Glycoengineered Antibodies - Towards The Next-Generation of Immunotherapeutics PDF

Glycoengineered Antibodies: Towards the Next-Generation of
Immunotherapeutics
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Renato Mastrangeli1, Wolf Palinsky2, Horst Bierau1
Author affiliations:
1
Biotech Development Programme, CMC Science & Intelligence, Merck Serono SpA, an affiliate of Merck
KgaA, Darmstadt, Germany. Via Luigi Einaudi, 11. 00012 Guidonia Montecelio (Roma) – Italy
2
Biotech Development Programme, Merck Biopharma, an affiliate of Merck KgaA, Darmstadt, Germany.
Zone Industrielle de l’Ouriettaz, Aubonne 1170, Switzerland
Corresponding author: Horst Bierau, Biotech Development Programme, CMC Science & Intelligence, Merck
Serono SpA, Via Luigi Einaudi, 11. 00012 Guidonia Montecelio (Roma), Italy. E-mail
horst.bierau@merckgroup.com, Phone number: +39 0774 350834.
Keywords: monoclonal antibody, effector functions, homogeneous glycosylation, glycoengineering,
biobetter
© The Author 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:
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1
Abstract
Monoclonal antibodies (mAbs) are currently the largest and fastest growing class of biopharmaceuticals,
and they address unmet medical needs e.g. in oncology and in auto-immune diseases. Their clinical efficacy

and safety is significantly affected by the structure and composition of their glycosylation profile which is
commonly heterogeneous, heavily dependent on the manufacturing process, and thus susceptible to
variations in the cell culture conditions. Glycosylation is therefore considered a critical quality attribute
(CQA) for mAbs. Commonly, in currently marketed therapeutic mAbs, the glycosylation profile is
suboptimal in terms of biological properties such as antibody-dependent cell-mediated cytotoxicity (ADCC)
or may give rise to safety concerns due to the presence of non-human glycans.
This article will review recent innovative developments in chemo-enzymatic glycoengineering, which allow
generating mAbs carrying single, well-defined, uniform Fc glycoforms, which confers the desired biological
properties for the target application. This approach offers significant benefits such as enhanced Fc effector
functions, improved safety profiles, higher batch-to-batch consistency, decreased risks related to
immunogenicity and manufacturing process changes, and the possibility to manufacture mAbs, in an
economical manner, in non-mammalian expression systems. Overall, this approach could facilitate and
reduce mAb manufacturing costs which in turn would translate into tangible benefits for both patients and
manufacturers. The first glycoengineered mAbs are about to enter clinical trials and it is expected that,
once glycoengineering reagents are available at affordable costs, and in-line with regulatory requirements,
that targeted remodelling of antibody Fc glycosylation will become an integral part in manufacturing the
next-generation of immunotherapeutics.
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Introduction
Recombinant monoclonal antibodies (mAbs) are one of the most important class of biotherapeutics. Rising
incidences of cancer and other chronic diseases are engendering a high demand for biologics, which is

serving as the key contributing factor for the growth of this industry. Since 1984, more than 60 mAbs and
fusion molecules have been approved by the US Food and Drug Administration (FDA) for over 30 targets in
cancer, autoimmune, and cardiovascular diseases (Shepard et al. 2017). In a recent market research study,
the global monoclonal antibody market was valued at USD 85.4 billion in 2015 and is expected to reach a
value of USD 138.6 billion by 2024 (http://www.grandviewresearch.com/pressrelease/global-monoclonal-
antibodies-market).
Historically, monoclonal antibody development has been driven by efforts to provide patients with safer
and more efficacious products through gradual improvements over time in terms of both reduced
immunogenicity and increased potency. The first generation mAbs were 100% murine and immunogenic in
human, and therefore not suited for chronic therapy. Successively, chimeric (approximately 35% murine),
humanized (approximately 7% murine) and fully human (0% murine) antibodies were developed over time
in order to decrease mAb immunogenicity. However, despite such efforts, even fully human antibodies
such as adalimumab, the best-selling antibody drug on the market, were still reported to be immunogenic
(Matucci et al. 2016). Moreover, a recent review on the evidence of benefits on the overall survival and the
quality of life of approved cancer drugs, including a number of mAbs, concluded that most of the drugs that
entered the market between 2009 and 2013 showed only marginal gains in terms of overall survival and
quality of life (Davis et al. 2017). A similar study on cancer treatments, approved by the FDA, concluded
that only a small proportion of these drugs unequivocally show benefits on the survival and the quality of
life (Kim and Prasad 2016). These studies therefore underscore the necessity to introduce new oncology
mAbs with maximized efficacy and safety profile which in turn translate into clear benefits such as the
survival and/or the quality of life of patients.
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Most of the mAb therapeutics are glycosylated, although non-glycosylated forms exist, e.g. when effector
functions are not desired. The present paper focusses on glycosylated mAbs.
IgG Fc glycosylation – structural and functional properties

Monoclonal antibodies are mainly based on the prototypic antibody structure of IgG1, the most abundant
serum subclass of -immunoglobulins. An IgG1 antibody is composed of two identical heavy (H) chains
(50 kDa) and two identical κ or λ light (L) chains (25 kDa), linked together by inter-molecular disulphide
bonds. Each heavy chain consists of an N-terminal variable domain (VH) followed by three constant
domains (CH1, CH2, CH3). The so-called hinge region, linking the CH1 and the CH2 domains, contains the
cysteines residues making up the four intermolecular disulphide bonds (two between the two heavy chains
H=H and two between the heavy chain and light chain 2H-L), and confers the typical Y-shaped quaternary
structure of IgG1s (Figure 1).
Each of the two CH2 domains of the heavy chains contains a conserved IgG-Fc N-glycan site (position Asn297
according to the EU numbering). The IgG-Fc overall shape resembles that of a horseshoe. Most of the
internal Fc space between the two CH2 domains is filled by the two oligosaccharide chains (Figure 1) which
engage in glycan-glycan and glycan-protein interactions (Figure 2).
The Fc-glycans dictate stability (Bowden et al. 2012), conformation and spacing between the two CH2 low
hinge regions. Consequently, they are implicated in the binding properties with complement factor (C1q)
and Fc gamma receptors (FcRs, including the activating receptors FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and
FcγRIIIb, and the inhibitory receptor FcγRIIb) (Idusogie et al. 2000, Krapp et al. 2003, Schneider and
Zacharias 2012). Furthermore, they impact on the opening/closure of the CH2-CH3 pivot region responsible
for the binding to the neonatal Fc receptor (FcRn) and the type II receptors (including DC-SIGN and CD23)
(Pincetic et al. 2014), and the susceptibility to enzymatic degradation (Falck et al. 2015, Zheng et al. 2014).
By masking hydrophobic residues, they also decrease aggregation propensity which was found to be higher
in non-glycosylated mAbs (Kayser et al. 2011). The manner in which the Fc glycans are embedded in the
protein structure gives rise to structural fluctuations which are more dynamic than previously assumed
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(Meier and Duus 2011); they allow various Fc conformational states (Frank et al. 2014) with the potential to
expose or shield large parts of the protein surface, and making a buried glycan accessible to processing
enzymes or binding proteins (Meier and Duus 2011). The Fc glycans are therefore crucial for the Fc

