T he Analysis of Multimeric Monoclonal Antibody Aggregates by Size-Exclusion UPLC

Significance of the Topic

Monoclonal antibody aggregation represents a critical quality attribute in biotherapeutic development and manufacturing. Soluble aggregates can arise at various stages from cell culture to storage, potentially compromising safety and efficacy. High-valency multimers pose heightened immunogenic risk due to their ability to trigger immune pathways independent of T cells.

Objectives and Study Overview

This study evaluates the performance of sub-3 µm BEH450 and BEH200 size-exclusion UPLC columns, alone and in series, for separating monomer to multimer antibody aggregates. Key goals include comparing UPLC to traditional HPLC SEC, expanding the useful molecular weight range up to 1.8 MDa, and assessing aggregate distribution in stressed monoclonal antibody samples.

Methodology

Sample Preparation

• IgM pentamer and dipentamer standards
• Cross-linked IgG aggregates via BS3 chemistry
• Trastuzumab freeze-thaw induced aggregation

Chromatographic Conditions

• Mobile phase 5 mM sodium phosphate, 250 mM NaCl, pH 6.8
• Isocratic flow at 0.35 mL/min, injection 5 µL
• Column temperature ambient, sample at 10 °C

Instrumentation Used

ACQUITY UPLC H-Class Bio System with 30 cm heater and TUV detector
Wyatt miniDAWN TREOS light scattering detector for SEC-MALS
BEH450 SEC columns (2.5 µm, 450 Å) and BEH200 SEC columns (1.7 µm, 200 Å)
Empower 3 and UNIFI data systems, Wyatt Astra software

Main Results and Discussion

• BEH450 UPLC delivered superior resolution and sensitivity for IgM pentamer versus HPLC, with clear separation of 900 kDa and 1.8 MDa species.
• Series configuration of BEH200 and BEH450 columns extended separation range from 10 kDa to 1.8 MDa, improving resolution of thyroglobulin dimer and small proteins.
• Cross-linked mAb sample showed improved monomer-dimer resolution on BEH200, while series setup revealed higher-order multimers beyond trimer.
• Freeze-thaw stressed trastuzumab analysis demonstrated rising levels of dimer, trimer, tetramer and larger aggregates; series columns enhanced separation of multimer distribution while preserving fragment resolution.

Benefits and Practical Applications

• Enhanced throughput and resolution compared to traditional SEC HPLC
• Broad molecular weight coverage from 10 kDa to nearly 2 MDa in a single analysis
• Detailed mapping of aggregate valency to support immunogenicity risk assessment
• Applicability in QC labs for stability testing, process monitoring, and formulation screening

Future Trends and Potential Applications

• Integration of SEC-UPLC with native MS or MALS for real-time characterization
• Development of even larger pore stationary phases to monitor ultra-high molecular weight complexes
• Two-dimensional separations combining different selectivities for detailed impurity profiling
• Machine learning algorithms for automated pattern recognition of aggregate profiles
• Expansion to other protein therapeutics such as bispecifics and fusion proteins

Conclusion

The combination of BEH450 and BEH200 SEC-UPLC columns on the ACQUITY UPLC H-Class Bio System delivers unparalleled resolution, sensitivity, and extended molecular weight range for monoclonal antibody aggregate analysis. This approach enhances aggregate detection, facilitates detailed valency profiling, and strengthens quality control across biotherapeutic pipelines.

References

1. Cromwell ME, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS Journal 2006;8:E572-9
2. Rosenberg AS. Effects of protein aggregates an immunologic perspective. AAPS Journal 2006;8:501-7
3. Koza S, Fountain KJ. Advances in size exclusion chromatography for macromolecular proteins. Waters Application Note 720004618EN 2013
4. Hong P, Koza S, Fountain KJ. Analysis of proteins by SEC-MS under non-denaturing conditions. Waters Application Note 720004254EN 2012