MODERNIZING BIOTHERAPEUTIC COMPENDIAL METHODS WITH A NEXT-GENERATION HPLC SYSTEM

Significance of the Topic

Modernizing compendial analytical methods for biotherapeutics addresses the limitations of legacy HPLC protocols by offering enhanced resolving power, faster throughput and alignment with current regulatory flexibility. This evolution supports more efficient quality control and biopharmaceutical development.

Objectives and Study Overview

The study set out to update USP general chapter methods for monoclonal antibody size exclusion chromatography and insulin peptide mapping. By comparing traditional workflows on a legacy HPLC system with next-generation instrumentation and sub-3 µm column technologies, the work evaluated gains in speed, resolution and resource consumption.

Methodology and Instrumentation

• Instrument: Alliance iS Bio HPLC System equipped with MaxPeak High Performance Surface technology to reduce biomolecule adsorption.
• SEC Columns: Legacy BioSuite Diol 250 Å, 5 µm, 7.8 × 300 mm versus XBridge Premier Protein SEC 250 Å, 2.5 µm columns at 4.6 × 150 mm and 7.8 × 150 mm.
• RPLC Columns: Legacy XSelect Peptide CSH C18 130 Å, 5 µm, 4.6 × 100 mm versus XSelect Premier Peptide CSH C18 130 Å, 2.5 µm, 4.6 × 50 mm.
• Mobile phases, flow rates and run conditions followed USP chapters with scaled adjustments to reduce solvent and sample volumes.

Main Results and Discussion

• SEC method runtime was reduced fourfold with the 7.8 mm ID column while the 4.6 mm ID column achieved a sixfold decrease in solvent and sample use, maintaining separation of low molecular weight species.
• Insulin peptide map analysis time decreased from 50 min to 12.5 min, meeting USP resolution (>3.4) and tailing (<1.5) criteria.
• Improved peak height to valley ratios (from 1.13 to 1.74) enhanced quantification of monoclonal antibody impurities.

Benefits and Practical Applications

The updated methods deliver significant reductions in analysis time, solvent consumption and sample requirements, boosting laboratory efficiency and lowering operating costs. The bio-inert design protects sensitive biomolecules and supports higher system pressures for modern columns, benefiting QC, process development and regulatory compliance in biopharmaceutical settings.

Future Trends and Potential Applications

• Adoption of sub-2 µm and core-shell particles to push speed and resolution further.
• Integration of automated sample preparation and on-line method validation for accelerated workflows.
• Coupling high-throughput separations with mass spectrometry for comprehensive biomolecule characterization.
• Leveraging data analytics and machine learning to optimize methods and monitor instrument performance in real time.

Conclusion

The combination of advanced instrumentation and optimized column technology successfully modernized compendial SEC and insulin peptide mapping methods, delivering faster, more sensitive and resource-efficient assays while maintaining compliance. This approach paves the way for future-proof analytical workflows in biotherapeutic development and quality control.

References

• USP. Chromatography General Chapter 621. USP-NF December 2022.
• USP. Physicochemical Analytical Procedures for Insulins General Chapter 121.1. USP-NF December 2016.
• Bigos P, Birdsall RE, Nyholm K. Modernizing Compendial SEC Methods for Biotherapeutics Using the Alliance iS Bio HPLC System. Waters Application Note April 2024. 720008290EN.