An Improved Workflow for Profiling and Quantitation of Sialic Acids in Biotherapeutics

Importance of Sialic Acid Profiling in Biotherapeutics

Terminal sialic acid residues on therapeutic glycoproteins influence immunogenicity, pharmacokinetics, and pharmacodynamics. Monitoring both absolute and relative levels of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) is critical for ensuring safety and efficacy of biotherapeutics.

Objectives and Study Overview

This work presents an improved high-throughput workflow based on the Agilent AdvanceBio Sialic Acid profiling and quantitation kit (p/n GS24-SAP). The method aims to release, label, and analyze terminal sialic acids in biotherapeutic glycoproteins and the NISTmAb, enabling both qualitative profiling and absolute quantitation.

Methodology

Sample preparation uses acid hydrolysis in a 96-well plate to release terminal sialic acids, followed by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB). DMB-labeled sialic acids are separated by reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) and detected by fluorescence (FLD) for quantitation, with optional high-resolution mass spectrometry (MS) confirmation.

Instrumentation Used

• Agilent 1290 Infinity II Quaternary LC system with Poroshell 120 EC-C18 column
• Agilent 1260 Infinity fluorescence detector (Ex 373 nm, Em 448 nm)
• Agilent 6545XT AdvanceBio LC/Q-TOF for MS confirmation
• 96-well thermocycler for release and labeling steps

Main Results and Discussion

The workflow successfully separated seven DMB-derivatized sialic acid species (Neu5Ac, Neu5Gc, and various acetylated derivatives) with consistent elution order. Calibration curves for Neu5Ac and Neu5Gc exhibited linearity (R² > 0.999) with limits of detection at 0.016 pmol and 0.012 pmol, respectively. Analysis of Rituxan and Enbrel revealed predominance of Neu5Ac, while NISTmAb and Erbitux contained mainly Neu5Gc. Quantitative results matched those from established kits and demonstrated improved sensitivity and reproducibility compared to earlier workflows.

Benefits and Practical Applications

• High-throughput processing with reduced sample preparation time and no dry-down step
• Enhanced sensitivity for low-abundance sialylation
• Combined qualitative profiling and absolute quantitation in a single workflow
• Robust quality control tool for biotherapeutic development and release testing

Future Trends and Potential Applications

Advancements may include further automation, integration with online MS for real-time monitoring, expansion of sialic acid reference panels, and application to diverse glycoprotein formats. Emerging labeling reagents and ultra-high-resolution separations will enhance sensitivity and expand analytical scope.

Conclusion

The improved AdvanceBio Sialic Acid profiling and quantitation kit offers a streamlined, sensitive, and reproducible workflow for profiling and quantifying terminal sialic acids in biotherapeutic glycoproteins. It supports critical quality attributes and facilitates rigorous control of glycosylation profiles.

Reference

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