How to Determine Glycan Profiles of Biopharmaceuticals from Peptide Mapping Data

Importance of the Topic

Glycosylation is a critical quality attribute of therapeutic proteins and influences their stability, activity, and immunogenicity. Traditional glycan analysis involves enzymatic release and labeling, which sacrifices site-specific information and requires specialized workflows.

Study Objectives and Overview

This work introduces a streamlined approach to determine glycan compositions directly from tryptic peptide mapping data, leveraging ion mobility PASEF and a novel heated electrospray source. The goals include:

• Retaining glycosylation site information
• Eliminating separate glycan release and labeling steps
• Utilizing standard peptide mapping datasets

Methodology

Sample Preparation
Proteins were reduced, alkylated, and digested with trypsin. The resulting peptides were separated by reversed-phase UHPLC and analyzed by timsTOF Pro using PASEF with a reduced precursor threshold.
Data Analysis
Initial peptide mapping was performed in BioPharma Compass, focusing on a 5–15 min retention time window enriched in Fc glycopeptides. MS/MS spectra were searched with GlycoQuest against the GlycO database, treating the tryptic peptide as a mass tag. Stringent thresholds (score > 40, fragmentation coverage > 40%, intensity coverage > 40%) ensured high-confidence assignments.

Used Instrumentation

• timsTOF Pro mass spectrometer
• VIP-HESI heated ESI source
• Elute UHPLC system
• Acquity CSH C18 column (2.1×100 mm, 1.7 μm)
• BioPharma Compass 2021b software
• GlycoQuest search engine

Main Results and Discussion

The workflow identified 36 distinct glycan compositions from NISTmAb tryptic digest, including complex, hybrid, and high-mannose structures. Neutral and acidic glycans eluted at different retention times. The most abundant glycan (G0F) was detected with high intensity, while low-abundance species were identified over a 100:1 dynamic range. Comparison with established labeled-glycan methods confirmed 27 of 30 expected compositions and revealed additional low-abundance structures.

Benefits and Practical Applications

• Generates comprehensive glycan profiles within standard peptide mapping runs
• Preserves site-specific glycosylation information
• Delivers high sensitivity and dynamic range using PASEF and VIP-HESI
• Accessible to non-experts for QC and R&D purposes

Future Trends and Applications

This approach can be extended to glycoproteins with multiple sites, such as viral spike proteins, to achieve site-specific glycan mapping in high throughput. Advances in isomer separation, enhanced fragmentation techniques, and automated data interpretation will further strengthen glycoproteomic analyses.

Conclusion

Direct glycopeptide analysis via PASEF-equipped timsTOF Pro and heated ESI simplifies glycan profiling, retains critical site information, and matches the performance of traditional workflows, offering a powerful tool for biopharmaceutical CQA.

Reference

1. Hinneburg et al J Am Soc Mass Spectrom 2016
2. Hilliard et al mAbs 2017
3. Meier et al Mol Cell Proteomics 2018
4. Gstöttner et al Anal Chem 2021