Effect of fucosylation and galactosylation on the conformation of an IgG1 antibody
In another experiment, a subtle change to the IgG1 glycan was made, and the conformation and dynamics of the IgG1 were investigated
using H/DX–MS. IgG1 samples were compared in various combinations with and without galactose and fucose (27). Previous published
work reported that decreased levels of fucose and increased levels of galactose increase effector function responses, and
that these effects appear to be additive (27–28, 32). It was also shown that neither fucose nor galactose is directly involved
in Fc receptor interactions (29, 33). If this is indeed the case, how is it that the addition of galactose or subtraction
of fucose affects the effector functionality? Experiments using H/DX–MS showed that the deuterium incorporation into the IgG1
backbone with or without fucose was essentially the same, indicating no difference in the conformation of the IgG1 backbone.
The fact that no change in H/DX–MS was seen when comparing the IgG1 with and without fucose indicates that the change in protein
conformation may involve the repositioning of amino-acid side chains, which cannot be detected by H/DX–MS. It is also possible
that segments of the protein's polypeptide backbone are repositioned to an environment that does not directly affect the solvent
exposure of the backbone amide. In H/DX–MS experiments comparing IgG1 with and without galactose, the only difference that
could be detected involved the reduction in deuterium incorporation in the IgG1 Fc heavy chain at residues 240–250 when galactose
was present. The direct involvement of these IgG1 Fc residues with Fc receptors has been shown to be unimportant (27). These
findings coupled with X–ray crystallography data that indicate that the Fc glycans form several contacts with a number of
amino acids on the IgG1 Fc (29) are puzzling.
To better understand these data, H/DX MS binding experiments were performed to investigate the interaction between an Fc receptor,
FcγRIIIa, and two IgG1 variants (27). In this work, an IgG1 with no fucose and complete galactosylation was found to interact
differently with FcγRIIIa relative to the native IgG1, which contained fucose and very little galactose (~15%). While no direct
evidence emerged to explain the molecular details on how galactose and fucose influence effector function, these data suggest
that a change to the IgG1 backbone conformation important for Fc functionality may not have occurred. In the case of fucose,
its presence may sterically interfere with optimal conformational changes required for the IgG1 Fc to bind the Fc receptor
with maximum interactions, leading to significantly weaker binding. Conversely, the presence of galactose may interact with
various amino–acid side chains on the IgG1 Fc to better facilitate a necessary conformational change that maximize IgG1 Fc
interaction with Fc effector proteins. As a result, the absence of fucose or the presence of galactose may promote conformational
flexibility within specific regions of the IgG1, which are favorable for effector function interactions. Such information
should be of interest to those involved in engineering Fc–specific effector activity, as this information provides insights
important for developing detailed models in explaining how carbohydrate–protein structure facilitates IgG1–effector function.
Early glycoprotein research has pointed to a general reduction in dynamic motion and increased thermal stability of a protein
with its coupling to a carbohydrate (34). However, details at the molecular level on how protein dynamics and stability arise
as a result of carbohydrate–protein interaction are not clear. X–ray crystallography is not always possible when a protein
is flexible and/or modified by an oligosaccharide. In addition, the view of the unique conformation captured in a crystal
may not be representative of those found in solution. In the case of NMR, the generally low sensitivity and potentially large
sources of overlapping signals, put restrictions on the size of the protein that can reasonably be studied. In addition, the
need to label proteins (usually using 15 N or 13 C) significantly limits routine sample analysis and throughput, which limits the widespread application of NMR.
H/DX–MS is a sensitive solution–based technique that offers spatial resolution to a few amino-acid residues, typically 5–10.
In fact, recent developments using electron transfer disassociation (ETD) offers an opportunity for H/DX–MS to reach single–residue
resolution with nearly complete sequence coverage (35). Given these attributes and those mentioned earlier in this report,
H/DX–MS is capable of providing useful information to answer many questions in the biopharmaceutical industry related to the
higher order structure of biopharmaceuticals. This capability was briefly demonstrated in this report by the ability to conduct
an array of comparability studies to reveal the impact of different oligosaccharides on the protein structure of an IgG1.
Such capabilities are useful to researchers responsible for designing new protein biopharmaceuticals, as well as those in
biopharmaceutical process development responsible for delivering a stable and consistent commercial drug product. As a result,
H/DX–MS should see a growing interest from a wide spectrum of biopharmaceutical scientists.