Available online at www.sciencedirect.
com
ScienceDirect
Customized protein glycosylation to improve
biopharmaceutical function and targeting
Linde Van Landuyt1,2,3, Chiara Lonigro1,2,3, Leander Meuris1,2 and
Nico Callewaert1,2
For a long time, glycoprotein production has been limited by the
inherent properties of production hosts. Glycosylation of
biopharmaceuticals has been regarded as a necessary evil,
often needed for protein folding or function, but also a source of
heterogeneity, complicating downstream processing and
product characterization. This has strongly determined the
choice of production hosts. Over the last few decades,
numerous glycoengineering efforts have helped solving this
problem. Moreover, insights from fundamental studies have
made it possible to improve therapeutic protein functionality
through careful glycoengineering. Here, we will focus on how
production host and in vitro glycoengineering approaches
allow to design biopharmaceuticals with glycans that impart
improved functionality. An important branch of research
explores how glycosylation can be tuned to improve
pharmacokinetics and reduce glycan heterogeneity of
therapeutics. Furthermore, antibody glycoengineering to
obtain homogeneous, defined glycan structures has been a
major focus. An example of this is the production of Fc glycans
without core fucose, exhibiting tremendously improved
Antibody-Dependent Cell Cytotoxicity (ADCC). In the last part,
glycoforms that allow for improved (subcellular) targeting and
cellular uptake, a field that opens possibilities for enzyme
replacement therapies and vaccine development, will be
highlighted.
Addresses
1
VIB-UGent Center for Medical Biotechnology, Belgium
2
Department of Biochemistry and Microbiology, Ghent University,
Technologiepark 927, B-9052 Gent, Belgium
Corresponding author: Callewaert, Nico (Nico.Callewaert@ugent.vib.be)
3
These authors contributed equally.
Current Opinion in Biotechnology 2019, 60:17–28
This review comes from a themed issue on Pharmaceutical
biotechnology
Edited by Yvonne Y Chen and James A Van Deventer
For a complete overview see the Issue and the Editorial
Available online 13th December 2018
https://doi.org/10.1016/j.copbio.2018.11.017
0958-1669/ã 2018 Elsevier Ltd. All rights reserved.
www.sciencedirect.com
Introduction
Over the first 40 years of its existence, the biopharma-
ceuticals sector has been settling on a limited toolbox of
expression hosts with validated scale-up and regulatory
track record. Broadly, Escherichia coli is used for easy-to-
fold proteins. Mammalian cell lines, in particular Chinese
Hamster Ovary (CHO) and to a lesser extent Human
Embryonic Kidney cells (HEK293) and some other hosts
are used for complex proteins. These proteins often
require human-type glycosylation. Yeasts are used for
proteins that benefit from a eukaryotic secretory system
folding environment, when glycans do not necessarily
need to be ‘human-type’. Insect cell lines have found
particular use for the production of virus-like particle
vaccines.
Protein glycosylation is the set of post-translational
modifications resulting from enzymatic addition of car-
bohydrates to proteins. It is the most widely occurring
modification on proteins that pass the secretory system.
The ER N-glycosylation pathway is conserved in all
eukaryotes currently used as biopharmaceutical protein
production hosts. It is involved in protein folding and
quality control, especially for more complex proteins
that contain disulphide bonds [1]. In the Golgi, a variety
of glycosyltransferases is responsible for cell-type-spe-
cific protein glycosylation. Given the dynamic nature of
the Golgi, resident glycosidases and glycosyltrans-
ferases often cannot modify all substrate molecules
before these are transported away or before competing
transferases modify the molecule. Consequently, Golgi
glycosylation pathways result in a heterogeneous mix-
ture of glycan intermediates on secreted (therapeutic)
proteins (Figure 1). Apart from being species-specific,
glycan profiles are also cell type-specific and influenced
by environmental parameters.
Until about 2000, the protein expression community
considered these cell type-specific (and often undesired)
glycan structures and heterogeneity as a given, coping
with it through appropriate choice of host combined with
process engineering and downstream purification opti-
mization, often at great cost. From the late 90s onwards,
biosynthetic engineering approaches allowed to over-
come the limitations imposed by glycosylation. Over
the past two decades, work has focused on customizing
glycan biosynthetic pathways to enhance therapeutic
protein functionality. This is a maturing research area,
with several glycoengineered products presently on the
Current Opinion in Biotechnology 2019, 60:17–28
18 Pharmaceutical biotechnology

Figure 1

mRNA

Ribosome Cytoplasm
ER lumen cis Golgi
LLO OST
assembly

N-glycans are
important in folding
and quality control

Mammalian Golgi Insect Golgi Plant Golgi Yeast Golgi

N-Acetylglucosamine (GlcNAc) Galactose (Gal) Phosphate

Mannose (Man) N-Acetylneuraminic acid (Neu5Ac)

Fucose (Fuc) Xylose (Xyl)

Current Opinion in Biotechnology

N-glycosylation pathway across the eukaryotic expression hosts.

