Leveraging the Alliance™ iS Bio HPLC System as a Modern HPLC for Peptide Drug Substances Analysis in QC Environments

Significance of the Topic

Peptide drug substances present analytical challenges in QC laboratories due to their bioreactivity and sensitivity to surface interactions. The Alliance iS Bio HPLC System, engineered with MaxPeak HPS technology, addresses these issues by minimizing sample adsorption and supporting robust, reproducible analyses.

Objectives and Study Overview

The study compared the performance of the modern Alliance iS Bio HPLC System against a legacy HPLC platform for routine QC testing of insulin, insulin analogs, glucagon, and GLP-1 peptides. Key aims included method migration, compendial compliance, and exploration of modern column chemistries to improve throughput and reduce costs.

Methodology and Instrumental Setup

• Instrumentation: Alliance iS Bio HPLC System with MaxPeak High Performance Surfaces technology; legacy HPLC System.
• Columns: XSelect CSH C18 and XSelect Premier Peptide CSH C18 columns with particle sizes of 5 µm and 2.5 µm in various lengths (100–150 mm) and 4.6 mm ID.
• Detection: Tunable UV detection at 214 nm.
• Mobile Phases: Phosphate or formic acid buffers, trifluoroacetic acid, ammonium formate systems; isocratic and gradient elution per compendial and development protocols.

Key Results and Discussion

• Intact Insulin Analysis: Both platforms met USP compendial criteria for resolution, peak tailing, and area precision. The Alliance system showed lower retention time RSD due to its 680 µL mixer and reduced dispersion.
• Peptide Mapping: Glu-C digestion of insulin and analogs yielded four diagnostic fragments. Method migration preserved resolution and reproducibility on both systems. Scaling to a 2.5 µm column reduced solvent and sample use by 50% and cut analysis time by 75% while satisfying USP <621> requirements.
• Glucagon Impurity Profiling: Forced degradation identified desamido impurities. The Alliance system resolved and quantified these impurities per USP limits. Vendor sample screening flagged failures to meet impurity thresholds.
• GLP-1 Panel Method Development: Using Premier columns and the Alliance system’s low dispersion, baseline noise was reduced and peak shapes improved. This enhanced detection of low-abundance impurities compared to the legacy HPLC.

Practical Benefits and Applications

• Smooth migration of legacy compendial methods with improved robustness and reproducibility.
• Enabling use of smaller-particle, shorter columns for faster, cost-efficient analyses.
• Enhanced QC throughput and data reliability in biopharmaceutical peptide testing.

Future Trends and Potential Applications

Adoption of bio-inert surface technologies and low-dispersion flow paths will continue to drive improvements in peptide and protein analytics. Integration with automated data systems, expansion to novel modalities (lipid-conjugated peptides, fusion proteins), and coupling with mass spectrometry are key future directions.

Conclusion

The Alliance iS Bio HPLC System demonstrates compatibility with compendial assays and offers modernization opportunities that reduce costs, increase throughput, and enhance data quality for peptide drug substance analysis in QC environments.

References

• USP. Insulin human. In: USP-NF. Rockville, MD: USP; Dec 1, 2015.
• USP. Physicochemical Analytical Procedures for Insulins <121.1>. In: USP-NF. Rockville, MD: USP; Dec 1, 2016.
• USP. Chromatography <621>. In: USP-NF. Rockville, MD: USP; Dec 1, 2022.
• USP. Glucagon for injection. In: USP-NF. Rockville, MD: USP; Feb 1, 2019.
• Clements B, Rainville P. Development of Separation Methods for GLP-1 Synthetic Peptides Utilizing a Systematic Protocol and MaxPeak High Performance Surface Technology. Waters Application Note. 720008267. 2024 April.