Improving UPLC-FLR-MS N-glycan Analysis of Disulfide-rich Fusion Proteins Through Optimization of Sample Preparation

Importance of the Topic

Fusion proteins are increasingly employed in biotherapeutics due to their extended plasma half-life and enhanced therapeutic activity. N-glycosylation profoundly influences safety, efficacy, and pharmacokinetics, making thorough glycan profiling essential for quality control and regulatory compliance, especially for fusion proteins with complex structures and abundant disulfide linkages.

Goals and Overview of the Study

This application note presents an optimized sample preparation workflow for UPLC-FLR-MS analysis of N-linked glycans released from disulfide-rich fusion proteins. The aim is to ensure complete glycan release, efficient labeling, and sensitive detection by introducing a reducing agent into the rapid denaturation step of the Waters GlycoWorks RapiFluor-MS N-glycan Kit and validating performance on a model fusion protein containing 28 disulfide bonds.

Methodology and Instrumentation

• Sample Preparation: Denature fusion protein with RapiGest SF detergent at 90 °C in the presence of 6 mM TCEP or DTT to reduce disulfide bonds, followed by 5 min digestion with Rapid PNGase F and RFMS labeling.
• Liquid Chromatography: ACQUITY UPLC I-Class PLUS with ACQUITY Glycan BEH Amide column (2.1×150 mm, 1.7 µm) at 60 °C; HILIC gradient using 100 mM ammonium formate in water and acetonitrile; injection volume 10 µL.
• Fluorescence Detection: ACQUITY FLR Detector (λexc = 265 nm, λem = 425 nm).
• Mass Spectrometry: ACQUITY RDa Mass Detector in positive ESI mode (50–2,000 m/z, 1.5 kV capillary, 45 V cone, 70–90 V fragmentation).
• Data Analysis: UNIFI v1.9.4 with “Glycan FLR with MS confirmation” workflow and custom glycan library.

Main Results and Discussion

Using the original mAb workflow, incomplete deglycosylation was evidenced by residual glycoforms in RPLC-MS. Incorporation of TCEP (or DTT) during a 3 min denaturation with RapiGest SF yielded full reduction of disulfide bonds, enabling complete PNGase F access and deglycosylation within 5 min. FLR–MS analysis identified 84 distinct glycans. Doubling RFMS reagent compensated for slight labeling inhibition by the reducing agent. Low-abundance glycans (e.g., Man7 at 0.08% relative abundance) exhibited high signal-to-noise in both FLR and MS channels.

Benefits and Practical Applications of the Method

• Rapid and streamlined sample preparation (< 30 min total) with no alkylation or buffer exchange.
• Complete glycan release from proteins with extensive disulfide networks.
• Enhanced FLR and MS sensitivity enabling detection of minor glycoforms.
• Compatibility with legacy 2-AB workflows and seamless transfer to modern RFMS labeling.
• Improved throughput and confidence in QC and comparability studies.

Future Trends and Potential Applications

Further automation of this workflow could support high-throughput glycomics in process development and manufacturing. Expanding the approach to other complex glycoprotein modalities—such as bispecifics or multi-domain fusion constructs—will enhance product characterization. Integration with real-time monitoring and AI-driven glycan annotation promises to accelerate biopharmaceutical development and regulatory submissions.

Conclusion

An optimized UPLC-FLR-MS sample preparation method incorporating an enzyme-friendly reducing agent and rapid denaturation achieves complete N-glycan release and high labeling efficiency for disulfide-rich fusion proteins. The protocol improves accuracy, reduces analysis time, and supports seamless migration from legacy glycan analysis platforms.

Reference

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• Zhang X et al. Increasing Productivity and Confidence for N-linked Glycan Analysis of Biosimilars Using the BioAccord System. Waters Application Note. 2019.
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