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).