Antennary Structures based on Exoglycosidase Sequencing
Detailed structures including core fucosylation, antennary composition, lactosamine extensions, and linkages were identified by exoglycosidase sequencing. The results of HILIC-HPLC analysis revealed 22 peaks (Figure S3, Table S2). Exoglycosidase treatment facilitated more accurate analysis of the N-glycan antennary structure of R27T (Figure 2). A summary of the results of exoglycosidase sequencing of N-glycans is included in Table S3. Data from sialidase (a368S), fucosidase (a34F, a6F), and galactosidase (a36G, b4G) exoglycosidase digestion can be used to quantify different types of antennary structures present within samples. A combination of enzymes can determine structures down to the core A2, A3, A3′, and A4 structures, for which the glucose unit (GU) values are documented (Table S3). Digestion with α-galactosidase (a36G) or α-3/4 fucosidase (a34F) generated identical chromatograms, indicating that neither outer arm fucose nor α-galactose moieties were present. Comparison of digestion with sialidase (a368S) alone and digestion with sialidase (a368S) plus both fucosidases (a34F and a6F) revealed that most peaks moved by ~0.4 GU units, in line with the loss of a core α1-6 fucose component. This means that core-fucosylated structures accounted for 94% of the total. A comparison of digestion with sialidase (a368S) plus fucosidases (a34F and a6F) and digestion with sialidase (a368S) plus fucosidases (a34F and a6F) plus beta-galactosidase (b4G) revealed the core antennary structures (A2, A3, A3′, and A4) for some peaks. However, a number of peaks did not digest and reveal the core structures until N -acetylglucosaminidase (sph) was also added, presumably because they contained lactosamine extension, which can be added to any antennae, resulting in diverse isomeric structures. Biantennary structures were most abundant. The N-glycan of R27T consists of a mixture of bi- (42.5%), tri- (17.2), and tetra-antennary glycans (8.8%), some of which have lactosamine extensions (29.3%). Most glycans in R27T (~94%) also have a core-fucosylated structure (Table 2). This could play a crucial role in stabilization of the R27T structure because the core fucose structure of the rhIFN-β carbohydrate may help to stabilize the rhIFN-β structure (Karpusas, Whitty, Runkel, & Hochman, 1998). In these structures, galactose α1-3 linked to β-galactose was not observed. This is also very important because the presence of α-galactose could lead to potential adverse reactions, immunogenic potential, and neutralization of the drug by anti-α-galactose antibodies (Eon-Duval, Broly, & Gleixner, 2012).