Sialic acids, which include N-acetylneuraminic acid and N-glycolylneuraminic acid, are a group of nine-carbon monosaccharides often found at the terminal position of glycoproteins and glycolipids. In the production of glycoprotein drug products, sialylation with N-acetylneuraminic acid ensures good circulatory half-life, biological activity, and solubility. However, inadvertent attachment of structurally similar N-glycolylneuraminic acid presents a safety concern, as this non-human sialic acid can trigger an immune response. Accurate determination of glycoprotein sialic acid composition is therefore essential in biopharmaceutical testing to ensure product safety and efficacy.
Reliable and efficient sialic acid analysis
The determination of glycoprotein sialic acids can be effectively achieved using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). HPAE chromatography using strong anion-exchange column chemistries can yield highly selective carbohydrate separations. Coupled with PAD, the technique enables direct quantification of analytes, avoiding time-consuming sample derivatization steps.
HPAE-PAD methods for sialic acid analysis often require high-purity sodium hydroxide and sodium acetate eluents. However, traditional methods for eluent preparation can be labor-intensive and a significant source of measurement inconsistency, as even small variations in eluent concentration can affect reproducibility.
Dual eluent generation cartridge (dual EGC) mode is an alternative operating approach for HPAE-PAD that employs a methanesulfonic acid EGC and potassium hydroxide EGC in series to generate an accurate and reproducible eluent gradient. As well as reducing method inconsistency, dual EGC mode operation also improves laboratory efficiency by helping busy teams save time during system setup.
Efficient separation and detection of sialic acids in glycoprotein samples
To demonstrate the potential of dual EGC mode HPAE-PAD for glycoprotein sialic acid analysis, we used a Thermo Scientific™ Dionex™ ICS-6000 HPIC™ system to determine sialic acid content in three glycoprotein samples, as reported in this application note. This dual EGC method improves upon our existing protocol for this application, and makes use of the Thermo Scientific™ Dionex™ CarboPac™ PA20-1 mm column, designed for use in dual EGC mode. This 1 mm column requires a flow rate approximately one-ninth that of the 3 mm version used in the earlier protocol, helping laboratories improve efficiency by reducing eluent waste.
The Dionex CarboPac PA20-1 mm column achieved efficient separation of standard solutions of N-acetylneuraminic acid and N-glycolylneuraminic acid, resulting in easily quantifiable peaks (Figure 1). Notably, the N-acetylneuraminic acid signal was well-separated from the void. This is particularly important when analyzing acid-hydrolyzed samples, which may contain poorly retained compounds. Retention times for both analytes were stable over 15 days of continuous injections of samples and standards, reflecting the effectiveness of the column rinsing step and the mobile phase purity achieved using dual EGC mode operation.
Calibration curves showed linearity in the range 0.1–25 μM for N-acetylneuraminic acid and 0.025–2.5 μM for N-glycolylneuraminic acid, with coefficients of determination (r2) greater than 0.999. The limit of detection, based on the signal-to-noise (S/N) ratio, was determined to be 20.9 nM for N-acetylneuraminic acid and 10.3 nM for N-glycolylneuraminic acid, highlighting the overall suitability of the method.
This method was then used to determine sialic acid content in three glycoprotein samples (bovine apo-transferrin, calf fetuin, and human alpha-1-acid glycoprotein). Prior to analysis, sialic acids were released from the glycoproteins by acetic acid hydrolysis, with samples prepared and analyzed in triplicate.
As expected, N-acetylneuraminic acid was detected in all three glycoprotein samples, whereas N-glycolylneuraminic acid was only present in the two non-human glycoproteins. Relative standard deviations for sialic acid content for the triplicate analyses ranged from 0.37 to 3.82%, reflecting the reproducibility of the sample preparation and analysis workflow.
Method accuracy was evaluated by determining recoveries of sialic acids spiked into each sample at 50–100% of the original amount. Recoveries for sialic acids in the three samples ranged from 95–105%, demonstrating the accuracy of the HPAE-PAD measurement technique. Inter-day precision was determined by triplicate injection of a calibration standard on three separate days. The calculated peak area precision varied from 0.83 to 1.04%, with a retention time precision of less than 0.2% for all target sialic acids, consistent with the excellent performance that can be achieved using dual EGC mode operation.
Dual EGC mode HPAE-PAD: Reliable results, with simplified operation
HPAE-PAD with dual EGC mode operation provides a reproducible and convenient approach for glycoprotein sialic acid analysis. By eliminating the variation in mobile phase concentration that can occur using traditional eluent preparation steps, dual ECG mode generates accurate and precise results while saving time and simplifying workflows.
Read more about dual EGC mode HPAE-PAD for the determination of sialic acids in glycoproteins here.
Dr. Jeffrey Rohrer, Director of Applications Development, contributed to this article.