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.