structure and the resulting multiple Fc effector functions (Jefferis 2012). They can have a direct influence,
when involved in binding (e.g. with FcRIIIa), or indirect influence, when involved in allosteric modulation of
the FcγRs/C1q/FcRn/type II receptor binding sites. The innate effector cells, namely NK, monocytes,
macrophages, dendritic cells, mast cells, neutrophils, eosinophils and B cells express one or several FcγRs as
well as complement factors and immune-lectin-like receptors (Euler and Alter 2015). The dynamic
modulation of the two CH2 low hinge regions, induced by the Fc glycans, plays a key role in the antibody
interaction with the Fc receptors and the C1q, allowing a fine-tuned communication with the immune
system cells, e.g. providing instructions to the innate immune system on how (by antibody-dependent cell-
mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-
dependent cytotoxicity (CDC) it should destroy the cells to which that antibody is bound. These effector
functions play a pivotal role in the mAbs used in oncology and contribute to the clearance of cancer cells.
For example, the abovementioned effector functions are all part of the mode of action (MoA) of rituximab,
the first mAb approved (1997) for cancer treatments (Jaglowski et al. 2010) .
The glycan residues on the 1-6 and 1-3 arm of each Fc-N-glycan are characterized by differential
interactions and mobility (Fortunato and Colina 2014, Krapp et al. 2003). The 1-6 arm is more rigid, while
the 1-3 arm is more flexible (Figure 2).
The galactose on the 1-3 arm has a high mobility and is more accessible to human alpha2-6 sialyltransferase
(ST6), leading to an innate 1-3 arm preferential sialylation also on released glycans (Barb, Brady, and
Prestegard 2009). On the contrary, galactose on the 1-6 arm is involved in four hydrogen bonds which
decrease the mobility and further stabilize the Fc structure (Raju 2008). It has been reported that
galactosylated mAbs have different properties in terms of Fab orientation (distribution of directional and
rotational vectors) as well as distribution of radii of gyration in comparison with agalactosylated mAbs
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(Fortunato and Colina 2014). This may further translate in a differential impact on the functional properties
of galactosylated in comparison with agalactosylated antibodies.
The Fc oligosaccharides and their 1-3 and 1-6 arm combination represent a special case among

glycoproteins with almost each of the oligosaccharide components directly or indirectly eliciting structural
or functional effects. The binding stoichiometry between mAbs and FcRs or C1q is 1:1, while between
mAbs and FcRn or type II receptors it is 1:2 (Pincetic et al. 2014).
Functional impact of sialylation and galactosylation. An intriguing aspect of Fc-glycans is the differential
impact of sialic acid linkage 2,6 versus 2,3 on the anti-inflammatory properties of antibodies(Anthony,
Nimmerjahn, et al. 2008, Anthony, Wermeling, et al. 2008, Sondermann et al. 2013). Human endogenous
Fc-glycans contain exclusively 2,6-linked sialic acid residues. Fc-2,6 sialylated glycans allow the Fc domain to
interconvert between an open and a closed conformation, thus increasing its conformational plasticity and
introducing novel functional properties, e.g. the capacity of the closed conformation to bind to the type II
receptors DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin) and
CD23 (Pincetic et al. 2014, Sondermann et al. 2013). In this manner, the 2,6 sialic acid confers anti-
inflammatory properties to IgG (Anthony and Ravetch 2010, Anthony, Wermeling, et al. 2008, Anthony,
Wermeling, and Ravetch 2012, Scanlan, Burton, and Dwek 2008). On the contrary, CHO cell lines (unless
genetically modified (El et al. 2013, Yang et al. 2015) cannot produce 2,6 sialylated glycans. Instead, they
produce only glycans with 2,3 sialic acid linkages, which however do not have anti-inflammatory properties
(Anthony, Nimmerjahn, et al. 2008, Anthony, Wermeling, et al. 2008). For intravenous immunoglobulins
(IVIGs), which are used for the treatment of different auto-immune diseases, and which commonly contain
comparatively low sialylation levels (<10%) (Huang et al. 2012), 2,6 sialylation has been reported to
improve anti-inflammatory properties (Anthony and Ravetch 2010).
Galactosylation is also implicated with anti-inflammatory properties. Highly galactosylated, as opposed to
agalactosylated immune-complexes, bind to the FcγRIIB and suppress the complement component 5a
receptor 1-related (C5aR) pro-inflammatory properties during complement activation (Karsten et al. 2012).
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Consequently, both galactosylated and 2,6 sialylated Fc-glycoforms can modulate the suppression of the
inflammatory responses. On the contrary, immune-complexes formed by agalactosylated Fc-glycoforms are
pro-inflammatory as they can only activate the activating FcγRs (Karsten et al. 2012, Nimmerjahn and

