Analysis of Sialylated Glycan Modification in Protein Therapeutics

Importance of Sialylated Glycan Analysis in Protein Therapeutics

Glycosylation, and in particular sialylation, profoundly influences the pharmacokinetics, safety, and efficacy of many biotherapeutics such as fusion proteins and erythropoietin. Precise quantitation of sialylated N-glycans is essential for ensuring product consistency and meeting regulatory quality attributes. However, conventional HILIC approaches often fail to resolve positional isomers, complicating the control of sialic acid variants in drug development and quality control.

Objectives and Study Overview

This application note presents a streamlined, high‐throughput approach for quantifying sialylated N-glycans in protein therapeutics. The method integrates Agilent AdvanceBio Gly-X N-glycan prep with InstantPC fluorescent labeling and anion exchange chromatography to achieve clear separation of glycan charge variants and reproducible quantitation in a single workflow.

Methodology and Instrumentation

The sample preparation begins with denaturation and enzymatic release of N-glycans from 1–40 µg protein samples using N-Glycanase at elevated temperature. Immediately after deglycosylation, InstantPC dye is added for a one-minute labeling reaction. Labeled glycans are purified via a cleanup plate to remove free dye. Chromatographic separation uses:

• Mobile Phase A: 10 mM Tris-HCl, pH 8.5
• Mobile Phase B: 10 mM Tris-HCl with 500 mM NaCl, pH 8.5
• Gradient: 0–24% B over 15 minutes, then to 100% B
• Flow rate and run time yielding 25 min separation plus 15 min re-equilibration

Used Instrumentation

• Agilent 1260 Infinity II LC system with quaternary pump
• Agilent 1260 FLD (λEx 285 nm, λEm 345 nm)
• Agilent Bio SAX column, NP5, 4.6 × 250 mm, 5 µm
• Agilent Buffer Advisor software
• Agilent AdvanceBio Gly-X InstantPC kit (GX96-IPC)

Main Results and Discussion

Optimization of mobile phase pH demonstrated that pH 8.5 provided optimal resolution between singly and doubly charged sialylated glycan peaks. Z-value calculation (Z = 1×SA1% + 2×SA2% + 3×SA3% + 4×SA4%) effectively summarizes the distribution of charge variants. Doubling the InstantPC dye increased peak areas by roughly 20% without altering Z-values, confirming unbiased labeling. Recovery tests using consecutive elution steps showed >95% recovery across all glycan forms. Reproducibility was validated by overlaying six successive injections, showing negligible retention time shifts and consistent peak profiles.

Benefits and Practical Applications

• High throughput: entire sample prep and labeling completed in ~1 hour
• Robust separation of sialylated isomers, facilitating accurate Z-value determination
• Reproducible quantitative data suitable for routine QC and comparability studies
• Compatibility with fluorescence detection and potential extension to LC-MS workflows

Future Trends and Potential Applications

Advancements in glycan labeling and separation promise even greater resolution of complex glycoforms. Integration with high-resolution mass spectrometry will enable detailed structural elucidation, while automated sample preparation platforms can further increase throughput. Emerging labels with enhanced sensitivity and multiplexing capabilities may drive next-generation glycoprofiling in biopharma R&D and QC.

Conclusion

The described InstantPC labeling coupled with anion exchange chromatography provides a rapid, reproducible, and high-throughput solution for quantifying sialylated N-glycans in therapeutic proteins. Its robust separation of charge variants and straightforward Z-value calculation make it well suited for routine quality control and comparability assessments.

References

1. Yuen CT, et al. Glycan Analysis of Glycoprotein Pharmaceuticals: Evaluation of Analytical Approaches to Z Number Determination in Pharmaceutical Erythropoietin Products. Biologicals 2011;39(6):396–403.
2. Agilent Technologies. Agilent AdvanceBio Gly-X N-Glycan Prep with InstantPC Kit, 96-ct User Manual, publication 5994-1231EN, 2019.
3. Pharmacopoeia of the People’s Republic of China: Volume III, 2020.
4. Yan J, Jones A. Streamlined Workflows for N-Glycan Analysis of Biotherapeutics Using Agilent AdvanceBio Gly-X InstantPC and 2-AB Express Sample Preparation with LC/FLD/MS. Agilent Technologies App Note 5994-1348EN, 2021.