Rapid High-Throughput Profiling and Quantitation of Sialic Acids in Biotherapeutics

Significance of the Topic

Biotherapeutic glycoprotein efficacy and safety are critically influenced by terminal sialic acids such as Neu5Ac and Neu5Gc. Neu5Gc, absent in human biosynthesis, can trigger immunogenic responses, while total sialylation affects clearance rates and effector functions. Fast, sensitive analysis of sialic acid content and composition is thus vital during drug development and quality control to ensure consistent product performance.

Objectives and Overview of the Study

This study introduces a streamlined high-throughput workflow for simultaneous profiling and quantitation of sialic acids in therapeutic glycoproteins. The method leverages a 96-well plate format with direct derivatization using 1,2-diamino-4,5-methylenedioxybenzene (DMB) and rapid reversed-phase UHPLC coupled to fluorescence and optional high-resolution mass spectrometry detection. Model proteins include rituximab (Rituxan), etanercept (Enbrel), NISTmAb, and cetuximab.

Methodology and Instrumentation

• Sample Preparation: Acid hydrolysis of 200 μg glycoprotein in 96-well plate, eliminating dry-down to reduce preparation time to 1–2 hours.
• DMB Labeling: Two-step reaction—desialylation at 80 °C for 2 h followed by DMB derivatization at 50 °C for 3 h in a thermocycler.
• Chromatography: Agilent 1290 Infinity II UHPLC with InfinityLab Poroshell 120 EC-C18 column (2.1×75 mm, 2.7 μm); 10 min gradient using methanol/acetonitrile/water and acetonitrile phases.
• Detection: Fluorescence detection (Ex 373 nm, Em 448 nm) on Agilent 1260 FLD; optional coupling to 6545XT AdvanceBio LC/Q-TOF MS for confirmatory analysis of m/z 400–1000.
• Quantitation: Calibration curves prepared from Neu5Ac and Neu5Gc standards; data processed with Agilent OpenLab CDS and MassHunter Qualitative Analysis.

Key Results and Discussion

• LOD and LOQ: Neu5Gc at 0.012 pmol/0.040 pmol and Neu5Ac at 0.016 pmol/0.053 pmol, demonstrating picomolar sensitivity.
• Sialic Acid Profiles:
  
  • Rituxan and Enbrel predominantly contained Neu5Ac with low Neu5Gc levels.
  • NISTmAb and Cetuximab showed higher Neu5Gc content.
• Reproducibility: Quantitative measurements of Neu5Ac and Neu5Gc yielded low %CV (<10%), confirming workflow robustness.
• Mass Confirmation: High-resolution MS spectra matched expected DMB derivatives, validating peak identities.

Benefits and Practical Applications

• High Throughput: 96-well format supports large sample sets with minimal handling.
• Time Efficiency: Elimination of drying steps reduces prep time.
• Sensitivity: Enables analysis of proteins with low sialylation levels, such as monoclonal antibodies.
• Comprehensive Data: Simultaneous qualitative profiling and absolute quantitation support critical quality attributes in biotherapeutic development and QC.

Future Trends and Opportunities

• Integration with automated robotic systems for end-to-end sample processing.
• Extension to other glycan modifications and linkage-specific analyses.
• Real-time process analytics during biomanufacturing using inline UHPLC-FLD/MS.
• Data-driven approaches and machine learning for glycosylation pattern recognition and predictive quality assessments.

Conclusion

This optimized DMB labeling and rapid UHPLC-FLD/MS workflow offers a robust, sensitive, and streamlined solution for high-throughput sialic acid analysis in biotherapeutics. By reducing sample prep time and maintaining high sensitivity, the method facilitates reliable monitoring of critical glycosylation attributes, supporting consistent drug quality and safety.

Reference

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