Deploying the Alliance™ iS Bio HPLC System as a Modern HPLC for Biopharmaceutical Analysis in QC Environments

Importance of the Topic

The analysis of biopharmaceuticals in QC environments demands robust, sensitive, and reproducible methods. Oligonucleotides in particular present challenges due to their phosphate backbones interacting with metal surfaces, leading to reduced recovery and peak tailing. A bio-inert HPLC platform with advanced surface technologies can streamline impurity profiling and improve data quality, supporting safe drug release.

Study Objectives and Overview

This study assessed the performance of the Alliance iS Bio HPLC System with MaxPeak High Performance Surfaces Technology against a legacy HPLC setup. Two test cases were chosen:

• MassPREP Oligonucleotide Standard (OST): a mixture of 15–35 mer oligodeoxythymidines.
• GEM91: a 25-mer fully thiolated phosphorothioate oligonucleotide.

Comparative metrics included analyte recovery, signal-to-noise for impurities, run time, solvent and sample consumption, and method transfer efficiency.

Methodology and Instrumentation

Both systems used ion-pair reversed-phase chromatography with hexylammonium acetate as the pairing agent. Key parameters:

• Columns: Legacy – XBridge BEH C18, 5 μm and 2.5 μm, 130 Å, 4.6×100 mm; Modern – XBridge Premier Oligonucleotide BEH C18, 2.5 μm, 130 Å, 4.6×50–150 mm.
• Mobile phases: 25 mM HAA in water (pH 7.0) and 25 mM HAA in 40/60 water/acetonitrile (pH 7.0), plus water and ACN for quaternary blending.
• Temperatures: Column at 60 °C, sample at 5 °C.
• Detection: UV at 260 nm.
• Software: Empower 3 FR4 for legacy; Empower 3.8.0 with Intelligent Method Translator App for Alliance iS Bio.

Key Results and Discussion

• Oligonucleotide recovery on the Alliance iS Bio system increased by up to 30% in peak height and 14% in peak area for the 20-mer relative to the legacy HPLC.
• Signal-to-noise for GEM91 impurities improved by up to 40%, enabling more accurate impurity quantitation.
• Method transfer using the Intelligent Method Translator App eliminated manual transcription errors and reproduced legacy chromatograms seamlessly.
• Scaling to 2.5 μm particle columns with lengths of 50–150 mm preserved selectivity while reducing run time, solvent, and sample use by up to threefold.
• Quaternary solvent blending allowed rapid scouting of ion-pair concentrations (25–100 mM) and gradient slopes, optimizing resolution and minimizing reagent waste.
• Reproducibility studies on GEM91 yielded RSD < 0.05% for retention time and < 3% for peak area across five injections.

Benefits and Practical Applications

The Alliance iS Bio HPLC System delivers:

• Enhanced recovery and sensitivity for metal-sensitive biopharmaceutical analytes.
• Streamlined method transfer and reduced error rates via intelligent software tools.
• Lower operating costs and environmental impact through reduced solvent and sample consumption.
• High throughput capabilities leveraging modern column chemistries at pressures up to 10,000 psi.

Used Instrumentation

• Alliance iS Bio HPLC System with MaxPeak HPS Technology
• XBridge Premier Oligonucleotide BEH C18 Columns (2.5 μm, 130 Å, lengths 50–150 mm)
• Legacy Waters Alliance HPLC System with XBridge BEH C18 Columns
• Empower 3 and Intelligent Method Translator App

Future Trends and Opportunities

Continuous innovation in bio-inert surfaces and high-pressure capable HPLC will support emerging modalities such as longer oligonucleotides, mRNA, and complex conjugates. Integration with AI-driven method development and greener solvent systems will further enhance lab efficiency, reduce environmental footprint, and accelerate biopharmaceutical QC workflows.

Conclusion

The Alliance iS Bio HPLC System proves to be a next-generation platform for biopharmaceutical QC, offering significant gains in recovery, sensitivity, throughput, and reproducibility. Its bio-inert design, advanced surface technology, and flexible solvent management provide a robust solution for routine and emerging analytical challenges.

References

1. Chen Z., Liu Z., Feng Y., et al. Global research on RNA vaccines for COVID-19 from 2019 to 2023: a bibliometric analysis. Vaccines Mol. Ther. 2024;15.
2. Koshel B.M., Birdsall R.E., Yu Y.Q. Improving Recovery and Quantitation of Oligonucleotide Impurities Using ACQUITY Premier with MaxPeak HPS Technology. Waters App. Note 720007238, April 2021.
3. Gilar M., Li Z., MacLean J. Improved Chromatographic Analysis of Oligonucleotides with ACQUITY Premier Oligonucleotide BEH C18 Columns. Waters App. Note 720007012, Aug. 2023.
4. Brennan K., Trudeau M., Donegan M., Rainville P.D. Improved Oligonucleotide SPE-LC-MS Analysis Using MaxPeak High Performance Technology. Waters App. Note 720007019, Sep. 2020.