Sialic acids, also referred to as neuraminic acids, are critical to glycoprotein bioavailability, function, stability, and metabolism. When present, these carbohydrates occupy terminal positions of oligosaccharides in glycosylated proteins, providing charged points of interaction essential in many biological pathways.
Although over 50 natural sialic acids have been identified, two forms are commonly determined in therapeutic glycoprotein products: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Due to loss of the enzyme CMP-N-acetylneuraminate monooxygenase, human tissue does not synthesize Neu5Gc, although it may be incorporated from dietary sources and has been studied as a disease marker.
Furthermore, Neu5Gc has been shown to be immunogenic, and the presence of this sialic acid in a therapeutic agent can potentially lead to an unwanted immune response. Therefore, total glycoprotein sialylation, and the identity of the sialic acids, play important roles in therapeutic protein efficacy, pharmacokinetics, and potential immunogenicity.
Glycoprotein sialylation analysis has been performed by many methods, ranging from colorimetric tests to sophisticated chromatographic methods. Chromatographic methods have the advantage of quantitatively differentiating sialic acids. The two chromatographic methods typically chosen for sialic acid determination are high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and sample derivatization followed by UHPLC with fluorescence detection (UHPLC-FLD). These two methods each detect Neu5Ac and Neu5Gc, but by different approaches. HPAE-PAD relies on oxidation of the carbohydrate at a gold working electrode.
This detection method is direct and specific to the oxidation potential of the analyte. UHPLC-FLD relies on derivatization of the sialic acids to form a fluorescent product. Specificity is controlled by the derivatization reaction. Separation is subsequently performed on a C18 column after derivatization. This method is indirect and relies on the derivatization reaction for both analyte separation and detection.
For purposes of illustration, this article compares two independent chromatographic assays developed for sialic acids in five model glycoproteins: calf fetuin, bovine apo-transferrin (b. apo-transferrin), human transferrin (h. transferrin), sheep α1-acid glycoprotein (s. AGP), and human α1-acid glycoprotein (h. AGP). Analyses by both HPAE-PAD and UHPLC-FLD are evaluated with acid hydrolyzates of these proteins.