Improving Released N-glycan Analysis in Biotherapeutic Development Using the ACQUITY Premier Solution with MaxPeak High Performance Surfaces (HPS) Technology

Significance of the Topic

Glycosylation plays a pivotal role in the safety, efficacy, and stability of biotherapeutic proteins. As a critical quality attribute, glycan profiling informs process consistency and product performance. Accurate assessment of released N-linked glycans, including neutral, sialylated, and acidic species, is essential for robust biopharmaceutical development and quality control.

Objectives and Study Overview

This application note evaluates an integrated LC-FLR-MS workflow enhanced by MaxPeak High Performance Surfaces (HPS) technology to improve recovery of metal-sensitive glycans. Key aims include:

• Comparing acidic glycan recovery using standard and ACQUITY Premier system configurations.
• Assessing phosphorylated glycan analysis from Cathepsin D as a model lysosomal enzyme.
• Demonstrating seamless transfer of existing UNIFI GU libraries across platforms.

Methodology and Instrumentation

N-linked glycans were enzymatically released and labeled with RapiFluor-MS. Separation used HILIC on ACQUITY Glycan BEH Amide Columns under two configurations: a standard BioAccord LC-MS platform with ACQUITY UPLC I-Class PLUS, and a BioAccord system with ACQUITY Premier BSM and MaxPeak HPS surfaces. Detection combined FLR and ESI-TOF MS (ACQUITY RDa, m/z 50–2000). Data processing employed waters_connect with UNIFI 1.9.4 and the “Glycan FLR with MS confirmation” workflow.

Main Results and Discussion

• Sialylated glycans exhibited a relative recovery improvement from 2.11% to 2.22% when using MaxPeak HPS technology, while neutral glycan responses remained consistent.
• Phosphorylated mannose-6-phosphate species from Cathepsin D showed up to threefold higher MS response with markedly sharper peak shapes, indicating reduced metal-mediated adsorption. Reproducibility over 15 injections yielded ~3.96% RSD.
• Glucose unit (GU) values for 19 standard glycans remained within ±0.001 GU across both configurations, validating GU library compatibility and method transferability.

Benefits and Practical Applications

The ACQUITY Premier Solution with MaxPeak HPS surfaces offers:

• Enhanced sensitivity and accuracy for acidic and phosphorylated glycans.
• Improved peak shape and quantitation reliability, especially for metal-interactive analytes.
• Automated peak assignment via existing UNIFI GU libraries, streamlining glycan profiling workflows.

Future Trends and Further Applications

Emerging directions include:

• Expansion of HPS-coated hardware in routine QA/QC and upstream process monitoring.
• Application to other metal-sensitive biomolecules such as phospholipids and metabolites.
• Integration with high-throughput platforms and advanced informatics for real-time glycan analytics.

Conclusion

The ACQUITY Premier Solution with MaxPeak HPS technology significantly enhances the recovery and analysis of released N-glycans, particularly acidic and phosphorylated species, without altering existing GU-based workflows. This robust, reproducible platform supports efficient biotherapeutic development, manufacturing, and release.

Reference

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