Size Exclusion Chromatography Method Transfer from a Standard HPLC to an Arc HPLC System

Significance of the Topic

Size-exclusion chromatography (SEC) is a critical analytical tool for characterizing biotherapeutic proteins by separating species based on hydrodynamic volume. It enables quantification of monomeric proteins, high molecular weight aggregates and low molecular weight degradants, which are key quality attributes in biopharmaceutical development and quality control.

Objectives and Study Overview

This application note illustrates the transfer of an SEC method for trastuzumab, a monoclonal antibody, from a standard HPLC system to a Waters Arc HPLC System. The goal was to compare retention time, peak area, resolution and repeatability across both platforms.

Methodology and Instrumentation

The SEC method employed the following configuration:

• Column: XBridge Protein BEH SEC, 200 Å, 2.5 µm, 7.8 mm × 300 mm
• Mobile phase: 20 mM phosphate buffer with 350 mM sodium chloride, pH 6.8
• Flow rate: 1.0 mL/min
• Sample: Trastuzumab at 10 mg/mL, injection volume 10 µL
• Systems compared: Industry-standard stainless steel HPLC and Waters Arc HPLC System

The method transfer included system flushing procedures to mitigate salt build-up on stainless steel components.

Main Results and Discussion

Both HPLC systems produced comparable chromatographic profiles with monomer, one high molecular weight (HMW) aggregate peak and two low molecular weight (LMW) degradant peaks baseline resolved from the main peak. Key findings included:

• Retention times differed by approximately 0.02 min, with repeatability within 0.1% RSD across five injections
• Peak area percentages for monomer, HMW and LMW species were within 0.01% between systems and showed ≤2% RSD
• The Arc HPLC System exhibited slightly narrower peak widths and lower tailing factors, resulting in an improved peak valley ratio (1.32 vs. 1.02), enhancing separation of the monomer shoulder (LMW1)

Benefits and Practical Applications of the Method

Transfer of SEC methods between HPLC platforms ensures flexibility in laboratory workflows and instrument utilization. The Arc HPLC System demonstrated robust performance for routine mAb size variant analysis, offering:

• Tight retention time and peak area repeatability
• Enhanced resolution between critical quality attribute peaks
• Compatibility with high-salt mobile phases

Future Trends and Potential Applications

Advances in chromatographic hardware and biocompatible surface treatments are expected to further improve SEC performance and system longevity. Integration with advanced data analysis software and automation will support high-throughput characterization of next-generation biotherapeutics, including bispecific antibodies and antibody–drug conjugates.

Conclusion

The SEC method for trastuzumab was successfully transferred from a standard HPLC system to the Arc HPLC System with minimal variation in analytical metrics. The Arc HPLC System delivered equal or superior chromatographic performance, validating its suitability for routine biotherapeutic size variant analysis.

References

1. Rosenberg AS. Effects of Protein Aggregates: An Immunologic Perspective. The AAPS Journal. 2006;8:501–507.
2. Xiang T, Lundell E, Sun Z, Liu H. Structural Effect of a Recombinant Monoclonal Antibody on Hinge Region Peptide Bond Hydrolysis. Journal of Chromatography B. 2007;858:254–262.
3. Koza S, Hong P, Fountain KJ. Size-Exclusion UltraPerformance Liquid Chromatography Method Development for the Analysis of the Degradation Products of the Trastuzumab Antibody. Waters Application Note. 2012;720004416EN.