N-glycosylation starts in the ER with lipid linked oligosaccharide (LLO) assembly, resulting in a precursor N-glycan that is transferred most often
cotranslationally onto an unfolded protein by an oligosaccharyltransferase (OST). After this, N-glycans assist in glycoprotein folding and folding
quality control, resulting in Man8GlcNAc2-containing proteins being exported to the cis Golgi (purple framework). Man8GlcNAc2 serves as a
substrate for mannosyltransferases in the Golgi of yeasts, leading to hypermannosylated structures. In other cells, like mammalian, insect or plant
cells, the protein’s Man8GlcNAc2 are first trimmed to Man5GlcNAc2 and are eventually modified with a first b1,2-N-acetylglucosamine in the cis
Golgi (red framework). This product is the starting point for the synthesis of complex type N-glycans in mammalian cells, paucimannosidic N-
glycans in insect cells or a1,3-fucose and b1,2-xylose-containing (paucimannosidic) N-glycans in plants. In mammalian cells, lysosomal enzymes
are modified with mannose-6-phosphate-containing N-glycans, which are important in transport of such enzymes to the lysosomes.

market. This review will thus focus on how glycans can Glycoengineering for improved
be customized beyond simply mimicking natural gly- pharmacokinetics or reduced heterogeneity
cans, to obtain defined glycans that improve therapeutic Much effort is presently being invested in developing new
function. biopharmaceuticals that allow reduced or more convenient

