Expanding Size-Exclusion Chromatography Platform Method Versatility for Monoclonal Antibody Analysis Using Waters XBridge Premier Protein SEC Columns

Significance of the Topic

Therapeutic proteins such as monoclonal antibodies must be rigorously monitored for size variants including aggregates and fragments, as these can influence immunogenicity, potency, and safety. Size-exclusion chromatography (SEC) is the standard method to resolve these critical quality attributes and ensure consistent product performance. Platform SEC methods enhance efficiency across similar molecules by minimizing method revalidation and training overhead.

Study Objectives and Overview

This work compares the performance and versatility of the new Waters XBridge Premier Protein SEC column against the prior BioResolve SEC mAb column for analyzing four biosimilar monoclonal antibodies. A quality by design (QbD) full factorial approach evaluated a matrix of mobile phase buffers at pH values from 5.8 to 7.6 and NaCl concentrations from 50 to 400 mM.

Methodology and Instrumentation

Four monoclonal antibody samples (bevacizumab, infliximab, rituximab, trastuzumab) were injected neat and analyzed under 16 eluent conditions at 0.75 mL/min on 7.8×300 mm columns packed with 2.5 μm particles. Data acquisition and mobile phase blending were performed with Empower 3 and Auto•Blend Plus.

• LC System: ACQUITY UPLC H-Class Bio with CH-30A APH Column Heater
• Detector: ACQUITY UPLC Tunable UV (280 nm and 214 nm)
• Columns: XBridge Premier Protein SEC (250 Å, 2.5 μm) and BioResolve SEC mAb (200 Å, 2.5 μm)
• Mobile phases: 20 mM sodium phosphate (pH 5.8–7.6) with 50–400 mM NaCl

Main Results and Discussion

Heat-map analysis of resolution and peak-area consistency revealed that the XBridge Premier column maintained effective quantification of high-molecular-weight (HMW) and low-molecular-weight (LMW) variants across a wider pH and ionic strength range than the BioResolve column. Both columns performed comparably at higher salt and pH ~7.4, but the Premier column excelled under low-ionic-strength and mildly acidic conditions, indicating reduced nonspecific interactions and enhanced versatility.

Benefits and Practical Applications

• Streamlined platform SEC methods with a single column and mobile phase across diverse mAb candidates
• Improved robustness and method transferability between HPLC, UHPLC, and UPLC systems
• Enhanced resolution for aggregate and fragment quantification under formulation-relevant conditions

Future Trends and Potential Applications

The inertness and broad pH stability (2.5–8.0) of BEH-PEO particles open opportunities for SEC analysis at physiological pH and ionic strengths, as well as unique formulation conditions. Future developments may include integration with automated method development tools, faster separations using sub-2 μm particle formats, and extending platform SEC to other protein therapeutics and complexes.

Conclusion

The Waters XBridge Premier Protein SEC column demonstrates significant advancement over the previous generation BEH-diol column by offering wider operating conditions, reduced protein-column interactions, and robust performance for mAb size-variant analysis. This enhanced versatility supports both platform and bespoke SEC method development for therapeutic protein QC and research.

References

• Wang X, An Z, Luo W, Xia N, Zhao Q. Molecular and Functional Analysis of Monoclonal Antibodies in Support of Biologics Development. Protein Cell. 2018;9(1):74–85.
• Moore R. Leveraging Platform Analytical Methods for Biopharma QbD. Pharma Manufacturing. 2017.
• Koza SM, Yang H, Yu YQ. Modern Size-Exclusion Chromatography Separations of Biosimilar Antibodies at Physiological pH and Ionic Strength. Waters Application Note. 2022.
• Yang R, Tang Y, Zhang B, Lu X, Liu A, Zhang YT. High Resolution Separation of Recombinant Monoclonal Antibodies by Size-Exclusion Ultra-High Performance Liquid Chromatography (SE-UHPLC). J Pharm Biomed Anal. 2015;109:52–61.
• Koza SM, Reed C, Chen W. Impact of LC System Dispersion on the Size-Exclusion Chromatography Analysis of Monoclonal IgG Antibody Aggregates and Fragments. Waters Application Note. 2019.
• Iraneta PC, Koza SM. High Resolution Size-Exclusion Chromatography Separations of Mab Aggregates, Monomers, and Fragments Using BioResolve SEC mAb Columns. Waters Application Note. 2020.