Molecular Characterization of Biotherapeutics

Significance of the Topic

Monoclonal antibodies represent a rapidly growing class of biotherapeutics with complex structures that require detailed molecular characterization to ensure safety, efficacy, and consistency. Size exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS) and dynamic light scattering (DLS) offers a robust platform to assess aggregation, molecular weight, and hydrodynamic size, critical quality attributes for both innovator biologics and biosimilars.

Objectives and Study Overview

This study compares the molecular properties of an innovator rituximab and its biosimilar counterpart using the Agilent 1260 Infinity Multi-Detector Bio-SEC Solution. Key goals included measuring molecular weight and hydrodynamic radius of monomeric and aggregated species, and evaluating aggregate formation under thermal stress (1 hour at 60 °C).

Methodology and Instrumentation

Size exclusion chromatography was performed on an Agilent Bio-SEC-3 column (300 Å, 7.8 × 300 mm, 3 µm) with PBS (pH 7.4) as mobile phase at 0.75 mL/min. Temperature control: autosampler at 5 °C and column at 30 °C. Detection employed UV (280 nm), static light scattering at 90°, and dynamic light scattering for hydrodynamic radius. Sample preparation included 0.2 µm filtration and injection volume of 5 µL.

Key Results and Discussion

Fresh innovator and biosimilar rituximab showed comparable monomer molecular weights (~156 kDa), hydrodynamic radii (~5 nm), retention times (~10.6 min), and excellent run-to-run precision. Thermal stress induced a distinct aggregate peak (~7.3 min) in both samples, representing ~9.8% of total species. Light scattering detection demonstrated markedly higher sensitivity for aggregates compared to UV alone, facilitating precise quantification of low-level aggregates and accurate determination of aggregate molecular weight (4.1–4.5 MDa) and size (~16–17 nm).

Benefits and Practical Applications

• Enhanced aggregate detection sensitivity using MALS over UV detection.
• Accurate determination of molecular weight and size for both monomer and aggregates.
• High precision and reproducibility support regulatory compliance for biosimilar comparability studies.

Future Trends and Opportunities

Integration of additional detectors (e.g., refractive index), expanded dynamic range for high-mass aggregates, and miniaturized SEC formats may further improve throughput and sensitivity. Emerging applications include real-time process monitoring and high-throughput screening of novel antibody formats.

Conclusion

The Agilent 1260 Infinity Multi-Detector Bio-SEC Solution successfully characterized rituximab innovator and biosimilar, revealing equivalent molecular properties and aggregate profiles under stress conditions. Light scattering detection provided superior sensitivity for aggregate analysis, underscoring its value in biopharmaceutical development and quality control.

Instrumentation Used

• Agilent 1260 Infinity Bio-inert Quaternary Pump
• Agilent 1260 Infinity Bio-inert Autosampler
• Agilent 1290 Infinity Thermostat (sample cooling)
• Agilent 1290 Infinity Thermostatted Column Compartment
• Agilent 1260 Infinity Diode Array Detector (280 nm)
• Agilent 1260 Infinity Multi-Detector Suite with dual-angle MALS and DLS

References

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5. Schneider S. Detailed aggregation characterization of monoclonal antibodies using the Agilent 1260 Infinity Multi-Detector Bio-SEC Solution with advanced light scattering detection. Agilent Technologies Application Note 5991-3954EN;2014.
6. Schneider S. Determination of protein molecular weight and size using the Agilent 1260 Infinity Multi-Detector Bio-SEC Solution with advanced light scattering detection. Agilent Technologies Application Note 5991-3955EN;2014.