Current Opinion in Biotechnology 2019, 60:17–28 www.sciencedirect.com

 Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 19
dosing, lowering costs and increasing patient comfort.
Many strategies have focused on improving bioavailability
and therapeutic response through increased serum half-
life. Traditionally, chemical conjugation to polyethylene
glycol (PEGylation) has been used, mainly for small pro-
teins, to increase their hydrodynamic size, thus reducing
renal clearance [2]. Besides this, the use of newly intro-
duced N-glycosylation sites has been exploited. N-glycans
are much larger per mass unit as compared to folded
proteins, making them effective for increasing a
therapeutic’s size. This approach for example lead to the
development of longer circulating forms of EPO [3].
More recently, glycosyltransferases have enabled site-
specific PEG conjugation to glycans (GlycoPEGylation)
[4]. O-glycans of several growth factors (G-CSF, IFN-
a2b, GM-CSF) and coagulation factors (Factor VIIa, VIII
and IX) have been modified with PEGylated N-acetyl-
neuraminic acid (NANA), using a-N-acetylgalactosami-
nide a2,6-sialyltransferase 1 (ST6GALNAC1) and a
PEG-modified CMP-NANA. Methods for (glyco)PEGy-
lation have been reviewed recently [5].
However, even if proteins are sufficiently large to escape
renal clearance, they can be readily cleared by receptors
on macrophages (mannose receptor) and hepatocytes
(asialoglycoprotein receptor) when their glycans are
incompletely sialylated. Extending biopharmaceuticals’
circulatory half-life by sialyltransferase coexpression in
various production hosts emerged soon after sequencing
of the first sialyltransferases [6–8].
Like PEGylation, poly-a2,8-sialylation (PSA) also
increases a therapeutic’s hydrodynamic radius, but with
the added advantage of not being foreign for the human
immune system. In a first approach, PSA of prokaryotic
origin was conjugated to lysine e-aminogroups of thera-
peutics. This was accomplished by PSA oxidation at the
non-reducing end, followed by reductive amination-
based coupling (PolyXEN, Xenetic Biosciences) [9].
Chemically polysialylated EPO and antibody fragments
were well tolerated in patients and had increased half-
lives [10–12]. However, glycoprotein stability, product
heterogeneity and process complexity remained a prob-
lem. Apart from chemical PSA conjugation, in vitro enzy-
matic conjugation has been demonstrated using recom-
binant Campylobacter jejuni Cst-II a2,8-sialyltransferase.
This enzyme allowed for disialylation of a-1-antitrypsin
(A1AT) N-glycans, that could then be completely con-
verted to PSA using Neisseria meningitidis a2,8-polysialyl-
transferase [13]. The main shortcomings of this approach
are the expensive reagents (enzymes and CMP-NANA)
and tightly controlled reaction conditions required to
prevent rehydrolysis of sialyl-units.
The first in vivo polysialylation strategy (Figure 2a)
introduced 35 NANA residues on a ScFv fusion,
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resulting in a threefold increased hydrodynamic volume
and 30-fold increased half-life [14]. This approach
required fusing the ScFv to an NCAM Ig5 domain
(which contains a polysialylation site) and a fibronectin
1 polysialyltransferase docking domain. Coexpression of
the fusion construct and the human poly-a2,8-sialyl-
transferase in HEK293S cells resulted in polysialylation
of the Ig5 domain.
Nicotiana benthamiana has also been extensively engi-
neered to obtain PSA in planta (Figure 2A). This required
introducing the biosynthesis pathway for sialylated com-
plex type N-glycans into a b1,2-xylosyl-fucosyltransferase
and core a1,3-fucosyltransferase knockout plant
(DXTFT) first. Transient expression of human sialyl-
transferases ST8SiaII and ST8SiaIV then allowed for in
vivo synthesis of PSA with more than 40 units, without
impacting protein yield and plant growth [15–17,18].
However, in vivo polysialylation in a eukaryotic host
always requires fusion of the therapeutic protein to one
of the naturally occurring polysialylated protein domains
(e.g. NCAM, Synaptic cell adhesion molecule, neuropilin
2) and only certain cell types can accomplish it. An
engineered E. coli strain (Figure 2b) allowed circumvent-
ing this problem: recombinant GFP with up to 50% of
GlcGalSia2–10 glycans and designed ankyrin repeat pro-
teins with up to 28-fold increased circulation times have
been generated [19]. Although promising, the strains
suffered from reduced bacterial growth and low protein
yield. Further strain engineering may solve this.
Several conceptually new in vivo approaches geared
towards reducing N-glycan and O-glycan (micro)hetero-
geneity have also been introduced during the last decade.
GlycoDelete technology offers a strategy to reduce N-
glycan heterogeneity. Starting from GnTI / HEK293 or
Arabidopsis thaliana, which produce high-mannose type
glycans, a Golgi-targeted ENGase, EndoT, allows for in
vivo removal of all N-glycans. The N-acetylglucosamine
(GlcNAc) stumps are then capped with galactose and
NANA, resulting in simple N-glycans. [20,21]. Genome
engineering of the glycosylation pathways can also result
in more defined glycosylation profiles [22].
Much more effort has gone into engineering N-glycans
than any other type of glycan. However, a significant
engineering effort towards reducing heterogeneity of
mucin-type glycosylation, the most common type of O-
glycosylation in mammals, has been made as well. In
human cells, the first N-acetylgalactosamine (GalNAc)
residue is transferred from UDP-GalNAc to Ser or Thr
residues by one of 20 different GalNAc transferases.
Further O-glycan synthesis usually starts with the action
of core 1 b1,3-galactosyltransferase (C1GalTI), which is
dependent on a specific chaperone, COSMC. A hugely
simplified O-glycosylation profile can be achieved by
knocking out COSMC (‘SimpleCell’ technology).
Current Opinion in Biotechnology 2019, 60:17–28
20 Pharmaceutical biotechnology

Figure 2

hCMAS hNANS hGNE

CST
n = ± 40

α-2,8
CMP α-2,8
α-2,6 α-2,6

hGalT hST6
N N N

hST8SiaII/IV

n = ±35

ScFv Ig5 FN1 ScFv Ig5 FN1

(a) Golgi of engineered ∆XTFT N. benthamiana and HEK293S cells

n = ±10
α-2,8
n=±3
α-2,8 α-2,8
α-2,3 α-2,3
β-1,4 β-1,4 β-1,4
ApNGT β1 LgtB β1
Cstll β1 PolyST β1
N N N N N

(b) Cytoplasm E. coli

Current Opinion in Biotechnology

Engineering strategies to allow for polysialylation of therapeutic proteins.

(a) In HEK293S cells, a polysialyltransferase and chain-initiating a2,8-sialyltransferase (PST/ST8SiaIV) were overexpressed to polysialylate the
immunoglobulin 5 (Ig5) domain of neural cell adhesion molecule (NCAM), which is fused to the protein of interest (ScFv) and to a docking domain
for the PST/ST8SiaIV (FN1). Approximately 35 NANA residues could be linked to the complex-type glycosylated (R) Ig5. In xylosyltransferase and
fucosyltransferase knockout (DXTFT) N. benthamiana cells, human UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (hGNE),
human NANA-phosphate-synthase (hNANS), human CMP-NANA synthetase (hCMAS), CMP-NANA transporter (CST), human b1,4-
galactosyltransfease (hGalT) and human a2,6-sialyltransferase (hST6) were stably overexpressed. Transient coexpression of human a2,8-
sialyltransferase (ST8SiaII/IV) in this background allowed to produce recombinant proteins containing PSA structures with a polymerization degree
of 40. (b) In E. coli, proteins could be glucosylated by the Actinobacillus pleuropneumonia cytoplasmic N-glycosyltransferase (ApNGT) and further
modified with Gal and NANA with a b1,4-galactosyltransferase from N. meningitides (LgtB) and an a2,3/a2,8-oligosialyltransferase from
Campylobacter jejuni (CstII). Recombinant proteins herein expressed had a PSA polymerization degree higher than 10.