Ravetch 2010, Collin and Ehlers 2013) but not the inhibitory FcγRIIB. Fc-glycan removal ablates both anti-
and pro-inflammatory responses (Kaneko, Nimmerjahn, and Ravetch 2006, Anthony and Nimmerjahn 2011)
(Albert et al. 2008). This underscores the relevance of the Fc-glycans in fine-tuning the interactions of the
Fc domain with its cognate receptors and thus, in their regulation (Pincetic et al. 2014). Consequently, both
2,6 sialylated (S2(α2,6)G2F) and galactosylated (G2F) Fc-glycans are contributing factors to the anti-
inflammatory properties of antibodies.
Differently from endogenous antibodies, which generally contain higher levels of galactosylated and 2,6
sialylated glycans, commercial antibody therapeutics commonly contain mainly agalactosylated glycans
(Batra and Rathore 2016, Jefferis 2012), and this can be expected to clearly differentiate their anti-
inflammatory and functional properties.
Generally, the Fc glycosylation is considered not to directly affect the mAb’s interaction with the FcRn
(Pawlowski et al. 2018). However, indirectly, specific glycan chains may indeed have a potential role on the
FcRn-mediated clearance given the Fab interaction with the FcRn (Jensen et al. 2017), the impact of the Fc-
glycan residues on the Fab orientation (Fortunato and Colina 2014), as well as the potential modulation of
opening/closure of the FcRn binding site (Chen et al. 2017). Also, patient-related factors influence the
impact of the Fc glycans on efficacy, safety and immunogenicity of therapeutic mAbs. There will be a
differential impact depending on the type (soluble or membrane-bound), and specificity of the target, the
indication, the patient’s FcγRs genetic polymorphism, and the potential presence of pre-existing antibodies
against certain glycoforms in a given patient population, e.g. anti-agalactosyl, anti-galactose-α-1,3-
galactose (anti--gal), anti-N-Glycolylneuraminic acid (anti-NGNA) (Lu et al. 2007, Zhu and Hurst 2002,
Macher and Galili 2008). Given the relatively high content of agalactosylated glycans in commercial, fully
human mAbs, such as adalimumab (Raju and Jordan 2012), it might be speculated that their observed
immunogenicity can, in part, be attributed to the presence of pre-existing antibodies against the
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agalactosylated glycoforms in RA patients (Lu et al. 2007), or to the immunogenic potential of the
agalactosylated/high-mannose glycoforms which have the ability to engage with mannose receptors on
dendritic cells (Dong, Storkus, and Salter 1999).

Regarding ADCC, controversial results have been reported on the impact of galactosylation, e.g. no impact
(Boyd, Lines, and Patel 1995, Shinkawa et al. 2003), or positive impact (Houde et al. 2010). The
controversial ADCC results may be explained by the differential impact of galactose on the two arms (1-6
and/or 1-3) of the Fc-glycan; the galactose on the 1-6 arm has been associated with the enhanced ADCC,
while the opposite has been observed when present on the 1-3 arm (Chung et al. 2014). Of interest, Houde
clearly showed the significantly higher binding to FcRIIIa, compared to the wild-type, when the mAb was
non-fucosylated and hypergalactosylated (approximately an 80-fold increase) in comparison to the non-
fucosylated agalactosylated glycoform (approximately a 40-fold increase) (Houde et al. 2010). Furthermore,
it has been suggested that a particular pairing of oligosaccharides is necessary for the optimum binding of
IgG to C1q, FcγRs. The apparent contradiction among some glycosylation data may thus partially be due to
the lack of information of the mode of pairing of the two oligosaccharides. The Fc portion can be
asymmetric as well as symmetric with respect to glycosylation, and the different pairing adds further
complexity to the functional studies. The information concerning the pairing of the Fc-glycan is also
relevant in understanding the biological significance of the heterogeneities of the IgG glycans (Masuda et
al. 2000).
The level of galactosylation was also found to be correlated with ADCP with higher galactosylation levels
resulting in stronger ADCP (Chung et al. 2014).
An enzymatic approach to study the functional impact of glycovariants has been reported by Roche in
which the same starting material can be degalactosylated, hypergalactosylated, and sialylated by using
commercially available enzymes and activated sugars (Thomann et al. 2015). The above study clearly shows
the relevance of galactosylation on ADCC which is higher in fucosylated hypergalactosylated glycans.
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Endogenous human IgG1 and differences with commercial, cell culture-derived mAbs. Regarding the overall
Fc-glycan composition, the endogenous IgG1 glycan structures are almost completely biantennary complex
glycans (99.4% complex, 0.6% hybrid, <0.1% HM) with an extensive micro-heterogeneity due to the

presence or absence of different terminal sugars: Sialic acid, Gal, GlcNAc, core Fuc and bisecting GlcNAc.
Total agalactosylated glycoforms (G0+G0F+G0FB) were found at a level of approximately ~ 16% (Flynn et al.
2010). However, elevated levels of agalactosylated glycans are found in humans under specific conditions
such as aging, inflammatory, and autoimmune diseases (Dall'Olio et al. 2013). Moreover, in rheumatoid
arthritis (RA) patients, elevated levels of anti-agalactosylated IgG and Rheumatoid Factors (RF) are present,
which bind preferentially to the agalactosylated glycoforms (Ichikawa et al. 1998).
During pregnancy, changes in the maternal immune adaptation are required to tolerate the foetus, and to
initiate and maintain a successful pregnancy. Changes include an increased level of galactosylation in the Fc
glycan profile of IgG in both healthy individuals and RA patients (Ruhaak et al. 2014). RA patients
experience amelioration of symptoms during pregnancy, followed by a worsening after delivery, which
leads to a decrease in the galactosylation level. In RA patients during pregnancy, galactosylation, but not
sialylation, is associated with lower disease activity (Bondt et al. 2013).
In the serum of patients suffering from rheumatoid arthritis and other autoimmune diseases,
agalactosylated IgGs are present in abundance. This, together with the observation that arthritis-like
symptoms could be induced by the infusion of agalactosylated IgG, but not by G2 IgG, into non-diseased
murine recipients, lead to the hypothesis that agalactosylated IgG play a potential role in the disease
pathogenesis (Dong, Storkus, and Salter 1999). A potential explanation suggested the involvement of the
mannose receptor, which is expressed on macrophages and dendritic cells, which binds glycans containing
exposed fucose, mannose, and N-acetylglucosamine residues (e.g. agalactosylated glycoforms). Ligand
uptake is continuous, due to receptor recycling, allowing the intracellular processing of large quantities of
ligands (Dong, Storkus, and Salter 1999). The dendritic cell uptake of agalactosylated IgG, but not G2 IgG,
was highly efficient (within 15 min after exposure of ligands), and was inhibited by mannose (Dong, Storkus,
and Salter 1999). The processing and the presentation of agalactosylated IgG-derived peptides could
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stimulate CD4+ T-cell responses. Therefore, the agalactosylated and the high mannose IgG glycoforms may
potentially alter function, safety, catabolism and antigenicity.
As mentioned above, most commercial monoclonal antibodies contain high levels of agalactosylated

