Mobile Phase Optimization in SEC Method Development

Importance of the Topic

Size exclusion chromatography (SEC) is a vital tool for evaluating aggregation in therapeutic proteins. Aggregates of monoclonal antibodies (mAbs) can compromise drug safety and efficacy, making precise detection essential in biopharmaceutical quality control and research.

Study Objectives and Overview

This application note aimed to optimize mobile phase conditions for Agilent AdvanceBio SEC columns. The focus was on buffer concentration, salt effects, and column configurations to enhance separation of monomers, dimers, and higher-order aggregates in protein samples including human IgG, ADC mimics, insulin, cytochrome c, and protein standards.

Methodology and Instrumentation

• Columns: Agilent AdvanceBio SEC 300 Å and 130 Å (7.8 × 300 mm, 2.7 μm; 300 Å and 130 Å pore sizes)
• LC System: Agilent 1260 Infinity bio-inert quaternary pump, autosampler, thermostatted column compartment, DAD detector
• Samples: IgG (human and bovine), ADC mimic, insulin, cytochrome c, myoglobin, gel filtration and pullulan standards
• Mobile Phases: Sodium phosphate buffers (50–600 mM, pH 7.0) with and without NaCl (50–500 mM); flow rate 1 mL/min; column at 25 °C; detector at 220 nm

Main Results and Discussion

Optimization of phosphate concentration showed that 150 mM phosphate buffer provided the lowest tailing and highest monomer–dimer resolution for IgG, with diminished secondary interactions and minimal induced aggregation. ADC and insulin also displayed improved peak shapes at 150–50 mM phosphate. Cytochrome c favored higher phosphate concentrations (400–600 mM) for optimal recovery and shape.

Salt addition experiments revealed that NaCl promoted aggregation in IgG, had minimal impact on ADC, and was unnecessary for insulin, while cytochrome c required 150–500 mM NaCl to reduce tailing factors. These findings suggest minimal salt addition unless specific proteins demand higher ionic strength.

Extending column length from 150 mm to 300 mm improved resolution at the cost of analysis time and pressure, and connecting two 300 mm columns in tandem yielded further resolution gains. Combining 130 Å and 300 Å pore sizes in series enhanced separation of medium-molecular-weight proteins and polysaccharide standards, though very large proteins were excluded from the smaller-pore column.

Benefits and Practical Applications

• Robust detection of monomers, dimers, and aggregates in therapeutic proteins
• Reliable method for QC/QA labs in biopharmaceutical industry
• Extended column lifetime through optimized buffer and minimal salt conditions
• Adjustable column configurations for tailored resolution and throughput

Future Trends and Opportunities

Anticipated developments include coupling SEC with multi-angle light scattering and mass spectrometry for absolute molecular-weight determination, miniaturized UHPLC-SEC formats for faster analysis, and AI-driven mobile phase optimization to further streamline method development.

Conclusion

Mobile phase composition critically influences SEC performance. A phosphate buffer of 150 mM at pH 7.0 and minimal salt addition generally yields optimal separation on AdvanceBio SEC columns. Column length and pore size combinations can be tailored to balance resolution, runtime, and backpressure requirements, supporting rigorous characterization of therapeutic proteins.

Reference

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