Enhanced SEC-MALS analysis of therapeutic mAbs, biosimilars and AAVs with XBridge Premier columns

Significance of the Topic

Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) is a cornerstone technology for characterizing biopharmaceutical products such as monoclonal antibodies, biosimilars and gene therapy vectors. It provides absolute molar mass determination, aggregate quantification and payload analysis in a robust, reagent-free assay suitable for QA/QC environments.

Objectives and Study Overview

This study evaluates the performance of Waters XBridge Premier Protein SEC and XBridge Premier GTx BEH SEC columns coupled to Wyatt DAWN MALS, Optilab dRI and UV detectors. The aim is to demonstrate enhanced separation, multi‐attribute quantitation of therapeutic antibodies, biosimilars and adeno‐associated viruses (AAVs) with minimal method development.

Methodology and Instrumentation

SEC separations were carried out on a Waters Arc Premier HPLC with 2998 PDA UV detector, Wyatt DAWN MALS with embedded QELS dynamic light scattering and Optilab dRI. Protein separations used XBridge Premier Protein SEC (250 Å, 7.8×300 mm) or generic silica‐bead columns with PBS mobile phase at 0.5 mL/min. AAV analyses employed XBridge Premier GTx BEH SEC (450 Å, 7.8×300 mm) or silica columns with SOP‐guided buffers at 0.5 mL/min. Data were processed in ASTRA software.

Main Results and Discussion

• Protein separations on XBridge Premier columns eliminated secondary interactions, resolving pertactin as a single monomer peak versus split peaks on generic silica. DLS confirmed elution order by hydrodynamic radius rather than molar mass.
• SEC-MALS delivered absolute molar mass for trastuzumab (Herceptin) and its biosimilar Kanjinti, revealing identical monomer/dimer profiles (~160 kDa and ~300 kDa) and subtle differences in low‐ and high‐molecular‐weight species between products.
• Gene therapy analyses quantified AAV capsid (~3.9 MDa), DNA (~1.27 MDa), particle concentration and full‐to‐total ratio in a single SEC‐UV‐MALS‐dRI run. The GTx column improved sensitivity below 5×10^10 particles/mL and fully resolved AAV aggregates.

Benefits and Practical Applications

• Platform columns reduce LC method development time and resources
• MALS provides direct molar mass measurements independent of elution artifacts
• Simultaneous multi‐attribute quantitation accelerates biopharmaceutical QA/QC
• Enhanced sensitivity conserves precious samples and improves data robustness

Future Trends and Potential Applications

• Integration of field‐flow fractionation (FFF‐MALS) for large aggregate analysis
• Extension to other viral vectors and gene therapy platforms
• Automation and high‐throughput SEC-MALS workflows in regulated environments
• Advanced light scattering detectors for improved spectral and size resolution

Conclusion

The combination of XBridge Premier SEC columns with SEC‐MALS offers a powerful, efficient and accurate approach to characterize therapeutic antibodies, biosimilars and AAVs. This platform enables high‐resolution separations, absolute molar mass determination and comprehensive multi‐attribute quantitation, supporting fast method deployment and robust QA/QC in biopharmaceutical development.

References

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