glycans (Batra and Rathore 2016, Jefferis 2012) as well as a discrete amount of high mannose glycans. This
is presumably attributable to decreased glycan processing, culture process parameters, e.g. low dissolved
oxygen (DO) concentration, or a reduced culture reduction potential (CRP), which have been reported to
decrease galactosylation (Dionne, Mishra, and Butler 2017). In addition, the presence of sialidases (Dorai
and Ganguly 2014) and other glycosidases (Gramer and Goochee 1993) in the cell culture medium may
further decrease the galactosylation/sialylation level of mAbs.
In commercial mAbs, the agalactosylated and the high-mannose glycans level may potentially confer an
inflammatory and immunogenic potential especially in autoimmune diseases. The inflammatory potential
may further be increased in the non-fucosylated agalactosylated component. In such conditions, it is likely
that mAb therapeutics with increased levels of galactosylation and 2,6 sialic acid contents, would exhibit
superior profiles in terms of safety, efficacy, PK, and immunogenicity.
In addition to the Fc-glycosylation, approximately 10-20% of endogenous antibodies are also glycosylated in
the variable domain of the Fab region, with glycans commonly having a different glycosylation profile than
the Fc-glycans (Mimura et al. 2007, Spiegelberg et al. 1970). The fact that the glycans in the variable
domain are generally more exposed than the Fc-glycans raises safety concerns when non-human glycan
residues such as N-Glycolylneuraminic acid (NGNA) and galactose-α-1,3-galactose (-gal) are involved
(Spiegelberg et al. 1970). Therefore, mAb glycosylation is a relevant CQA (Reusch and Tejada 2015) to be
tightly monitored in the NBE development, in comparability studies following manufacturing changes, and
in the biosimilar development (comparability with the reference medicinal product).
In summary, several concerns associated with commercial mAb preparations could be addressed by closer
mimicking physiological, human-like glycosylation patterns.
Therapeutic mAb manufacturing - current state of the art
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The development of therapeutic mAbs is a lengthy and expensive process. Manufacturers are facing
increasing demands in product quality and affordable drugs by authorities. Moreover, the emergence of
biosimilars exerts additional cost pressure. In conventional biopharmaceutical manufacturing processes, it

is generally accepted that the process itself defines the product which is characterized by numerous quality
attributes. Among these, glycosylation is an important subset as it has profound effects in terms of efficacy
and safety.
Mammalian host systems are the preferred expression platforms because they allow for the highest level of
posttranslational processing and functional activity of the protein. The most prominent host cell lines
currently used for marketed products are Chinese Hamster Ovary (CHO) or mouse myeloma (NS0, SP2/0)
cell lines (Dumont et al. 2016, Kunert and Reinhart 2016). However, as a consequence, their glycosylation
profiles may differ significantly from those produced in human cells, raising concerns regarding
immunogenicity and safety, e.g. due to the potential presence of non-human α-Gal or NGNA residues
introduced by mouse cells (Chung et al. 2008, Jefferis 2009) and, at lower levels, also in CHO (Bosques et al.
2010, Montjovent et al. 2017). For example, administration of mAbs containing α-Gal in patients with pre-
existing IgE antibodies directed against the α-Gal epitope can lead to hypersensitivity reactions (Platts-Mills
et al. 2015).
When antibodies are produced at industrial scale, the N-glycosylation machinery of the host cell lines
define the key Fc-glycan structure (Dell and Morris 2001). The glycosylation processing within the cells
involves a complex series of enzymatic reactions catalyzed by membrane-bound glycosidases (stepwise
trimming) and glycosyltransferases (stepwise addition of new sugar) (Helenius and Aebi 2001). Attached
key glycans depend on the cell type and the physiological status of the cell; the expression and the activity
of the cellular enzymes are exquisitely sensitive to events which take place within the cell. Depending on
the extension of processing, the N-glycans are diversified in the Golgi apparatus in high mannose, hybrid
and complex type (Shi and Goudar 2014) (Figure 3), and the transition from hybrid- to complex-type further
stabilizes the Fc region (Bowden et al. 2012).
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Culture media components and process parameters such as pH, temperature, etc. may also affect glycan
composition (Batra and Rathore 2016, Liu, Nowak, et al. 2016). In addition, once a glycoprotein is secreted,
glycosidases (e.g. sialidases) released by lysed cells, especially at a later stage of the bioprocess, may

