Immunoglobulin G (IgG) (as a glycoprotein) is pres�
ent in the serum of healthy humans as numerous glyco�
forms due to the presence of complex oligosaccharides in
the Fab� and Fc�fragments of the antibody. Whereas the
oligosaccharides in the Fab�fragment determine binding
of antibody with antigen, the presence of oligosaccharides
in the Fc�fragment does not affect antigen binding, but it
has a great impact on biological mechanisms that are
activated by immune complexes formed with participa�
tion of Fc�domains. Recombinant monoclonal antibody
technology, which currently is the basis for creating ther�
apeutic monoclonal antibodies (TMA), helps to elucidate
the role of carbohydrate residues in the functions of anti�
bodies in normalcy and pathology.
Commercially available TMAs are produced in ani�
mal cells mainly in any of three mammalian cell lines:
Chinese hamster ovary cells (CHO), murine NS0 (non�
secreting mouse myeloma cells), or SP2/0 (mouse myelo�
ma cells). The majority of more than 40 TMAs used in
clinics is chimeras (mouse/human) or humanized mouse
antibodies of IgG1 subclass [1, 2]. The TMAs are glyco�
proteins with glycosylation defining structural character�
istics of Fab� and Fc�fragments of the antibody, which in
turn affects binding of the antibody with antigen, as well
ISSN 0006�2979, Biochemistry (Moscow), 2016, Vol. 81, No. 8, pp. 835�857. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © Y. L. Dorokhov, E. V. Sheshukova, E. N. Kosobokova, A. V. Shindyapina, V. S. Kosorukov, T. V. Komarova, 2016, published in Biokhimiya, 2016,
Vol. 81, No. 8, pp. 1069�1090.
REVIEW
835
Abbreviations: ADCC, antibody�dependent cell cytotoxicity; ADCP, antibody�dependent cellular phagocytosis; Asn297, asparagine
at position 297 of amino acid sequence of the IgG heavy chain; CDC, complement�dependent cytotoxicity; CH, constant domain
of the heavy chain; CL, constant domain of the light chain; CMP�Neu5Ac, CMP�acetylneuraminic acid; EMA, European
Medicines Agency; ER, endoplasmic reticulum; Fab�fragment, fragment antigen binding; Fc, fragment crystallizable; FDA,
United States Food and Drug Administration; Fuc, fucose; FUT8, α1,6�fucosyltransferase; α�Gal, galactose�α1,3�galactose;
GalT, β�N�acetylglycopeptide β�1,4�galactosyltransferase; GalNAc, N�acetylgalactosamine; GCS I and II, mannosyl�oligosac�
charide glycosidase I and II; Glc, glucose; GlcNAc, N�acetylglucosamine; GM II, Golgi α�mannosidase II; GNT I, α�1,3�man�
nosyl�glucoprotein 2�β�N�acetylglucosaminyltransferase; GNT II, α�1,6�mannosyl�glucoprotein 2�β�N�acetylglucosaminyltrans�
ferase; HER2, human epidermal growth factor receptor 2; HR, hinge region; HVR, hypervariable region; Ig, immunoglobulin;
IgG, immunoglobulin class G; Man, mannose; MNS, mannosidase; Neu5Gc, glycolylneuraminic acid; NK, natural killer cells;
OST, oligosaccharyltransferase; SIAT, β�galactoside α�2,3/6�sialyltransferase; TMA, therapeutic monoclonal antibodies; TNFα,
tumor necrosis factor α; VH, variable domain of the heavy chain; VL, variable domain of the light chain; ZFN, zinc finger nuclease.
* To whom correspondence should be addressed.
Functional Role of Carbohydrate Residues in HumanImmunoglobulin G and Therapeutic Monoclonal Antibodies
Y. L. Dorokhov1,2*, E. V. Sheshukova1, E. N. Kosobokova1,A. V. Shindyapina1,2, V. S. Kosorukov1, and T. V. Komarova1,2
1Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia; fax: +7 (499) 132�89622Belozersky Institute of Physico�Chemical Biology, Lomonosov Moscow State University,
119991 Moscow, Russia; fax: +7 (495) 939�3181; E�mail: [email protected]
Received April 28, 2016
Revision received May 27, 2016
Abstract—Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previous�
ly considered untreatable. Currently more than 40 full�size TMAs created primarily based on immunoglobulin G1 are wide�
ly used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological
activity, effector functions, immunogenicity, and half�life in the patient’s serum. The importance of carbohydrate residues
for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with empha�
sis given to N�glycosylation of the Fc fragment of the antibody.