Originally developed to sequence-map the human and
CHO O-GalNAc glycoproteome [23], it could prove
useful in the future for homogenizing O-glycans on ther-
apeutics. However, the resulting single GalNAc residues
may have immunogenic potential, as such O-glycan trun-
cations on proteins like MUC1 generates neo-epitopes.
Alternatively, as each cell type only expresses a limited
number of protein-O-GalNAc transferases, CRISPR-Cas
knockout is likely feasible.
In vitro glycoengineering approaches have also made it
possible to obtain highly homogeneous glycosylation, for
example to study IgG–FcgR interactions. For such pur-
poses, several companies offer a portfolio of well

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 Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 21
characterized enzymes and nucleotide sugars (e.g.:
Roche, CustomBiotech) [24]. Clearly, in vitro glycan
(re-)construction has become economically feasible
for research purposes but we have yet to see its
industrial application in biopharmaceutical manufactur-
ing, in the light of its relatively high cost and process
complexity [25].
Antibody glycoengineering
Glycoengineering for altered effector functionality:
afucosylated and aglycosylated IgG
N-glycans linked to Asn297 of the Crystallizable Frag-
ment (Fc) of IgG monoclonal antibodies (mAbs) strongly
influence binding to Fcg receptors (FcgR) on immune
cells and complement proteins, thus modulating their
biological activity. Decrypting the ‘code’ of these inter-
actions to some extent [26], has enabled effector func-
tion-adapted glycoengineering. Antibody-Dependent
Cellular Cytotoxicity (ADCC)-based therapies have been
improved by the discovery that antibodies with Fc gly-
cans lacking a core a1,6-fucose (Fuc) (‘afucosylated anti-
bodies’) exhibit an increased effector function [27]. They
bind much stronger to FcgRIIIa on Natural Killer cells
(NK), triggering activation. Afucosylated mAbs outcom-
pete fucosylated human IgGs in in vitro FcgRIIIa binding
assays and also seem to enhance Antibody-Dependent
Cellular Phagocytosis (ADCP) in vivo. These enhanced
functionalities are important for treating tumors [28,29]
and possibly also to clear virally infected cells [30].
In CHO cells, afucosylated antibody production technol-
ogy interferes with the GDP-Fuc and glycan biosynthesis
pathways (Figure 3). The first afucosyl antibody (Moga-
lizumab-kpkc) for treatment of relapsed or refractory
CCR4+ adult T-cell leukemia/lymphoma was approved
in Japan in 2012. It is produced in a CHO core-a1,6-
fucosyltransferase (FUT8) knock-out cell line (Kyowa
Hakko Kirin, POTELLIGENT1, Figure 3b-4). Benra-
lizumab (also POTELLIGENT1), was approved by
FDA in 2017. Interfering with GDP-Fuc biosynthesis
is an alternative strategy used in GlymaxX1 technology
(ProBioGen, Figure 3b-3). In yet another approach,
Roche-GlycArt (GlycoMAb1) overexpressed N-acetyl-
glucosaminyltransferase III (GnTIII) to produce glycans
carrying a bisecting GlcNAc, inhibiting core fucosylation.
Obinutuzumab, an anti-CD20 mAb produced with Gly-
cArt technology, shows enhanced FcgRIIIa binding,
ADCC and antitumor activity. It also binds a different
epitope on CD20 than its comparator antibody (Ritux-
imab), making it less straightforward to assign the
enhanced efficacy solely to glycosylation engineering
[31]. Further optimization encompassed co-overexpres-
sing the Golgi a-mannosidase together with GnTIII,
resulting in complex-type afucosylated bisected glycans
(Figure 3b-7) [32]. Many other afucosylated antibodies
are now in clinical trials [33].
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Since FUT8 has limited activity on Man5–9 N-glycans,
these are also largely afucosyl glycans. The a-D-manno-
sidase inhibitors kifunensine and deoxymannojirimycin
have therefore been used to block the N-glycosylation
pathway at the Man8 or Man9 stage, or GnTI / to block
at the Man5 stage, all decreasing core fucosylation in
CHO cells (Figure 3b-8) [34–36]. Oligomannose glyco-
proteins are expected to be rapidly cleared through the
mannose receptors (MR), which could possibly make
them less useful as biopharmaceuticals. However, results
in this field are controversial [35,37].
CHO cells are currently the ‘workhorse’ for mAb produc-
tion. Nevertheless, yeasts and plants are gradually being
introduced as alternatives. Yeasts naturally produce afu-
cosyl glycans and glycoengineered Komagataella phaffii
(Pichia pastoris) producing afucosylated humanized com-
plex or oligomannose N-glycans has been developed for
antibody production (Merck/GlycoFi and GlycoSwitch)
[38,39]. However, completely inactivating the endoge-
nous N-glycosylation and O-glycosylation pathways with-
out affecting cell growth remains challenging, because
these glycans are a functionally important part of the yeast
cell wall. While ‘humanized’ strains produce mostly
human-type N-glycans, a certain fraction of yeast struc-
tures remains. This is often overlooked as it only becomes
clear upon thorough analysis. Such problems can be
partially overcome through stacking of multiple glycosyl-
transferase knockouts, but this is highly complex [40].
Research is ongoing to overcome these remaining chal-
lenges and it is likely that a feasible, scalable solution will
soon be available (Callewaert lab, unpublished results).
Recently reported 2nd generation glycan ‘humanization’
in insect cells, is also highly promising [41].
As mentioned before, also plants have been glycoengi-
neered (i.e. DXTFT). As an example, Lemna minor
DXTFT produces antibodies with GlcNAc2Man3Glc-
NAc2 glycans, which show enhanced ADCC as compared
to fucosylated CHO produced antibody [42].
Completely removing glycans is a radically different
approach to circumvent elaborate glycoengineering strat-
egies. Apart from homogeneity, simplified downstream
processing and the possibility to use microbial
manufacturing make aglycosylated antibodies attractive
[43]. This is particularly true when no Fc effector func-
tionality is required and when the Fc mostly provides a
dimerization and pharmacokinetics-enhancing platform
for the antigen-binding domains. Indeed, importantly,
FcRn binding (which mediates IgG’s very long circula-
tory half-life) is Fc glycosylation-independent. Initial
concerns over stability and immunogenicity have largely
subsided after several aglycosylated antibodies have
entered the clinic without showing problems related to
stability or immunogenicity so far [44]. The furthest
advanced of these non-glycosylated antibodies, further
Current Opinion in Biotechnology 2019, 60:17–28
22 Pharmaceutical biotechnology

