM for 4 mm column, 50 mM for 7.8 mm column), pH 6.8, 300 mM NaCl.
• Buffers filtered through 0.2 µm membranes.

Chromatographic setup:

• Columns: MAbPac SEC-1, 4.0 × 300 mm and 7.8 × 300 mm.
• UHPLC system: Thermo Scientific Vanquish Flex Quaternary (quaternary pump, split sampler, column compartment, DAD detector).
• Flow rates: 0.25 mL/min (4 mm), 0.8 mL/min (7.8 mm); column temperature 30 °C.
• Injection volumes: 2 µL of 2.5 µg/µL (4 mm), 0.5 µL of 2.5 µg/µL (7.8 mm).
• Detection: 280 nm (both columns) and 214 nm (4 mm only).
• Data handled with Thermo Chromeleon 7.2 SR4 CDS.

Key results and discussion

4.0 × 300 mm column:

• Over 1,953 on-column injections (2,000 total), monomer retention time varied by only 0.043 min.
• Peak asymmetry remained between 0.88 and 0.93 throughout.
• Column efficiency (European Pharmacopoeia plates) stayed above 85% of initial value for the first 1,866 bevacizumab injections.
• After 1,866 injections, efficiency began to decline, though aggregation profiles remained unchanged.

7.8 × 300 mm column:

• Up to 1,292 on-column injections maintained strong performance.
• Retention times and peak symmetry remained stable; initial efficiency (>11,000 plates) dropped to 6,300 plates by the last injection (~55% of initial).
• Periodic efficiency dips between injections 300–500 were traced to sample aging rather than column degradation.

Benefits and practical applications

Extended column lifetime reduces consumable costs and downtime in routine mAb aggregate monitoring. The biocompatible UHPLC system tolerates high-salt, potentially corrosive mobile phases and supports continuous operation. Narrow-bore columns with low-dispersion transfer tubing minimize sample and solvent usage while delivering superior peak shape.

Future trends and potential applications

Advances may include further miniaturization of columns, novel stationary-phase chemistries to extend lifetimes, integration with real-time monitoring and data analytics, and application to diverse biotherapeutics beyond monoclonal antibodies.

Conclusion

Thermo Scientific MAbPac SEC-1 columns coupled with a Vanquish Flex UHPLC platform demonstrate exceptional long-term stability for protein aggregate analysis of monoclonal antibodies. Both narrow-bore (4 mm) and standard (7.8 mm) formats maintain consistent retention, symmetry, and efficiency over hundreds to thousands of injections, outperforming alternative SEC columns.

References

1. Thermo Scientific Application Note AN21602: The importance of correct UHPLC instrument set-up for protein aggregate analysis by size-exclusion chromatography, 2016.