Separate and Quantify Rituximab Aggregates and Fragments with High-Resolution SEC

Importance of the Topic

Monoclonal antibodies (mAbs) have become cornerstone therapeutics in oncology and autoimmune diseases. Aggregation and fragmentation of these proteins can compromise safety and efficacy by provoking immunogenic responses or altering pharmacokinetics. High-resolution size exclusion chromatography (SEC) is essential for robust quality control and comparability studies during development and manufacturing of mAb products.

Study Objectives and Overview

This application note demonstrates a reliable SEC method for separating and quantifying intact rituximab, its aggregates, and fragments in both innovator and biosimilar samples. The work focuses on forced-stress degradation analysis, evaluating column performance, method precision, and the ability to distinguish closely related therapeutic proteins and antibody drug conjugates (ADCs).

Methodology

Column performance was calibrated using a mixture of protein standards spanning 1.35–670 kDa. Forced-stress degradation involved repeated pH cycling between pH 1.0 and 10.0 and incubation at 60 °C for 1 hour to generate aggregates and fragments. Samples of intact and stressed mAbs were analyzed in six replicates. Mobile phase conditions employed phosphate-buffered saline (50 mM sodium phosphate, 150 mM NaCl, pH 7.4), a 0.8 mL/min flow rate, ambient column compartment temperature, and UV detection at 220 and 280 nm.

Instrumentation

• Agilent 1260 Infinity Bio-inert Quaternary LC Pump and Autosampler modules
• Thermostatted Column Compartment with bioinert heating
• AdvanceBio SEC 300Å, 7.8×300 mm, 2.7 μm column
• Diode array detector (DAD) with bioinert flow cell
• Agilent ChemStation software for data acquisition and processing

Main Results and Discussion

Calibration yielded a linear log(molecular weight) vs elution volume plot (R2=0.997), defining the exclusion limit at 670 kDa. The method resolved monomeric rituximab at ~8.3 minutes with sharp, symmetrical peaks and no nonspecific interactions. Retention time and area reproducibility were excellent (RSD <0.04% and <1%, respectively) for both innovator and biosimilar samples. Overlays of intact samples confirmed >99% purity and high similarity between products. Stress studies revealed an increase in aggregates for the innovator (up to ~23% area) compared to biosimilar (~24%), while both exhibited predominant fragment peaks eluting at ~12.8 minutes. The column also distinguished other therapeutic mAbs (cetuximab, trastuzumab) and an ADC based on molecular mass differences.

Benefits and Practical Applications

The developed SEC method provides rapid and sensitive monitoring of mAb variants, enabling accurate quantification of aggregates and fragments in quality control workflows. Its high resolution supports biosimilar comparability assessments and formulation stability studies. The bioinert system minimizes sample adsorption, preserving protein integrity.

Future Trends and Opportunities

Advances in SEC media and particle technology will continue to improve resolution and speed for large biomolecules. Integration with mass spectrometry and multiangle light scattering can provide complementary molecular weight confirmation. Automated high-throughput SEC platforms and inline fraction collection will further accelerate biopharmaceutical characterization.

Conclusion

The Agilent AdvanceBio SEC 300Å column coupled with the 1260 Infinity Bio-inert Quaternary LC system delivers high-resolution, sensitive separation of intact mAbs, aggregates, and fragments. The method shows outstanding precision and applicability for biosimilar comparability and forced-stress degradation studies, making it a valuable tool for biopharmaceutical development and quality control.

References

1. Bıazak Kükrer et al. Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography. Pharm. Res. 2010, 27, 2197–2204.
2. Rodriquez-Diaz R.; Wehr T. Use of Size Exclusion Chromatography in Biopharmaceutical Development. In Analytical Techniques for Biopharmaceutical Development; CRC Press: New York, 2005.