Figure 3

(a)
CHO cell glycosylation
GDP- GMD GDP
FX
SLC35C1

GDP

GDP GDP

ManI GnTI FUT8 Manll GnTII GaIT R= RR

Golgi
Cytoplasm

(b)

Glyco-engineering strategies GDP-

3 RMD

GKDM
GDP 1 GMD GDP- 5-alkynyl-fucose
2 FX 6
5 2-fluorofucose
SLC35C1

8 DMJ
Kifunensine GDP-
7 R=
GDP GDP
ManI 9 GnTI 4 FUT8 Manll GnTII GaIT 8-9 R=

RR
GnTIII+ 1-2-3 R= ADCC
7 4-5-6
ManII FcyIIIa
Glycans resulting from different stratagies NK ceII

Golgi

Cytoplasm

Current Opinion in Biotechnology

CHO cell line glycoengineering strategies for the production of antibodies with customized glycans.
The Golgi glycosylation pathway in CHO cells and a typical biantennary N-glycan as it occurs on the Fc domain of monoclonal antibodies
produced in WT CHO cells (a). This pathway has been extensively engineered (b) to inhibit or reduce antibody fucosylation in order to enhance
FcgRIIIA binding and ADCC. The different glyco-engineering strategies are numbered from 1 to 9. Enzyme knock-out is depicted in red and (over)
expression in green. The glycan structures resulting from these strategies are shown at the right side of panel b with numbering matching to the
engineering strategies. (1) GDP-mannose 5,6-dehydratase (GMD) and (2) GDP-keto-6-deoxymannose 3,5 epimerase-4-reductase (FX) are key
enzymes in de novo GDP-fucose biosynthesis. Their inactivation blocks all cellular fucosylation [74,75]. (3) The bacterial GDP-4-keto-6-
deoxymannose reductase (RMD) can be expressed in CHO cells to convert GDP-4-keto-6-deoxymannose (GKDM), an intermediate of the GDP-
fucose synthesis pathway, to GDP-rhamnose and block GDP-fucose biosynthesis (GlymaxX, ProBioGen) [76]. Knock-out of the fucosyltransferase
FUT8 (4) is at the basis of the Potelligent technology (BioWa, Lonza) [77]. Knock-out of the GDP-fucose transporter SLC35C1 (5), which transports
GDP-fucose from the cytoplasm to the Golgi, has also been reported to eliminate Fc-fucosylation [78]. Recently, metabolic inhibition strategies
with small molecules have been developed, such as 2-fluorofucose and 5-alkynyl-Fuc (SEA technology, 6) [79]. Overexpression of GnTIII and Golgi
a-mannosidase II (7) results in bisecting GlcNAc-modified, non-fucosylated glycans (GlycArt, Roche), which show enhanced ADCC and antitumor
activity. Oligomannose Fc-linked glycans also lack the core fucose and mediate enhanced ADCC. Therefore, the a-D-mannosidase inhibitors (8)
kifunensine and deoxymannojirimycin (DMJ) have been used to block the N-glycosylation pathway at the Man8 or Man9 oligomannose stage and
decrease the fucosylation in CHO cells [34,35]. GnTI / (9) CHO cells express Man5GlcNAc2-modified IgG1 with a low degree of core fucose,
which also shows higher anti-tumour activity [36].

