Size Exclusion Chromatography Method Development of NIST mAb Using an Agilent AdvanceBio SEC 200 Å 1.9 μm Column

Importance of the Topic

Size exclusion chromatography is a cornerstone technique for characterizing size variants and aggregate species in biotherapeutic proteins such as monoclonal antibodies.
Accurate separation and quantitation of monomer, dimer, and higher molecular weight species are essential for ensuring product safety and efficacy.
Efficient method development requires systematic evaluation of buffer composition, salt concentration, and pH.

Study Objectives and Overview

This study aimed to develop a robust SEC method for the NIST monoclonal antibody (RM 8671) using an Agilent AdvanceBio SEC 200 Å 1.9 µm column.
By leveraging the Agilent 1260 Infinity II bio-inert LC system with quaternary pump and Buffer Advisor software, a wide range of mobile phase conditions was screened rapidly.
Key parameters included phosphate buffer concentration, NaCl addition, and pH to balance peak symmetry, resolution, and quantitation accuracy of high molecular weight species.

Methodology and Instrumentation

Experiments were performed on an Agilent 1260 Infinity II bio-inert LC system with the following components:

• Bio-inert quaternary pump for online blending of buffer and salt stock solutions
• Variable wavelength UV detector at 280 nm
• Multicolumn thermostat maintained at 25 °C
• AdvanceBio SEC 200 Å 1.9 µm, 4.6 × 300 mm column

Mobile phases were prepared using Agilent Buffer Advisor software from stock solutions of 1 M NaCl, 245 mM NaH2PO4, and 420 mM Na2HPO4.
Screening covered phosphate buffer concentrations from 25 to 350 mM, NaCl from 0 to 250 mM, and pH range 6.6 to 7.4.

Key Results and Discussion

• At 150 mM phosphate without NaCl, pH 7.4 delivered the best peak shape (As ≈ 1.41), resolution (Dimer/Monomer ≈ 2.78) and accurate HMW quantitation (~ 3 %).
• Inclusion of 250 mM NaCl enabled lower phosphate concentrations while maintaining symmetry and resolution; 50 mM phosphate with 250 mM NaCl at pH 6.8 achieved As 1.33 and Rs 2.86.
• Increasing phosphate above 300 mM improved peak symmetry but degraded dimer/monomer resolution.
• The optimal balance of peak shape, resolution, and HMW accuracy was found with 50 mM phosphate, 250 mM NaCl at pH 6.8.

Benefits and Practical Applications

The optimized SEC method enables reliable quantitation of monomer and aggregate levels in antibody preparations.
Combining bio-inert LC hardware with Buffer Advisor software streamlines mobile phase development, reducing method setup time.

Future Trends and Applications

Integration of automated buffer calculation tools with next-generation LC systems will further accelerate biotherapeutic characterization workflows.
Applying this approach to other protein classes and column chemistries can enhance understanding of aggregation mechanisms.
High-throughput method screening and machine learning guidance may offer predictive optimization for SEC assays.

Conclusion

A systematic mobile phase optimization strategy using Agilent’s bio-inert LC platform and Buffer Advisor software yielded an SEC method for NIST mAb with excellent peak symmetry, resolution, and aggregate quantitation accuracy.
The final conditions of 50 mM phosphate, 250 mM NaCl at pH 6.8 provide a robust assay for monitoring size variants in monoclonal antibodies.

Reference

1. Schiel J. E. et al. The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability. Anal. Bioanal. Chem. 2018, 410, 2127–2139.