remove terminal glycan residues (Dorai and Ganguly 2014, Gramer and Goochee 1993).
Depending on the cell type and process, the principle structures emerging are high mannose, hybrid, and
complex type (see Figure 3). Within complex type glycans, the overall composition and structure may vary
significantly, e.g. through the presence/absence of core fucose, the degree of terminal galactosylation (G0,
G1, G2), the presence/absence and type of terminal sialic acid substitution N-Acetylneuraminic acid
(NANA), NGNA, the presence/absence of -gal. The resulting glycan profile may lead to a differential
impact on structure, stability, biological functions, pharmacokinetics, immunogenicity, and safety.
One way to improve the antibody properties is to introduce a better control on the composition of the Fc-
glycosylation, in particular, the reduction of fucosylation with the aim to increase ADCC. Current
optimization efforts focus on host cell engineering and the cell culture conditions (El et al. 2013, Imai-
Nishiya et al. 2007, Yang et al. 2015, Bruhlmann et al. 2017, Bruhlmann et al. 2015). For example, to
improve ADCC, a significant improvement through cell-based glycoengineering has been previously
reported with the first approved mAbs mogamulizumab and obinutuzumab. Mogamulizumab (POTELIGEO®,
KW-0761) is a humanized mAb derived from Kyowa Hakko Kirin's POTELLIGENT (®) technology which uses a
α-1,6-fucosyltransferase (FUT8) knockout CHO cell line to produce mAbs with non-fucosylated glycan
mixtures (Beck and Reichert 2012). Obinutuzumab (Gazyva™, GA-101) is derived from Roche GlycoMAb®
technology which overexpresses β1,4-Nacetylglucosaminyltransferase III (GnTIII). Once the GnT-III adds a
bisecting GlcNAc to an oligosaccharide, the core-fucosylation is inhibited (Cameron and McCormack 2014).
Both technologies produce therapeutic mAbs with enhanced ADCC activity. Furthermore, the use of
ﬂuorinated fucose analogues as a speciﬁc inhibitor of fucosyltransferase was reported to substantially
reduce core fucosylation of N-linked glycans in CHO cells (Brühlmann et al. 2015, Rillahan et al. 2012) and
can therefore be expected to enhance ADCC activity of mAbs through rational media design.
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However, there are still some limitations with regards to maximized ADCC properties that can be obtained
for oncology mAbs. Although this approach allows more control over the fucosylation level, these platforms
still produce heterogeneous mixtures of the non-fucosylated glycoforms containing predominantly

agalactosylated glycoforms, as reported for mogamulizumab. Elevated levels of the agalactosylated glycan
variants result from impaired glycan processing since processing largely depends on the presence of core
fucose and, consequently, non-fucosylated forms tend to be less processed (Castilho et al. 2015, Mimura et
al. 2018).
From a process economic point of view, the volumetric yields of mammalian cells are comparatively low
compared to expression in other eukaryotic systems such as yeast. Yeast expression systems (e.g.
Saccharomyces cerevisiae and Pichia pastoris) achieve rapid cell growth and high-protein yields with facile
production scalability (Gerngross 2004), and have most of the subcellular machinery for posttranslational
modification present in higher eukaryotes. However, a key challenge associated with yeast expression is
their production of high mannose glycans which may confer a short half-life and render proteins less
efficacious and even immunogenic in humans (Dean 1999, Gemmill and Trimble 1999). Therefore, cell line
development efforts in yeast have focused on genetic modifications to reduce the high mannose content,
and the production of glycoproteins, with humanized sialylated glycans, has been reported (Hamilton and
Gerngross 2007).
In summary, the glycan distribution defines to a large extent, the functional (with regards to effector
functions) and safety profile of a manufactured mAb. The pain points resulting from current manufacturing
processes may be summarized as follows. For patients, the efficacy and safety may be suboptimal, and
manufacturers struggle with time-consuming process development, and inefficient manufacturing
processes prone to process changes and batch-to-batch variability.
Current approaches to in-vitro glycoengineering. Enzymatic glycoengineering refers to an in-vitro
modification of glycans with the aim to reduce glycan heterogeneity. The protocols typically employ
enzymes targeting terminal sugar residues (neuraminidase, β(1,4) galactosidase, β(1,4)
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galactosyltransferase, and (2,6) sialyltransferase) and suitable activated sugars (e.g. UDP-gal, CMP-NANA)
to increase the galactosylation and/or sialylation level on mAbs. Thomann et al. describes the workflow of
the in-solution modification of a monoclonal antibody at medium scale (Thomann et al. 2015). Similarly,

Tayi reports the sequential enzymatic transformation on a solid-state matrix, e.g. protein A and G columns
(Tayi et al 2018). The immobilisation of the antibody in the latter approach facilitates the processing by
allowing the enzymes and buffers to be washed out in repeated cycles of enzymatic reactions. The use of
affinity resins commonly used in mAb purification may be further optimized for the implementation in
large-scale manufacturing processes. However, although the end products may be derived from the same
starting material, and despite the possibility to introduce “human-like” sialic acid with 2,6 linkages, such in-
vitro glycoengineering approaches are not able to deliver a truly homogeneous glycosylation. For example,
this approach does not allow modifying high mannose glycans or transforming hybrid into complex glycans.
Therefore, although enrichment of a desired complex glycan variant can be achieved, a certain overall
heterogeneity arising from the presence of high-mannose and hybrid variants will remain in the final
product. As a consequence, this methodology, in its current format, is not applicable to proteins expressed
in wild type yeast due to the high content of high mannose glycans. As a further limitation, although the
target glycan is enriched, the final products contain also still heterogeneous mixtures of monosialylated and
disialylated glycans and several rounds of enzymatic reactions may be required in order to reach the
desired degree of sialylation (Thomann et al. 2015, Tagy et al. 2018). Furthermore, some enzymatic
reactions may lead to an increased level of Asn deamidation and Asp isomerization (Thomann et al. 2015).
Homogeneous glycans and the “universal” glycan
The functional properties (e.g. ADCC) in therapeutic mAbs may be enhanced through Fc-glycoforms that are
both non-fucosylated and hyper-galactosylated. However, the enhanced potency carries also the potential
risk of an immunogenic reaction towards non-diseased cells also bearing the antibody target, underlining
the importance of target specificity (Dekkers et al. 2017).
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Wong et al. coined the term “glycoantibody”, referring to a homogeneous population of mAbs having a
single, uniform glycoform on the Fc region. The same group also coined the term “universal” glycan for the
non-fucosylated, fully sialylated biantennary glycan chain (S2(α2,6)G2) which, when homogeneously added

