Sialic acids are derivatives of the nine-carbon sugar neuraminic acid. Two important sialic acids are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) and these can be found in many biomolecules including glycoproteins, proteoglycans, and glycolipids. The presence of Neu5Gc in a therapeutic glycoprotein can potentially cause an immune response to humans because humans generally do not produce Neu5Gc. Therefore, it is important to determine the sialic acid content of a glycoprotein when assaying for therapeutic efficacy and safety.<br/>Sialic acids can be determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) after releasing them from the glycoprotein by acid hydrolysis or enzymatic digestion. HPAE-PAD features direct detection; and therefore, eliminates errors associated with analyte derivatization. The eluent can be manually prepared sodium hydroxide/sodium acetate.<br/>In this work, the method was evaluated with the same column often used for HPAE-PAD sialic acid determinations, but in a 1 mm format and with HPAE-PAD run in dual eluent generation cartridge mode in which the chromatography system automatically prepares the eluent. The 1-mm column requires a flow rate approximately 9 times less than the 3 mm column, and thus significantly reduces eluent consumption and waste generation. Dual EGC mode replaces the manual preparation of the sodium hydroxide/sodium acetate eluents. This mode uses a methanesulfonic acid (MSA) eluent generation cartridge (EGC) and a potassium hydroxide (KOH) EGC in series to generate an extremely reproducible and accurate KOH/KMSA eluent gradient needed for the chromatography of many carbohydrates. The separation, linearity, and reproducibility were excellent.  Comparison with a traditional HPAE-PAD determination of sialic acids using sodium hydroxide/sodium acetate eluents showed that the Dual EGC method delivers equivalent results and improves retention time precision, while simplifying operation (no eluent preparation).

Determination of glycoprotein sialic acid composition using HPAE-PAD in dual eluent generation cartridge mode

ACS Spring 2022

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