Monitoring Impurities Using a High Throughput Focused Gradient With the Alliance™ iS Bio System

Importance of the Topic

Monoclonal antibody therapeutics demand rigorous impurity profiling to ensure safety, efficacy, and regulatory compliance. High-throughput UV-based assays are vital in QC labs to monitor low-abundance modifications such as peptide oxidation, enabling rapid decision making in biomanufacturing.

Objectives and Overview

This study illustrates the development and transfer of a targeted reversed-phase LC-UV assay for the methionine-containing T21 peptide and its oxidized form. A full peptide mapping method was condensed into a 20-minute focused gradient on the Alliance iS Bio HPLC System to demonstrate high throughput and robust impurity monitoring.

Methodology and Instrumentation

• Platform: Alliance iS Bio HPLC System equipped with a 690 µL Titanium diffusion bonded mixer, integrated sample metering pump, and large-volume needle loop. Controlled via Empower 3 CDS.
• Mobile Phases: A—0.1 % difluoroacetic acid (DFA) in water; B—0.1 % DFA in acetonitrile.
• Column: XSelect Premier Peptide CSH C18, 130 Å, 2.5 µm, 4.6×100 mm at 60 °C.
• Detection: UV at 220 nm; mass confirmation during development on Arc Premier System with ACQUITY QDa™ detector.
• Gradient Development: Transitioned from an 80-minute MS-enabled peptide mapping gradient to a 20-minute focused gradient targeting the native and oxidized T21 peptides.
• Injection Volumes: 30 to 100 µL to assess load linearity and sensitivity.

Main Results and Discussion

• DFA provided improved peak shape and moderate ion suppression, facilitating both UV detection and MS method development.
• The 20-minute gradient fully resolved native and oxidized T21 peptides; mass spectra confirmed peak identity and purity across leading, apex, and trailing regions.
• The diffusion bonded mixer delivered a stable baseline over 49 blanks and 17 sample injections across 30 hours, enhancing confidence in low-abundance impurity detection.
• Peak area scaled linearly with injection volumes from 10 to 100 µL (5–50 µg on column), leveraging precise high-volume injections without external loops.
• An 18-sample sequence (90 injections total) over 30 hours showed consistent oxidized T21 levels (mean ≤2 % area, RSD ≅12 %), demonstrating robustness for routine QC.

Benefits and Practical Applications

• High-throughput operation reduces run time and increases laboratory efficiency.
• Improved baseline stability and sensitivity enhance detection of low-level impurities.
• Integrated large-volume injections streamline workflow and minimize dispersion.
• Simplified transfer from MS-based development to UV-based QC assays.

Future Trends and Applications

Integration of AI-driven method optimization and advanced data analytics will further accelerate assay development. Novel ion-pairing reagents and mixer technologies could enhance sensitivity. Expansion of focused gradients to other critical quality attributes will broaden applicability in biologics QC.

Conclusion

The Alliance iS Bio HPLC System, combined with a focused gradient and Titanium diffusion bonded mixer, offers a robust, efficient platform for UV-based monitoring of peptide impurities. Its high throughput, reliable baseline performance, and precise large-volume injections make it ideal for QC environments in biopharmaceutical manufacturing.

Reference

• Nyugen JM, Liu X, Lauber MA. Comparing Mobile Phase Additives for the Separation of mAb Tryptic Peptides. Waters Application Note, 2019.
• Han D, Birdsall RE, Nyholm K. Migrating Peptide Mapping Methods to the Alliance iS Bio System. Waters Application Note, 2024.
• Birdsall RE, Du X, Nyholm K. Extending Biopharmaceutical QC Analytics with the ACQUITY QDa II Mass Detector. Waters Application Note, 2024.
• Martin K, Gauthier L, Hong P. Improved Sensitivity for TFA Gradients on Alliance iS HPLC. Waters Application Note, 2024.
• Waters Corporation. Ti Diffusion Bonded Mixer Installation Guide. User Manual, 2024.