Future-proofing the Biopharmaceutical QC Laboratory: Using the ACQUITY UPLC H-Class Bio System to Run SEC-HPLC and SEC-UPLC

Significance of the Topic

Size exclusion chromatography (SEC) is a pivotal analytical technique in biopharmaceutical quality control to detect and quantify protein aggregation. Aggregates can compromise safety and efficacy of therapeutic proteins, making accurate, high-resolution assessment essential for ensuring product integrity.

Aims and Overview of the Study

This study demonstrates the transfer of a conventional SEC-HPLC method for monoclonal antibody aggregation analysis onto the ACQUITY UPLC H-Class Bio System. It compares performance metrics between legacy HPLC and UPLC modalities and adapts the method for UPLC using a sub-2 µm BEH SEC column to enhance resolution and throughput.

Applied Methodology and Instrumentation

SEC separations were performed under isocratic conditions using 20 mM phosphate buffer, 200 mM NaCl, pH 6.8. For HPLC, a Biosuite SEC 250 Å, 10 µm, 7.5 × 300 mm column at 0.4 mL/min over 35 min was deployed. The UPLC adaptation employed a BEH Protein SEC 200 Å, 1.7 µm, 4.6 × 150 mm column at 0.885 mL/min over 3 min. Both assays utilized the ACQUITY UPLC H-Class Bio System.
Used instrumentation:

• ACQUITY UPLC H-Class Bio System with Quaternary Solvent Manager and Sample Manager
• ACQUITY UPLC Tunable UV Detector (214 nm)
• 100 µL extension loop

Key Results and Discussion

• Method transfer from HPLC to the UPLC system yielded equivalent retention times and peak area reproducibility for protein standards and infliximab monomer/dimer species.
• Transition to a sub-2 µm BEH SEC column reduced run time from 35 to 3 minutes while maintaining quantitative accuracy.
• UPLC increased chromatographic efficiency, revealing minor aggregate species undetected by HPLC and improving resolution by two- to three-fold across peak pairs.

Benefits and Practical Applications

• Streamlined QC workflows through a unified HPLC/UPLC platform
• Enhanced sensitivity to low-abundance aggregates
• High-throughput capability reducing analysis time by over 90%
• Seamless method migration without major parameter adjustments

Future Trends and Applications

Advancements in SEC-UPLC are expected to include integration with multi-attribute mass spectrometry for comprehensive characterization, microflow platforms to further reduce solvent consumption, and automated data analysis enabled by machine learning to streamline decision-making in QC laboratories.

Conclusion

The ACQUITY UPLC H-Class Bio System effectively supports both legacy SEC-HPLC methods and modern SEC-UPLC workflows. By enabling rapid, high-resolution separations without compromising quantitative integrity, it future-proofs biopharmaceutical QC laboratories and aligns routine testing with next-generation analytical capabilities.

Reference

Cosgrave EFJ, McCarthy SM. Future-proofing the Biopharmaceutical QC Laboratory: Using the ACQUITY UPLC H-Class Bio System to Run SEC-HPLC and SEC-UPLC. Waters Corporation; 2014.