Size Exclusion Chromatography Analysis of a Monoclonal Antibody and Antibody Drug Conjugate

Importance of the Topic

Size exclusion chromatography (SEC) is a critical tool for characterizing size variants such as aggregates and fragments in biotherapeutic proteins. Accurate separation of high and low molecular weight species is essential to ensure product efficacy and safety and to monitor critical quality attributes during development, manufacturing, and storage.

Objectives and Overview

This study evaluates the performance of the Agilent AdvanceBio SEC 200 Å 1.9 µm column against two vendor sub-2 µm SEC columns for the analysis of a monoclonal antibody (mAb) and an antibody drug conjugate (ADC) mimic. The goal is to compare peak shape, resolution, and secondary interactions under identical chromatographic conditions.

Methodology

Reagents included a universal antibody standard and an ADC mimic at 1 mg/mL, mobile phase consisting of 50 mM sodium phosphate and 200 mM NaCl at pH 7.0. Chromatographic parameters were uniform: 4.6 × 300 mm columns, 0.35 mL/min flow rate, 25 °C, and UV detection at 220 nm.

Used Instrumentation

• Agilent 1260 Infinity II bio-inert quaternary pump
• Agilent 1260 Infinity II bio-inert multisampler with cooler
• Agilent 1260 Infinity II multicolumn thermostat with heat exchanger
• Agilent 1260 Infinity II variable wavelength detector

Results and Discussion

The AdvanceBio SEC column delivered the narrowest peak widths, superior dimer/monomer and monomer/fragment resolution for the mAb, and reduced backpressure compared to Vendor A and B columns. For the ADC mimic, it minimized hydrophobic secondary interactions, yielding a sharp, symmetric peak and baseline separation of low molecular weight fragments (Rs = 2.55), whereas Vendor A showed peak splitting and Vendor B only partial resolution.

Benefits and Practical Applications

• Enhanced resolution of HMW and LMW variants
• Sharp peak shapes with minimal nonspecific binding
• Reliable analysis of hydrophobic ADC payloads
• Compatibility with high-throughput workflows due to manageable backpressure

Future Trends and Possibilities

Advancements may include integration with mass spectrometry, further miniaturization and UHPLC adaptation, development of new stationary phase chemistries tailored to hydrophobic bioconjugates, and enhanced automation for routine QC applications.

Conclusion

The Agilent AdvanceBio SEC 200 Å 1.9 µm column outperforms comparable sub-2 µm columns for both mAb and ADC separations, offering superior resolution, peak shape, and reduced secondary interactions critical for biotherapeutic analysis.

Reference

1. Rosenberg A. S. Effects of Protein Aggregates: an Immunologic Perspective. AAPS J. 2006;8(3):E501-507.
2. De Groot A. S.; Scott D. W. Immunogenicity of Protein Therapeutics. Trends Immunol. 2007;28(11):482-490.