Method Optimization for the Analysis of Monoclonal Antibodies by Size-Exclusion Chromatography

Significance of the Topic

Monoclonal antibodies are critical in modern biopharmaceuticals due to their high specificity and affinity for target molecules. However, aggregates formed during production and storage can reduce therapeutic efficacy and trigger adverse immune responses. Reliable monitoring of these aggregates is therefore essential for ensuring safety and compliance with regulatory guidelines.

Objectives and Study Overview

This study presents the optimization of a size-exclusion chromatography (SEC) method for the analysis of monoclonal antibody aggregates and fragments. It investigates the influence of mobile phase salt concentration, flow rate, and pH on chromatographic separation, and demonstrates the use of dedicated method scouting software to streamline development and improve performance.

Methodology and Instrumentation

The optimized method employs a Shimadzu Nexera XS inert UHPLC system with a Shim-pack Bio Diol-300 column (150 mm×4.6 mm, 2 µm). A phosphate buffer (100 mmol/L, pH 7.0) containing 100 mmol/L NaCl was used as the mobile phase at a flow rate of 0.25 mL/min and column temperature of 25 °C. Detection was performed at 280 nm with a 5 µL injection. LabSolutions MD software automated solvent blending and systematic screening of salt concentration and pH.

Key Results and Discussion

• Salt concentration: 100 mmol/L NaCl provided optimal monomer peak symmetry and resolution from aggregates.
• Flow rate: 0.25 mL/min balanced analysis time (under 15 minutes) and chromatographic resolution.
• pH: Neutral pH 7.2 minimized electrostatic interactions and peak tailing, yielding the best separation.
• Column comparison: The 150 mm Bio Diol-300 column achieved superior monomer–fragment separation versus a 300 mm commercial SEC column.

Benefits and Practical Applications

Optimized SEC conditions enable rapid, high-resolution profiling of antibody aggregates, supporting stringent ICH-Q6B impurity testing. Automated method scouting accelerates development workflows and allows tailored analytical protocols for diverse antibody therapeutics.

Future Trends and Opportunities

• Adoption of sub-2 µm SEC packing materials to further reduce analysis times.
• Coupling SEC with mass spectrometry for detailed aggregate characterization.
• Advanced software-driven platforms for real-time method optimization in quality control labs.

Conclusion

By systematically optimizing mobile phase composition, flow rate, and pH on an inert UHPLC platform, this work delivers a robust, high-throughput SEC method for monoclonal antibody aggregate analysis. Tailored parameter selection is key to accurate impurity quantification and consistent antibody drug quality.

Instrumentation

• Shimadzu Nexera XS inert UHPLC system
• Shim-pack Bio Diol-300 column (150 mm×4.6 mm, 2 µm)
• SPD-M40 inert UV flow cell (280 nm)
• LabSolutions MD method scouting software
• TORAST-H glass vials
• LC-40D XSi and SIL-40C XSi components for high corrosion resistance

References

1. ICH-Q6B. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. 2001.
2. Niki E, Watanabe N. Introduction to High Performance Liquid Chromatography. Journal of Japan Oil Chemists’ Society. 1980;29(2):127–134.
3. Shimadzu Corporation. Shim-pack Bio Diol technical details. 2022.