Sialic Acid Content and Variation
Since sialic acids are terminal, negatively charged components that can determine the serum half-life and influence immunogenicity and biological activity, they are typically considered glycosylation CQAs. The abundance and terminal capping ratio of sialic acids on galactose are very important because they impact the function and biological half-life of biopharmaceuticals, since neutral glycans without sialic acid are cleared by asialoglycoprotein receptors in the liver (Achord, Brot, & Sly, 1977; Dobryszycka & Kukral, 1970; Morell, Irvine, Sternlieb, Scheinberg, & Ashwell, 1968).
Overall charge profiles for R27T was shown according to the number of sialic acids (Figure S2). The 2S component was the most abundant (35.6%) followed by 1S (26.1%), 3S (21.7%), and 4S (16.6%). The absolute quantitation of sialic acid residues of R27T was also measured as the molar ratio of sialic acid per R27T molecule (Table 1). The sialic acid content of R27T (2.81 mol/mol protein) was, as expected, much greater (3-fold) than that of Rebif (0.77 mol/mol protein), and the value was consistent with that in our previous study (Song et al., 2014). Sufficient sialylation can prolong the half-life, as demonstrated in our previous study (Song et al., 2014).Galactose monosaccharide analysis was performed to investigate the sialic acid capping ratio and evaluate the quality of R27T and Rebif (Table 1). The terminal sialic acid capping ratio on galactose was ~0.28−0.31 for both R27T and Rebif samples.
Another important aspect of sialylation is variation, which mainly includes N -acetyl-neuraminic acid (Neu5Ac) andN -glycolyl-neuraminic acid (Neu5Gc). Since Neu5Gc cannot be synthesized in humans, it may be recognized as a foreign epitope with immunogenic potential(Ghaderi, Taylor, Padler-Karavani, Diaz, & Varki, 2010; Hokke et al., 1990). Numerous studies have illustrated the necessity to minimize the relative content of Neu5Gc sialic acids in glycoprotein biopharmaceuticals(Ghaderi et al., 2010). Both sialic acid major variants, Neu5Ac and Neu5Gc were more abundant in R27T samples (2.75, 0.06 mol/mol protein, respectively) than in Rebif (0.77 mol/mol protein, not detected) (Table 1). The relative Neu5Gc percentage of total sialylation for R27T was 2.1%, but this was not detected for Rebif. In Chinese hamster ovary (CHO) cells, ~3% of sialic acids were identified as Neu5Gc for recombinant plasminogen activator, follicle-stimulating hormone (FSH), and erythropoietin (EPO)(Hokke et al., 1990). In addition, EPO containing 1% Neu5Gc induces a negligible immunogenic response, whereas EPO with ~7% Neu5Gc elicits a clinically significant immunogenic response (Noguchi, Mukuria, Suzuki, & Naiki, 1995). However, due to differences in products, as well as dosage and frequency for clinical use, it is difficult to generalize criteria values for Neu5Gc content in biopharmaceutical products. Various factors can be conveniently used to minimize or maintain Neu5Gc levels below 1% during the manufacturing of R27T (Borys et al., 2010).