to a mAbs, confers several improved properties in terms of humanization of sialic acid linkage, significantly
improved binding to the FcγRIIIa (both VV and FF polymorphisms), increased ADCC (while maintaining the
binding properties to other receptors such as FcγRs and C1q), increased ADCC of S2(α2,6)G2 in comparison
to S2(α2,3)G2, and a higher conformational plasticity between the open and closed Fc conformation (Chen
et al. 2017, Lin et al. 2015). The closed conformation is suggested to optimize the binding properties to
FcRn (Chen et al. 2017), this in turn could result in a decreased clearance rate of mAbs. The latter feature
can represent a differentiating factor between the glycan chains (S2(α2,6)G2) and G2. Further studies
would be necessary to establish if the closed conformation of non-fucosylated (S2(α2,6)G2) enhance the
binding properties with both the FcRn and the type II Fc receptors. The type II Fc receptors are known to
confer anti-inflammatory properties to the closed conformation of fucosylated (S2(α2,6)G2F) antibodies
(Sondermann et al. 2013).
It is worth noting that non-fucosylated glycoengineered mAbs not only maximize ADCC “in vivo”, where a
high amount of approximately 10mg/mL of endogenous antibodies compete for the FcRs but they can also
enhance macrophage/monocyte induced ADCP activity (Herter et al. 2014). ADCP is a well-recognized and
relevant mechanism of action of therapeutic antibodies used in oncology (Weiskopf and Weissman 2015).
Chemoenzymatic glycoengineering to obtain homogeneously glycosylated antibodies. Chemoenzymatic
glycoengineering refers to an in-vitro remodelling of glycans, employing enzymes such as endoglycosidases
and glycosynthase. Details on such a platform can be found in the recent review by Mimura (Mimura et al.
2018) as well as in the protocol by Tang (Tang, Wang, and Huang 2017) on chemoenzymatic synthesis of
glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates (ADCs). Briefly,
chemoenzymatic glycoengineering is based on two enzymatic steps: the trimming of all the heterogeneous
N-glycans by an endoglycosidase leaving the first GlcNac residue and the adding back a well-defined N-
glycan with desired properties en bloc by glycosyntase mutants which, as a substrate, use a synthetic N-
15
glycan core oxazoline. By co-expressing endoglycosidases (EndoH), the first step can potentially be omitted,
as the deglycosylation occurs in-vivo (Bennett et al. 2018). This platform has a high flexibility with the
potential to produce non-fucosylated, fucosylated, and functionalized Fc glycans.

The platform may be applied to both mAbs, antibody mixtures such as IVIGs (e.g. to obtain highly sialylated
(>90%) IVGs), and as a functionalization platform to produce ADCs or other labelling modifications. The
feasibility of this approach has been reported by several groups (see next section). Furthermore, it has been
shown that this approach, apart from the specific transformation of the mAb glycans, does not affect other
posttranslational modifications (Liu, Giddens, et al. 2016). Until now, this platform has mainly been
employed to generate symmetric and homogeneous glycosylation populations for structural and functional
studies (Kurogochi et al. 2015, Lin et al. 2015).
Case studies. Chemoenzymatic glycoengineering platforms were successfully employed by several groups,
amongst which Academia Sinica, National Taiwan University, CHO Pharma Inc. (Taiwan) and the University
of Maryland, Rockefeller University (USA) have produced pivotal papers (Huang et al. 2012, Li, Zhu, et al.
2017, Lin et al. 2015, Tang, Wang, and Huang 2017, Tang et al. 2016, Zou et al. 2011). A few of these
published studies use commercial mAbs as reference models, e.g. Rituxan (rituximab) (Li, DiLillo, et al.
2017, Lin et al. 2015), Herceptin (trastuzumab) (Chen et al. 2017, Kurogochi et al. 2015, Li, DiLillo, et al.
2017, Lin et al. 2015, Liu et al. 2018) and Humira (adalimumab) (Tsai et al. 2017).
A common important finding reported in these studies, although the actual fold increase differed, was that
the glycoengineered mAbs with a homogeneous glycosylation showed clearly enhanced FcRIIIa binding
compared to heterogeneously glycosylated reference samples (wild type), (Li, DiLillo, et al. 2017, Lin et al.
2015). Moreover, FcRIIIa binding was found to be consistently higher in the non-fucosylated form of the
molecule (both G2 and S2(α2,6)G2) in comparison not only to wildtype but also in comparison to the
glycoengineered mAbs which contain homogeneous non-fucosylated agalactosylated and M3 glycoforms
(Lin et al. 2015) (Figure 4).
Some patients may become resistant to rituximab due to high dosage and long-term use. It has been shown
“in vitro” that rituximab resistant cells are, however, still sensitive to glycoengineered mAb (S2(α2,6)G2)
16
presumably due to their higher affinity (Lin et al. 2015). The S2(α2,6)G2 and the G2 glycoengineered mAbs
maximize ADCC but can also enhance ADCP (Herter et al. 2014). The benefits of glycoengineered mAbs in
terms of enhanced ADCC and ADCP activity in presence of competitive endogenous antibodies due to their

enhanced binding to FcRIII are worth underlining; in addition, Chen demonstrated the differential binding
activity to FcRIIIa of trastuzumab in absence/presence of the target (Chen et al. 2017). In presence of the
target, a strong decrease in affinity was observed for wildtype trastuzumab (Kd = 24 nM  Kd = 169 nM),
while the decrease was minimal for the glycoengineered trastuzumab (S2(α2,6)G2) (Kd = 10 nM Kd = 20
nM). Due to the Fc conformational plasticity (open/closed conformation) induced by S2(α2,6)G2, and/or
differential impact of G2 vs. G0 on Fabs orientation (distribution of directional and rotational vectors), one
can speculate that, similarly to the FcRIII binding site, the binding sites for the other FcRs and C1q, may
also be less affected by the presence of the target ligands. If confirmed, this may translate in higher effector
function (ADCC, ADCP and CDC) in the tumor microenvironment of S2(α2,6)G2and G2 glycoengineered
mAbs.
Glycoengineering was also applied to IVIGs with the aim of increasing the 2,6-sialic acid content (from <10%
to >90%). Furthermore, the platform is also applicable to the generation of functionalized glycans, suitable
for click-chemistry for the targeted introduction of payloads, e.g. cytotoxic drug development (e.g.
antibody-drug conjugates, ADCs), or for labelling and tracers for in-vitro and in-vivo cell imaging,
immunohistochemistry and western blotting (Huang et al. 2012, Tang, Wang, and Huang 2017).
Finally, first clinical trials are underway applying glycoengineered mAbs. CHO Pharma Inc., a Taiwan-based
start-up company, has applied its proprietary glycan-based technology platforms to develop truly
homogeneously glycosylated therapeutics that are intended as "biobetters" of currently marketed
antibodies. A Phase I open-label, multiple-dose study of a glycoengineered anti-CD20 antibody (Trial ID:
NCT03221348) has been initiated (March 2018) for Refractory or Relapsed Follicular Lymphoma indication
while others are in pre-clinical development phases.
Some suppliers already commercialize reagents that are required for glycan engineering. For example,
Fushimi Pharmaceutical Co. Ltd. (Japan) offers enzymes and substrates for the targeted remodelling of
17
protein glycans at lab scale. Genovis (Sweden) commercializes soluble and immobilized enzymes required
for deglycosylation of mAbs (up to 100 mg scale), as well as kits with broader applications such as cytotoxic
drug development, in-vitro and in-vivo cell imaging, immunohistochemistry and western blotting.

