METHOD MODERNIZATION OF COMPENDIAL METHODS USING USP <621> WITH A NEXT-GENERATION HPLC SYSTEM

Significance of the Topic

As analytical requirements for biotherapeutics become more stringent, legacy compendial HPLC methods guided by USP general chapters face limitations in throughput and resolution. Modern high-performance systems allow faster analyses and lower resource consumption while meeting regulatory criteria.

Objectives and Study Overview

This study evaluates the application of USP <129> SEC and USP <121.1> insulin peptide mapping methods using a next-generation Alliance iS Bio HPLC System versus a traditional HPLC platform. The aim is to reduce run times and solvent/sample usage without compromising resolution and system suitability.

Methodology and Instrumentation

• Legacy System: BioSuite Diol SEC column (250 Å, 5 µm, 7.8 × 300 mm) at 0.5 mL/min, 30 min run; XSelect Peptide CSH C18 column (130 Å, 5 µm, 4.6 × 100 mm) for peptide mapping at 1 mL/min, 50 min run.
• Modernized System: Alliance iS Bio HPLC with MaxPeak HPS surfaces; XBridge Premier Protein SEC columns (250 Å, 2.5 µm, 7.8 × 150 mm and 4.6 × 150 mm) at 0.35 mL/min, 7.5 min run; XSelect Premier Peptide CSH C18 column (130 Å, 2.5 µm, 4.6 × 50 mm) at 2 mL/min, 7.5 min run.
• Mobile Phases and Conditions: SEC with 0.20 M potassium phosphate/0.25 M KCl, pH 6.2 at 30 °C; peptide mapping with H2O/ACN/(NH4)2SO4 buffers at 40 °C detection at 214 nm.

Key Results and Discussion

• Size Exclusion Chromatography: A 7.8 mm ID, 2.5 µm column delivered four-fold faster separations and maintained resolution for low molecular weight species, while a 4.6 mm ID column achieved a six-fold reduction in solvent and sample usage with acceptable performance.
• Monoclonal Antibody Analysis: Scaling to 2.5 µm particles preserved critical resolution (RS ≥ 3.4) and tailing factor (≤ 1.5) according to USP <129> on the Alliance iS Bio HPLC System.
• Insulin Peptide Mapping: Glu-C digestion yields four diagnostic fragments; modernization reduced analysis time from 50 to 7.5 minutes and maintained reproducible peak area and retention times across both systems.

Benefits and Practical Applications

• Significant gains in throughput support high-volume QC workflows.
• Reduced solvent and sample consumption lowers operating costs and environmental impact.
• Retention of compendial system suitability and separation criteria ensures regulatory compliance.
• Applicability spans biotherapeutic size variant analysis and peptide characterization.

Future Trends and Potential Applications

Adoption of ultrahigh-pressure systems and sub-2 µm particles will further shorten methods. Integration with mass spectrometry and automated data-driven optimization can enhance sensitivity and robustness. Emphasis on sustainability will drive greener mobile phases and microflow formats.

Conclusion

Modernizing compendial SEC and peptide mapping methods using the Alliance iS Bio HPLC System and MaxPeak HPS surfaces yields faster assays with lower resource use while meeting USP <621> performance requirements. This approach aligns with current and future needs in biopharmaceutical quality control.

Reference

• USP. Chromatography <621>. In: USP-NF. Rockville, MD: USP; Dec 1, 2022.
• USP. Physicochemical Analytical Procedures for Insulins <121.1>. In: USP-NF. Rockville, MD: USP; Dec 1, 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.
• Han D, Birdsall RE, Nyholm K. Leveraging the Alliance iS Bio HPLC System as a Modern HPLC for Peptide Drug Substances Analysis in QC Environments. Waters Application Note. May 2024. 720008345EN.