Size Exclusion Chromatography Analysis of Antibody Drug Conjugates

Significance of ADC Aggregation Analysis

Aggregation of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) poses critical challenges for therapeutic efficacy, safety, and regulatory compliance. Aggregates can reduce binding activity, increase immunogenicity, and complicate downstream processing. Reliable size exclusion chromatography (SEC) methods are therefore essential for monitoring product quality throughout development, manufacturing, and storage.

Objectives and Study Overview

This study evaluated the performance of the Agilent AdvanceBio SEC 300 Å column in comparison with the older Agilent Bio SEC-3 column, using the Agilent 1260 Infinity II Bio-inert LC system. The aim was to determine which combination provides sharper, more symmetric peaks and baseline separation of aggregates for both mAbs and hydrophobic ADCs.

Methodology and Instrumentation

The Agilent 1260 Infinity II Bio-inert LC system featured a bio-inert pump, multisampler with cooler, multicolumn thermostat with inert flowpath, and a diode array detector. Two SEC columns (7.8 × 300 mm) were tested: AdvanceBio SEC (2.7 µm) and Bio SEC-3 (3 µm). Mobile phase was phosphate-buffered saline (pH 7.4), flow rate 0.8 mL/min, column temperature 30 °C, injection 10 µL, detection at 280 nm.

Main Results and Discussion

Analysis of trastuzumab (Herceptin) monomer showed similar sharp peaks on both columns, indicating minimal nonspecific interactions for simple mAbs. However, trastuzumab emtansine (T-DM1) and brentuximab vedotin exhibited severe peak tailing and poor resolution on the Bio SEC-3 column due to hydrophobic interactions. In contrast, the AdvanceBio SEC column delivered narrow, symmetrical peaks with baseline separation of monomer and aggregate for both ADCs. This difference stems from the hydrophilic polymer coating and bio-inert flowpath that prevent nonspecific adsorption.

Benefits and Practical Applications

• Accurate quantitation of aggregate levels in mAbs and ADCs
• Improved peak symmetry and resolution reduce method development time
• Bio-inert flowpath supports high-salt and low-pH buffers without corrosion
• Robust platform for QA/QC in biopharmaceutical production

Future Trends and Potential Applications

Emerging directions include integration of SEC with multi-angle light scattering (MALS) and mass spectrometry for direct molecular weight determination, development of higher throughput bio-inert LC systems, and application of advanced column chemistries to accommodate increasingly complex biotherapeutics such as bispecific antibodies and Fc-fusion proteins.

Conclusion

The combination of the Agilent 1260 Infinity II Bio-inert LC system and the AdvanceBio SEC column offers superior performance for aggregate analysis of both mAbs and ADCs. Its inert flowpath and optimized column coating ensure pure size-based separations, enabling accurate quantitation and reliable QC of complex biotherapeutics.

References

• Wakankar A.; et al. Analytical methods for physiochemical characterization of antibody drug conjugates. mAbs 2011, 3(2), 161–172.
• Agilent Technologies. Advantages of Agilent AdvanceBio SEC Columns for Biopharmaceutical Analysis. Technical Overview, publication number 5991-6474EN, 2015.
• Schneider S.; Jegle U.; Dickhut C. Size Exclusion Chromatography Analysis of Antibody Drug Conjugates Using the Agilent 1260 Infinity II Bio-inert LC System. Application Note, Agilent Technologies, publication number 5991-8244EN, 2017.