Chemoenzymatic glycoengineering – advantages. The pain points associated with current manufacturing
processes, i.e. the suboptimal safety and efficacy, and batch-to-batch inconsistencies, could collectively be
addressed by having maximum control over the Fc-glycosylation of the end products. For example, instead
of relying on the host cell machinery, the chemoenzymatic glycoengineering approach is a radically novel
effort in which the glycans that are attached during the cell culture phase are entirely remodelled by
replacing them with those optimal for the target application, e.g. humanized N-glycans, maximized efficacy.
In this manner, the final product can be produced with precisely defined and desired glycan profiles, e.g.
homogeneous, human-like glycans with enhanced biological properties. Moreover, the resulting glycan
profile is also uncoupled from the process, i.e. independent from the expression host, and unaffected by
process variations and changes.
The chemoenzymatic glycoengineering approach offers thus several significant advantages.
 Maximized efficacy – Studying homogeneous glycan populations in Rituximab, Lin et al. (Lin et al.
2015) has shown that non-fucosylated and completely galactosylated (G2) or human-like sialylated
mAbs (S2(α2,6)G2) are superior (approx. 4-6 fold higher FcγRIIIa binding compared with non-
fucosylated agalactosylated glycans (G0) and high mannose variants. Compared to the
heterogeneous wildtype glycan population, an increase in terms of the FcγRIIIa binding of up to 20-
fold was observed for the homogeneous S2(α2,6)G2 variant. Analogous to Li et al. 2017, who
observed a correlation between in-vitro binding and enhanced in-vivo activity (Li et al. 2017), it can
be expected that the maximized binding translates into maximised in vivo efficacy. This emphasizes
that an antibody, which is chemo-enzymatically engineered for a particular homogeneous glycan
population, is not only superior to currently marketed mAbs carrying a conventional,
18
heterogeneous glycan population, but also superior compared with low-fucose products derived
from processes with glycosylation improvements on the cell-line level.
 Maximized safety – Chemoenzymatic glycoengineering allows precise control over the glycan

structures present in a product. Therefore, any potential presence of non-human variants, high
mannose, and undesired agalactosylated glycans can be remodelled. Moreover, the precise tuning
of the glycan content allows avoiding immunogenicity and adverse reactions due to pre-existing
antibodies in patients, e.g. anti--Gal, anti-NGNA, anti-agalactosyl (Ichikawa et al. 1998, Nishijima,
Sato, and Takehara 2001, Lu et al. 2007, Zhu and Hurst 2002, Macher and Galili 2008, Teranishi et
al. 2002).
 Increased process flexibility and efficiency –
o Facilitated process development – Development is shortened timewise because less time
would be required to fine-tune the fermentation conditions to achieve a desired
glycosylation profile. The pivotal role of the intracellular functions of N-linked glycans in
terms of protein folding, oligomerization, quality control, sorting, and transport is retained
with this platform (Helenius and Aebi 2001). Differently glycosylated end products can be
produced from the same intermediate bulk. Furthermore, the approach can facilitate also
functional studies when applied to current products.
o Free choice of expression host – The production of glycosylated mAbs is no longer
restricted to mammalian cells. Instead, other eukaryotic expression hosts or even plant
cells could thus be selected for maximum productivity (Bennett et al. 2018, Liu et al. 2018).
o More robust processes and increased batch-to-batch consistency – During the re-
modelling of the glycan profile, after the fermentation, any change in glycosylation,
because of a process change, will be normalized.
Chemoenzymatic glycoengineering – challenges and limitations. Current manufacturing plants and
analytical methods are not optimised for the application of this platform and should be redesigned to apply
this procedure in the most effective way. Additional processing steps, glycan removal and re-attachment
19
will be required for glycan remodeling. The additional use of enzymes and oligosaccharide reagents poses
the risk to introduce additional HCPs or process related impurities. This will necessitate additional analytical
methods to monitor their clearance and may require additional purification steps to remove newly

introduced process related impurities. The handling times, additional equipment and reagents will add to
the overall manufacturing costs, although implementation of chemoenzymatic glycoengineering may be
facilitated by the integration of additional processing steps into existing operations, e.g. enzymatic
reactions on solid-phases like protein A columns (Tayi et al 2018).
Currently, enzymes and oligosaccharide reagents are not yet commercially available at relevant production
scales. However, once dedicated equipment and reagents will be produced for mAb manufacturing, their
unit costs will go down with scale.
Conclusions and perspectives
Chemoenzymatic glycoengineering is emerging as a radical and attractive approach to gain unprecedented
control over the glycosylation profile of mAbs. It therefore offers the possibility to generate mAbs with
precisely defined and desired glycans, resulting in superior product quality and safety, or in other targeted
functionalisation such as ADCs.
Additional processing costs will be offset by a significant cost-saving potential, due to the possibility of
more economic process formats with significantly higher volumetric productivities (Kurogochi et al. 2015,
Liu et al. 2018).
Currently, this platform is focused on Fc-glycans, however, by using suitable reagents and enzymes, it may
become generally applicable to glycoproteins as a platform for glycan remodelling to generate superior
products. For example, it has been suggested that among interferon-beta-1a glycoforms those with higher-
antennarity could better sustain bioactivity over time (Mastrangeli et al. 2014).
20
Once the first glycoengineered antibody, obtained by this approach, will demonstrate to be superior in the
clinic use, and customized reagents and equipment will be commercially available at affordable costs and
in-line with regulatory requirements, the platform is likely to become a breakthrough, paving the way for

the development and production of the next-generation of immunotherapeutics.
Acknowledgements: The authors would like to thank Maria Concetta Audino for support regarding
literature searches and Hervé Broly for critically reviewing the manuscript.
21
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Legends to figures

