Quantitation of mAb and ADC Aggregation Using SEC and an Aqueous Mobile Phase

Importance of the Topic

Aggregation and degradation of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) represent critical quality attributes in biotherapeutic development. Unchecked aggregation can reduce efficacy, increase immunogenicity, and compromise patient safety. Reliable quantitation of monomers, aggregates, and fragments is essential for both research and quality control in the biopharmaceutical industry.

Objectives and Overview of the Study

This application note describes the development of a sensitive and robust size exclusion chromatography (SEC) method to quantify aggregates and degradation products in a model mAb (trastuzumab) and an ADC (T-DM1). The study evaluates an Agilent AdvanceBio SEC 300 Å, 2.7 µm column coupled to an Agilent 1260 Infinity Bio-inert Quaternary LC system using a single aqueous mobile phase.

Methodology and Instrumentation

The separation was performed on an AdvanceBio SEC 300 Å, 7.8 × 300 mm, 2.7 µm column with phosphate-buffered saline (50 mM phosphate, 150 mM NaCl, pH 7.4) at 0.8 mL/min and ambient column temperature. UV detection was conducted at 220 nm and 280 nm. The Agilent 1260 Infinity Bio-inert Quaternary LC comprised a quaternary pump, bio-inert autosampler, thermostatted column compartment, and diode-array detector. Linearity, precision, limit of detection (LOD), and limit of quantitation (LOQ) were assessed across eight concentration levels (15.6–2000 µg/mL). Stress conditions (pH shifts and 60 °C incubation) generated aggregates and fragments for method validation.

Main Results and Discussion

Excellent baseline resolution of monomer, dimer, and higher aggregates was achieved within 15 minutes for both trastuzumab and T-DM1 using only aqueous mobile phase. Retention time RSDs were below 0.04 %, and area RSDs were under 1 %, demonstrating high reproducibility. Calibration curves were linear (R² = 1) from 15.6 to 2000 µg/mL. LOD and LOQ values were 15 µg/mL and 31 µg/mL, respectively. Stress-induced samples showed clear separation of aggregates eluting earlier and fragments eluting later than the monomer peak. Aggregate levels increased to 19–28 % under forced-stress conditions.

Benefits and Practical Applications of the Method

• Single aqueous mobile phase for hydrophilic mAbs and hydrophobic ADCs without organic modifiers
• High-resolution, reproducible separation suitable for QA/QC laboratories
• Rapid analysis time (<15 min) for routine batch testing
• Bio-inert LC hardware resists corrosion and reduces nonspecific interactions

Future Trends and Applications

Advances in SEC column chemistries and bio-inert systems will support even higher throughput and sensitivity for next-generation biologics. Integration with mass spectrometry and multi-angle light scattering will enable simultaneous molecular weight determination and more detailed aggregate characterization. Automated data processing and in-line monitoring could facilitate real-time quality control in continuous biomanufacturing.

Conclusion

The described SEC method using Agilent AdvanceBio SEC 300 Å on a bio-inert LC platform offers a simple, sensitive, and reproducible approach to quantify mAb and ADC aggregation and degradation. Its robustness and use of a single aqueous mobile phase make it ideal for routine biopharma QA/QC analysis.

References

1. Ratanji KD, Derrick JP, James S, et al. J Immunotoxicol. 2014;11(2):99–109.
2. Wakankar A, Chen Y, Gokarn Y, Jacobson F. MAbs. 2011;3(2):161–172.
3. Başak Kükrer B, Filipe V, van Duijn E, et al. Pharm Res. 2010;27:2197–2204.
4. Rodriguez-Diaz R, Wehr T. In: Analytical Techniques for Biopharmaceutical Development. CRC Press; 2005.