protein-engineered for stability and to suppress FcgR generate variants with improved FcgRIIIa binding. How-
activation, is likely ALD403/Eptinezumab (Alder Bio- ever, FcgRIIIa binding affinity could only be restored to
pharmaceuticals, Bothell, WA), which is produced in P. the level of fucosylated antibodies [45]. Despite this, the
pastoris. Furthermore, yeast surface display of mutant ADCC activity turned out to be lower than for the glyco-
libraries has generated aglycosylated Fc’s that are able to sylated antibody Herceptin. The authors speculated that
engage different FcgRs both in vitro and in vivo. Directed this is possibly due to enhanced affinity for the inhibitory
evolution of the Fc part of anti-HER2 antibodies allowed to Fcg receptor, FcgRIIb [46].

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 Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 23

Galactosylation and sialylation of mAb Fc N-glycans
Contrary to afucosyl antibodies, the role of Fc galactosy-
lation and sialylation remains more controversial, partially
due to overall smaller effects on antibody functionality. In
vitro results suggest that high Fc N-glycan galactosylation
levels may enhance some effector functions [24]. On the
other hand, decreased antibody galactosylation is associ-
ated with pathogenic autoantibodies in immune diseases
such as rheumatoid arthritis [47]. It is unclear whether this
is a consequence of disease rather than a functionally
important alteration. Some evidence supports an anti-
inflammatory effect of increased presence of terminal
a-2,6-NANA on fucosylated IgGs when these are admin-
istrated to treat autoimmune and inflammatory diseases.
The mechanisms are still under debate but reduced
FcgRIIIA and C1q binding and increased expression of
the inhibitory FcgRIIB on effector macrophages seem to
be involved [48].
Since CHO cells lack a functional ST6GAL1, they do not
cap their glycans with a-2,6-NANA. Co-expression of
B4GalT1 and ST6GAL1 resulted in increased Trastuzu-
mab galactosylation and sialylation [49]. Alternatively, in
vitro chemical and chemoenzymatic strategies can be
used (Figure 4). Recently, Roche developed a workflow
relying on B4GalT1 and ST6Gal1 for the generation of
defined Fc glycans (Figure 4a) [11]. In an entirely differ-
ent approach, sialylation of the mAbs Rituximab and
Trastuzumab was increased by removing the Fc glycan
with WT EndoS2 and transglycosylating a defined sialy-
lated biantennary glycan onto the remaining protein-
linked (core fucosylated) GlcNAc with mutant EndoS2

Figure 4

(a)

R=
B4GALT1 ST6GAL1

UDP UDP CMP CMP

(b)

R= EndoS2 EndoS D184M

(c)

H NH
N O
O
N
EndoS2 GalT(Y289L) click chemistry
R= N3 N H
UDP- N3 UDP

H H
O
H
O N

Current Opinion in Biotechnology

Chemoenzymatic glycoengineering for the production of antibodies with customized glycans.

(a) B4GalT1 and ST6GAL1 (Roche) can be used on heterogeneous ‘bulk’ antibody glycans for in vitro galactosylation and sialylation [24,25]. (b)
Heterogeneous Fc N-glycans are first trimmed by the wild type EndoS2. The remaining protein-linked core-fucosylated GlcNAc is then used as
acceptor for the transglycosylation of the desired N-glycans from the oxazoline substrate, catalysed by EndoS D184M [50]. (c) Similarly, enzymatic
trimming with EndoS2 is used to cleave the Fc-glycans in the chitobiose core, leaving a single GlcNAc, which is further modified by the mutant
galactosyltransferase (GalT Y289L) and UDP-GalNAz. The GalNaz moiety can then be used to couple small molecule payload (red star) with an
azide cyclooctyne click chemistry reaction. This is used for antibody labelling or preparing antibody drug-conjugates [56].