Figure 1. Human IgG1 3D structure
The heavy chains are shown in blue and light blue, the light chains are shown in red and light red,
respectively. The 2 N-glycans, present at each of the two heavy chains, are illustrated in a solid sphere
representation with different colors (grey and yellow). The illustration was generated by rendering the PDB
entry 1hzh by using the PyMOL Molecular Graphic System version 1.8.6.0. Schrödinger, LLC. Each light chain
consists of an N-terminal variable domain (VL) and a constant domain (CL). Two heavy chain–light chain
heterodimers combine into a single antibody molecule (H2L2). The light chain is associated to the VH and
the CH1 domains to form the fragment antigen binding (Fab) region. The C-terminal part of the antibody,
formed by (CH2-CH3)2 domains, is called fragment crystallisable (Fc) region. The variable region of the Fab
is responsible for the capability of immunoglobulins of binding to an antigen, while the Fc part, which
contains the binding sites for the complement component 1q (C1q) and the Fc--receptors (FcRs), is
responsible for the antibody effector functions such as the antibody-dependent cell-mediated cytotoxicity
(ADCC), the antibody-dependent cell-mediated phagocytosis (ADCP), and the complement-dependent
cytotoxicity (CDC). In addition, the Fc region contains two Fc neonatal receptor (FcRn) binding sites which
are responsible for the long antibody half-life (Quast et al. 2017).
Figure 2. Biantennary complex glycan structure found in the Fc region of human endogenous IgG.
Structural implications and differential interactions and mobility of α1,6 and α1,3 arms of the Fc glycan.
Figure 3. IgG1 Fc-glycan microheterogeneities
A) human IgG1, B) CHO, and C) Mouse cells (NS0/SP2). The double-dash (- -) symbol between residues
indicates a potential truncation contributing to the overall high Fc-glycan microheterogeneity. Complex
33
type glycans containing 0, 1 or 2 galactose residues are referred as G0F, G1F and G2F glycoform when core-
fucosylated and G0, G1 and G2 when core-fucose is absent. Non-human glycans are highly expressed by
mouse cells and at low levels, by CHO cells.

Figure 4. Relative FcγRIIIa binding increase of non-fucosylated homogeneous Fc-glycans
The figure illustrates the fold increase, in terms of FcγRIIIa binding, observed for non-fucosylated,
homogeneous Fc glycan variants compared with the heterogenous wildtype population as a reference. The
homogeneous glycan populations, using rituximab and trastuzumab as model proteins, were obtained by
chemo-enzymatic glycoengineering (Lin et al. 2015).
34
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Glycoengineered Antibodies: Towards the Next-Generation of

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Zone Industrielle de l’Ouriettaz, Aubonne 1170, Switzerland

horst.bierau@merckgroup.com, Phone number: +39 0774 350834.

Keywords: monoclonal antibody, effector functions, homogeneous glycosylation, glycoengineering,

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suboptimal in terms of biological properties such as antibody-dependent cell-mediated cytotoxicity (ADCC)

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value of USD 138.6 billion by 2024 (http://www.grandviewresearch.com/pressrelease/global-monoclonal-

survival and/or the quality of life of patients.

IgG Fc glycosylation – structural and functional properties

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structure of IgG1s (Figure 1).

engage in glycan-glycan and glycan-protein interactions (Figure 2).

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mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-

the 1-3 arm is more flexible (Figure 2).

of galactosylated in comparison with agalactosylated antibodies.

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mAbs and FcRn or type II receptors it is 1:2 (Pincetic et al. 2014).

receptors DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin) and

improve anti-inflammatory properties (Anthony and Ravetch 2010).

Galactosylation is also implicated with anti-inflammatory properties. Highly galactosylated, as opposed to

inflammatory responses. On the contrary, immune-complexes formed by agalactosylated Fc-glycoforms are

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inflammatory properties of antibodies.

inflammatory and functional properties.

against certain glycoforms in a given patient population, e.g. anti-agalactosyl, anti-galactose-α-1,3-

dendritic cells (Dong, Storkus, and Salter 1999).

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non-fucosylated and hypergalactosylated (approximately an 80-fold increase) in comparison to the non-

resulting in stronger ADCP (Chung et al. 2014).

the relevance of galactosylation on ADCC which is higher in fucosylated hypergalactosylated glycans.

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which bind preferentially to the agalactosylated glycoforms (Ichikawa et al. 1998).

sialylation, is associated with lower disease activity (Bondt et al. 2013).

potentially alter function, safety, catabolism and antigenicity.

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further decrease the galactosylation/sialylation level of mAbs.

superior profiles in terms of safety, efficacy, PK, and immunogenicity.

in the biosimilar development (comparability with the reference medicinal product).

mimicking physiological, human-like glycosylation patterns.

Therapeutic mAb manufacturing - current state of the art

biosimilars exerts additional cost pressure. In conventional biopharmaceutical manufacturing processes, it

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involves a complex series of enzymatic reactions catalyzed by membrane-bound glycosidases (stepwise

stabilizes the Fc region (Bowden et al. 2012).

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impact on structure, stability, biological functions, pharmacokinetics, immunogenicity, and safety.

ﬂuorinated fucose analogues as a speciﬁc inhibitor of fucosyltransferase was reported to substantially

still produce heterogeneous mixtures of the non-fucosylated glycoforms containing predominantly

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manufacturers struggle with time-consuming process development, and inefficient manufacturing

processes prone to process changes and batch-to-batch variability.

Current approaches to in-vitro glycoengineering. Enzymatic glycoengineering refers to an in-vitro

enzymes targeting terminal sugar residues (neuraminidase, β(1,4) galactosidase, β(1,4)

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Homogeneous glycans and the “universal” glycan

the importance of target specificity (Dekkers et al. 2017).

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confer anti-inflammatory properties to the closed conformation of fucosylated (S2(α2,6)G2F) antibodies

(Sondermann et al. 2013).

Chemoenzymatic glycoengineering to obtain homogeneously glycosylated antibodies. Chemoenzymatic