N-glycan Profiles based on AEX- and HILIC-HPLC
Since sialic acid is the most common CQA for biopharmaceutical products, it can be useful to separate N-glycans on the basis of charge by WAX-HPLC, enabling more accurate relative quantification of multi-sialylated structures. R27T glycans were separated on a WAX-HPLC column, and 17 peak fractions were collected (Figure S4A). Fractions were combined (F2+F3, F4+F5, and F7+F8) when peaks were not clearly separated. Each fraction containing zero (F1), one (F4+F5), two (F7+F8), three (F9), or four (F10−13) sialic acids was then analyzed by HILIC-HPLC (Figure S4B). Fractions 2, 3, 6, and 14−17 did not contain any glycans. Fractions containing charged glycans were then digested with sialidase and re-run on the HILIC-HPLC column (Figure 3). Structures were identified from the GU values in relation to neutral structures identified by exoglycosidase sequencing. Fraction 1 contained neutral glycans (biantennary structures A2G2 and FA2G2). Fraction 4+5 contained mono-sialylated, core-fucosylated bi-, tri-, and tetra-antennary (with zero, one, or two lactosamine repeats) glycans. Fraction 7+8 contained di-sialylated glycans, and both major peaks (GU 8.4 and 8.9) digested to FA2G2 at GU 7.5. α2-3-linked sialic acid adds less to the GU value than α2-6-linked sialic acid. Thus, we can conclude that the largest peak (GU 8.4) includes an α2-3-linked sialic acid while the second peak (GU 8.9) has an α2-6-linked sialic acid. A small proportion of triantennary glycans were also di-sialylated. Fraction 9 contained tri-sialylated, core-fucosylated bi-, tri-, and tetra-antennary glycans with zero, one, or two lactosamine repeats. Both forms of triantennary glycans were identified: A3G3, in which the third GlcNAc is β1-4-linked to the 3-linked mannose, and A3′G3, in which the third GlcNAc is β1-6-linked to the 6-linked mannose. There were also some tri-sialylated tetra-antennary glycans without core fucose. Fraction 10 contained tetra-sialylated, core-fucosylated tetra-antennary glycans with two or three lactosamine repeats. Fraction 11 contained tetra-sialylated, core-fucosylated tetra-antennary glycans with two lactosamine repeats. Both peaks digested to the same GU value after removal of sialic acids, suggesting that the second smaller peak contained glycans with α2-6-linked sialic acid(s). Fraction 12 contained tetra-sialylated, core-fucosylated tetra-antennary glycans with one lactosamine repeat, as well as tetra-sialylated tetra-antennary glycans with and without core fucose. Fraction 13 contained tetra-sialylated, core-fucosylated tetra-antennary glycans. All peaks digested to the same GU value after removal of sialic acids, suggesting that the smaller peaks contained glycans with α2-6-linked sialic acid(s). The lactosamine extension adds approximately the same value to the GU value as the addition of an extra antenna (both are Gal-GlcNAc).
A summary of the identified glycans matched to peaks for the whole pool is given in Table 3 and Figure S5. The GU values changed slightly over time (this is normal for sialylated glycans); hence peaks in fractions were matched to a profile of the whole undigested pool run at the same time. The major N-glycan, which accounts for ~42% of total N-glycans, is a di-sialylated, core-fucosylated biantennary structure. However, R27T also exhibited considerable variability in its glycoprofile, with tri- and tetra-antennary glycans, as well as biantennary glycan forms being detected. Surprisingly, rhIFN-β containing a larger proportion of higher antennary glycoforms showed more sustained bioactivity over time (Mastrangeli et al., 2015). Indeed, in our previous study, R27T exhibited more prolonged signaling than the mono-glycosylated rhIFN-β, with altered receptor-binding kinetics (Lee et al., 2018). A larger portion of higher antennary components in R27T may therefore influence cellular signaling effects.