A universal chromatography method for aggregate analysis of monoclonal antibodies

Importance of Aggregate Analysis in Biotherapeutics

Protein aggregation is a critical quality attribute for therapeutic monoclonal antibodies (mAbs), as aggregates can trigger immunogenic responses and reduce efficacy. Reliable quantification of monomers, dimers and higher-order species ensures patient safety and supports regulatory compliance during development, manufacturing and storage of biopharmaceuticals.

Goals and Study Overview

This application note demonstrates a single, universal size-exclusion chromatography (SEC) method on the Thermo Scientific™ MAbPac™ SEC-1 column for aggregate analysis of five structurally diverse mAbs. The objectives were:

• Develop a globally applicable SEC protocol for bevacizumab, cetuximab, infliximab, rituximab and trastuzumab
• Achieve symmetrical monomer peaks indicating minimal secondary interactions
• Maintain high monomer-dimer resolution for accurate quantitation

Methodology and Instrumentation

Samples of formulated mAbs were reconstituted and directly injected (1 µL) onto a MAbPac SEC-1 column (7.8×300 mm) using a Thermo Scientific™ Vanquish™ Flex Quaternary UHPLC system. The mobile phase consisted of 100 mM phosphate buffer (pH 6.8) with 0.2 M NaCl at 0.3 mL/min and 25 °C. Detection was performed at 214 nm. Data were acquired and processed with Chromeleon™ CDS v7.2 SR4.

Main Results and Discussion

All five mAbs eluted in order roughly correlating with molecular weight (cetuximab first at ~8.37 min to bevacizumab last at ~8.84 min) with monomer peaks exhibiting asymmetry factors of 1.07–1.13, except infliximab (2.20) which retained tailing under standard conditions. Aggregate content ranged from 0.10 % (infliximab) to 2.99 % (bevacizumab). Overlay of chromatograms confirmed consistent retention times and sharp peak shapes across diverse glycosylation patterns and isoelectric points.

Benefits and Practical Application of the Method

The MAbPac SEC-1 protocol delivers high separation power for monomer-dimer pairs, minimal nonspecific interactions and robustness against sample variability. A single buffer and column configuration can be applied globally for QC testing, stability studies and comparability assessments, reducing method development time and easing regulatory filings.

Future Trends and Potential Applications

Advances may include coupling SEC with multi-angle light scattering or mass spectrometry for orthogonal aggregate characterization. High-throughput, automated workflows and novel stationary phases with optimized pore structures will further improve resolution and speed. Integration of real-time monitoring in bioreactors using SEC-derived data is also an emerging trend.

Conclusion

The study validates the MAbPac SEC-1 column with a common high-salt mobile phase as a universal solution for accurate aggregate profiling of five key therapeutic mAbs. It offers consistent retention, symmetrical peaks and reliable quantitation across diverse antibody formats.

References

1. 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.
2. Arakawa T., Philo J.S., Ejima D., Tsumoto K., Arisaka F. Aggregation Analysis of Therapeutic Proteins. BioProcess Int., 2006;4(10):42–43.
3. Rao S., Pohl C. Reversible interference of Fe3+ with monoclonal antibody analysis in cation exchange columns. Anal. Biochem., 2011;409:293–295.
4. Thermo Fisher Scientific. Application Note AN21602: The Importance of Correct UHPLC Instrument Setup for Protein Aggregate Analysis by Size-Exclusion Chromatography. 2016.