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24 Pharmaceutical biotechnology
D184M (Figure 4b) [50]. Transglycosylation to generate
sialylated glycans has also been performed with WT
EndoS, starting with P. pastoris GlycoSwitch Super-
Man5-produced Trastuzumab (Biogrammatics) [51].
An outstanding issue in these remodelling approaches
is where to source bulk quantities of the to-be-transgly-
cosylated N-glycan structures. Chemical synthesis is now
feasible for relatively limited quantities for some of the
desired structures [52,53,54], and a number of natural
glycoprotein sources are available (e.g. from egg yolk)
[55]. However, synthesis or purification from a natural
source in sufficient quantities for synthesizing kilograms
of IgG (the scale required for most real-life therapeutic
applications) affordably and at cGMP-compliant quality
is likely to remain a major challenge. Significant R&D
attention is required in order to make it competitive with
in cellulo glyco-optimization, which has the very important
advantage of only requiring the manufacturing unit pro-
cesses commonly used in the biopharmaceutical industry.
In another application of Fc glycoengineering, EndoS-
type enzymes generate trimmed Fc glycans, which can
then be specifically conjugated with chemically modified
sugars to enable production of Antibody Drug Conjugates
and antibody labelling via click chemistry (GlycoCon-
nect, Synaffix, Figure 4c) [56].
Glycoengineering for (subcellular) targeting
Glycans can target therapeutic proteins to their desired site
of action. The most prominent and extensively studied
targets for deliverable glycoengineered proteins are the P-
type lectins. These Man-6-phosphate (M6P)-binding
receptors (MPR) are present in the Golgi, endosomes,
lysosomes and on the plasma membrane of many cell types.
Increasing the M6P content in enzymes for replacement
therapy (ERT) in various lysosomal storage diseases
enhances cellular uptake and lysosomal delivery. Over-
expression of an engineered GlcNAc phosphotransferase in
CHO cells led to 2.1–4.9-fold increased M6P levels on acid
a-mannosidase and acid a-glucosidase (hGAA) and better
MPR binding [57]. More recently, chemical conjugation of
natural and synthetic M6P-containing glycans to enzymes
for ERT are being investigated. After the first generation of
chemically conjugated M6P carbohydrates [58], isosteric
analogues of M6P functionalized at the anomeric position
(AMFAs), have been coupled to hGAA produced in Sf9
insect [59] and CHO cells [60]. In our laboratory, boosting
the native yeast pathways that synthesize Man-Pi-6-Man
glycotopes resulted in production of human lysosomal
enzymes with >85% of phosphorylated glycans. Subse-
quently, removing the phosphate-capping Man residues
using a newly discovered a-mannosidase, generated M6P-
modified enzymes with strongly enhanced uptake in
patient cells [61].
In a similar approach as described for antibodies, a wide
range of (mutant) ENGases (reviewed in Ref. [62]) is
Current Opinion in Biotechnology 2019, 60:17–28
used to generate single GlcNAc N-glycans and subse-
quently transglycosylate M6P glycan oxazolines onto
them. Most noteworthy, (mutant) EndoA is capable of
transglycosylating oxazolines with M6P moieties in vary-
ing numbers and positions onto GlcNAc acceptor sites
[63,64]. However, production scalability remains chal-
lenging. Moreover, efficacy of the resulting drug candi-
dates has not yet been tested in vivo.
In Gaucher disease, mainly macrophages are affected. In
these cells, recombinant glucocerebrosidase for ERT is
targeted to the endocytotic macrophage receptor (MR) by
virtue of various oligomannose N-glycans. In Cerezyme
(Genzyme), presently the most used clinical product,
exoglycosidases are used to trim down native CHO N-
glycans to Man3GlcNAc2. Alternative approaches include
the use of mannosidase inhibitors or GnTI / CHO and
HEK cells. More recently, an a-1,2-mannosidase triple
knockout HEK293S cell line allowed to produce Man8/9-
GlcNAc2-modified enzymes [65]. Plants produce pauci-
mannosidic N-glycans by nature. Building on this, gluco-
cerebrosidase from transgenic carrot cells (Taliglucerase
a, Elelyso) is now available as an alternative to Cerezyme
for the treatment of Gaucher’s. Furthermore, a complex
glycan deficient A. thaliana mutant [66] and Physcomitrella
patens GnTI / mutant [67] were created to produce
Man5GlcNAc2 modified lysosomal enzymes.
Mannose binding receptors on dendritic cells, macro-
phages and B cells play a key role in pathogen recogni-
tion. Lectins with other glycan specificities (sialic acid-
binding Siglecs and Gal-binding lectins) are expressed on
specific subpopulations of immune regulatory cells.
