DMB-Labeled Sialic Acid Analyses Using HPLC-, UHPLC-, and UPLC-Based, BEH C18 Columns

Importance of the Topic

In biopharmaceutical development, sialic acids represent critical quality attributes of glycoproteins, influencing therapeutic half-life, immunogenicity, and biological activity. Accurate profiling of sialic acid variants such as Neu5Ac and Neu5Gc is essential for ensuring product safety, efficacy, and regulatory compliance.

Objectives and Study Overview

This study aimed to evaluate a pre-column DMB (1,2-diamino-4,5-methylenedioxybenzene) derivatization approach coupled to reversed-phase BEH C18 chromatography. The goal was to demonstrate consistent baseline resolution of six sialic acid derivatives using HPLC, UHPLC, and UPLC systems through geometric scaling of column dimensions and particle sizes.

Methodology and Instrumentation

The workflow began with mild acid hydrolysis of glycoproteins to release sialic acids, followed by DMB labeling under light-protected conditions. Samples were separated under isocratic conditions using Waters BEH C18 columns of varying configurations. Instrumentation included:

• ACQUITY UPLC System with fluorescence detection (Ex 373 nm, Em 448 nm)
• XBridge BEH C18, 130Å, 3.5 µm, 4.6 × 100 mm (HPLC)
• XBridge BEH C18, 130Å, 2.5 µm XP, 3.0 × 75 mm (UHPLC)
• ACQUITY UPLC BEH C18, 130Å, 1.7 µm, 2.1 × 50 mm (UPLC)

Eluent composition was acetonitrile : methanol : water (9 : 7 : 84), with column and sample temperatures maintained at 30 °C and 4 °C, respectively.

Main Results and Discussion

An isocratic mobile phase provided baseline separation of six DMB-labeled sialic acid standards without gradient steps. Run times decreased geometrically from approximately 23 minutes on the HPLC column to 12 minutes on the UHPLC column and 5.7 minutes on the UPLC column, while maintaining resolution and peak quality. This scaling approach reduced analysis time and solvent usage without compromising data quality.

Benefits and Practical Applications

• Regulatory alignment: method supports ICH Q6B requirements for sialic acid quantitation.
• High throughput: shorter run times improve sample processing capacity.
• Resource efficiency: decreased solvent consumption and waste.
• Versatility: compatible with existing HPLC, UHPLC, and UPLC platforms.
• Quality control: robust monitoring of critical glyco-attributes across development stages.

Future Trends and Potential Applications

Emerging opportunities include integration with mass spectrometry for structural elucidation, automation for real-time in-process monitoring, and miniaturized or microfluidic LC platforms to further reduce sample and solvent requirements. Advances in data analytics and digital chromatography are likely to enhance glycoform profiling in biomanufacturing.

Conclusion

This work demonstrates a unified approach for efficient, high-quality analysis of DMB-labeled sialic acids using geometrically scaled BEH C18 columns. The method delivers reliable resolution and significant gains in throughput, supporting rigorous quality assessment throughout biopharmaceutical development.

References

1. Wang B, Brand-Miller J. The role and potential of sialic acid in human nutrition. Eur J Clin Nutr. 2003;57:1351–1369.
2. Nguyen DH, Tangvoranuntakul P, Varki A. Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells. J Immunol. 2005;175:228–236.
3. ICH Q6B. Specifications: test procedures and acceptance criteria for biotechnological/biological products. Fed Regist. 1999;64:44928.
4. Rohrer JS. Analyzing sialic acids using high-performance anion-exchange chromatography with pulsed amperometric detection. Anal Biochem. 2000;283:3–9.
5. Hara S, Yamaguchi M, Nakamura M. Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography. Anal Biochem. 1989;179:162–166.