Efficient Method Development of Monoclonal Antibody Size Variants by Size Exclusion Chromatography

Importance of the Topic

Monoclonal antibodies (mAbs) are key biotherapeutics prone to aggregation and fragmentation, which can compromise safety and efficacy. Regulatory guidelines demand accurate quantification of these variants. Size-exclusion chromatography (SEC) remains the gold standard for monitoring mAb size variants, but requires careful optimization to achieve both high resolution and robust performance under varying conditions.

Objectives and Study Overview

This study demonstrates an analytical quality by design (AQbD) approach for rapid method development of mAb size-variant separation. Using LabSolutions MD software, the workflow focused on:

• Optimizing mobile phase salt concentration
• Screening pH, flow rate, and column temperature
• Building design spaces to predict optimal conditions independent of operator expertise

Materials and Methods

An isocratic SEC method was employed:

• Column: TSKgel UP-SW3000, 150 × 4.6 mm, 2 µm
• Mobile phases: phosphate buffer with varied NaCl (50–300 mmol/L)
• Analytical parameters screened: buffer pH (6.1–7.3), flow rate (0.15–0.25 mL/min), oven temperature (20–30 °C)
• Detection: UV at 280 nm

Used Instrumentation

• Nexera XS inert UHPLC system with high salt corrosion resistance
• pH monitor pHM-40 for real-time mobile phase pH tracking
• LabSolutions MD software for automated method scouting and design-space generation
• SPD-M40 inert flow cell detector

Main Results and Discussion

• Salt concentration optimization revealed 150 mmol/L NaCl as the best compromise between resolution of aggregate/monomer and monomer/fragment peak-to-valley ratio.
• Design-space maps identified optimal conditions at pH 7.3, 0.15 mL/min flow rate, and 30 °C, improving electrostatic interactions near the mAb isoelectric point.
• Lower flow rates and higher temperatures enhanced mass transfer, yielding sharper peaks and higher resolution.
• Continuous pH monitoring confirmed mobile phase stability throughout analyses.

Benefits and Practical Applications

The AQbD approach delivered a robust SEC method with minimal trial-and-error. Nexera XS inert ensures reliable operation with high-salt buffers, while LabSolutions MD simplifies optimization, reduces development time, and supports regulatory compliance in biopharmaceutical QC laboratories.

Future Trends and Opportunities

• Integration of machine-learning algorithms for predictive method optimization.
• Expansion to other biotherapeutics, including fusion proteins and ADCs.
• Real-time PAT sensors for in-line quality monitoring.
• High-throughput screening platforms combining multi-dimensional design spaces.

Conclusion

An AQbD-driven workflow using LabSolutions MD, pH monitoring, and a corrosion-resistant UHPLC system enabled efficient and reproducible SEC method development for mAb size variants. The resulting method offers high resolution, robust performance, and compliance with regulatory expectations.

Reference

1. Efficient Method Development by automated pH Screening with LabSolutions MD (C190-0563)