Therefore, glycosylation of protein antigens to influence
immune responses has been undertaken [68]. Virus-like
particles containing hepatitis B virus small envelope
protein (HBsAgS VLPs), engineered to contain extra
glycans, showed an accelerated B-cell immune response
when compared to non-glycosylated HBsAgS N146Q
mutant and WT glycosylated VLPs. Both the hypergly-
cosylated and WT variants carried a similar type of core-
fucosylated bisecting glycan [69]. In another study, b1,2-
xylosyltransferase from Nicotiana tabacum was co-
expressed with the extracellular part of the RSV-F surface
protein in CHO cells. Xylosylated RSV-F resulted in
improved vaccination efficacy in a human artificial lymph
node model [70]. Plant glycan modifications can be aller-
genic in mammals [71], so future will tell whether such a
strategy of enhancing immunity will be sufficiently safe to
be deployed at large. Introduction of insect-type glycans
on the Hepatitis C virus E2 protein by production in
Drosophila S2 cells induced a high titre of broadly neu-
tralizing antibodies that were able to protect humanized
mice and non-human primates against HCV challenge
[72,73]. The latter two examples indicate that the use of
non-human glycans to increase immunogenicity repre-
sents an interesting strategy in vaccine design, although
www.sciencedirect.com
 Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 25
clinical development is still needed to assess safety, as
always.
Conclusion
Early in vivo glycoengineering focused on the production of
more homogeneous glycoproteins by pushing the biosyn-
thetic pathways in mammalian cells towards the desired
type of glycan. Alternative approaches aimed at recon-
structing the human pathways into other eukaryotic cells
like yeasts, insect cells and plants. Such strategies have
enabled scalable production of biopharmaceuticals with
more defined glycans and improved functionalities. How-
ever, due to the complexity and intricacies of the lengthy
biosynthetic pathways, often some unwanted structures
remain. Consequently, producing completely homoge-
neous glycoproteins for now remains an issue, although
strong progress has been made through the use of genome
engineering tools [22]. Currently, a promising trend in in
vivo glycoengineering is to minimize N-glycan and O-
glycan complexity, aiming to get the most useful yet as
small as possible structure, or to make them completely
obsolete through protein engineering, whenever feasible. A
related trend is the integrated engineering of in vivo gly-
cosylation pathways in the production host, coupled with in
vitro enzymatic remodelling of glycans in downstream
processing. Remodelling with hydrolytic enzymes (remov-
ing undesired glyco-structures) is simple and can be per-
formed at scale cost-effectively. On the other hand, remo-
delling using transferases (adding desired glyco-structures)
is more complex due to the requirement for activated
glycosylation donor substrates. The costs involved have
so far not been warranted by functionality gains versus
proteins produced straight from (glycoengineered or ‘wild
type’) cells with only partially ‘correct’ glycans. To our
knowledge, no biopharmaceuticals produced using glyco-
syltransferase ‘polishing’ are as yet available, though some
are in (pre)clinical development. The cost of sugar nucleo-
tides is decreasing, so this may be a matter of time.
In conclusion, the field of biopharmaceutical glycoengi-
neering is maturing. With the advent of glycoengineered
biopharmaceuticals on the market in 2012, glycoengi-
neering is now fully in the focus of the biopharma sector.
A new generation of thoroughly developed technologies
will likely give rise to start-up biotechnology enterprises
in the next few years to build on the great opportunities to
design new and improved biopharmaceutical agents.
Glycoengineered production hosts will clearly keep play-
ing an increasingly important role in biopharmaceutical
production for years to come.
Conflict of interest statement
Nothing declared.
Acknowledgements
L. Van Landuyt and C. Lonigro hold a strategic basic research fellowship of
the Flanders Fund for Scientific Research (FWO-Vlaanderen) [application
www.sciencedirect.com
number: 1S54817N and 1S11418N] and Dr. L. Meuris is a doctor assistant
of Ghent University. This work was in part funded by an ERC consolidator
grant ‘GlycoTarget’ [application number: 616966], by Ghent University
projects [application number: GOA01G01412 and BOF17-GOA-018] and by
FWO Vlaanderen [application number: G041417N]. The Callewaert lab is
partly funded by VIB. We also acknowledge C. De Wachter for proofreading
this manuscript.
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