DOI: 10.1134/S0006297916080058
Key words: monoclonal antibody, immunoglobulin G, glycosylation, antibody�dependent cell cytotoxicity, Chinese hamster
ovary cells, immunotherapy, biosimilarity
836 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
as its effector functions [3]. Therapeutic activity of TMA
can depend on the attached oligosaccharide. In spite of
extensive studies on the structure–function relationship
of antibodies conducted in recent years [4, 5], the role of
carbohydrate residues in pharmacodynamics and phar�
macokinetics of TMA remains incompletely understood.
Despite the high accuracy of protein synthesis in cell cul�
ture, TMAs are formed with slight variations in the car�
bohydrate composition. The glycosylation profile of a
TMA and its carbohydrate composition are defined (to a
great extent) by the cell line together with the procedure
and conditions of cell cultivation [4].
An idea has been suggested on the possibility of cre�
ating glycomodified TMA with enhanced efficiency in
treatment of oncological diseases [6, 7]. However, the
high efficiency of glycomodified TMA was proven only
for malignant blood cells [obinutuzumab (Gazyva®) and
mogamulizumab (Poteligeo®)], but not for solid and
metastatic tumors [3]. The effect of glycomodification of
antibodies on their pharmacodynamics and pharmacoki�
netic properties as well as on compliance with critical
requirements for pharmaceutical products have been
reviewed [7].
SHORT ESSAY ON HUMAN ANTIBODIES:
TYPES OF IMMUNOGLOBULINS, STRUCTURE
OF IgGs, AND THEIR EFFECTOR FUNCTIONS
A short description of IgG structure and its effector
functions is presented here to facilitate better understand�
ing of the role of carbohydrate residues in functioning of
antibodies, although a large number of reviews have been
devoted to this issue.
Types of immunoglobulins. Five classes of immuno�
globulins (IgM, IgG, IgE, IgD, and IgA) and several sub�
classes – two in IgA and four in IgG – are present in the
human body [8, 9] (Table 1). B�lymphocytes release pre�
dominately IgG molecules following secondary immu�
nization. Unlike other immunoglobulins, IgG can cross
the placenta and other extravascular spaces and is secret�
ed (together with IgA) in breast milk. The IgG molecules
consist of two heavy 50�kDa γ�chains and two 25�kDa
light chains belonging of two types – kappa (κ) and lamb�
da (λ) (Table 1). Subdivision of IgG into four subclasses is
determined by their relative concentration in normal
human serum, where IgG1, IgG2, IgG3, and IgG4 com�
prise ∼66, 23, 7, and 4% of total content of serum IgG,
respectively [10]. The human IgG1 subclass provides the
basis for creating therapeutic antibodies, because this type
of immunoglobulins (i) has long blood half�life (Table 1)
and (ii) exhibits more pronounced effector functions in
comparison with other classes and subclasses of human
immunoglobulins [9, 16]. Among the other antibodies,
IgG has a relatively simple structure. It is a homodimer
with molecular mass of 150 kDa, in which each monomer
consists of two polypeptide chains – heavy and light –
bound to each other via one interchain disulfide bond.
These two monomers are combined into one full�size IgG
via disulfide bonds between the heavy chains (Fig. 1).
Three structural units are identified in IgG [8, 9, 16]: (i)
Fab�fragment (“fragment, antigen binding” or “antigen
binding fragment”) comprising the antigen binding struc�
ture consisting of variable domains (VL) of two light chains
and variable domains (VH) of two heavy chains forming
paratopes, as well as two constant domains of the light and
heavy chains (CH1 and CL1); (ii) C�terminal structure
denoted as the Fc�fragment (fragment crystallizable) that
includes constant domains of the heavy chain CH2 and
CH3; (iii) hinge region (HR) that ensures mobility of anti�
body fragments relative to each other due to its flexible
structure. The length and flexibility of the hinge region
vary greatly among the IgG subclasses, which affects the
Fab conformation relative to the Fc domain. The HR of
IgG1 consists of 15 amino acids and is very flexible. The
IgG heavy chains in this region are covalently bound via
disulfide bonds (two in IgG1 and IgG4, four in IgG2, and
11 in IgG3), while the region of the CH2 and CH3 domains
contains noncovalent bonds between chains. Depending
on the isotype, the dimerization of two halves of an anti�
body suggests formation of from 2 to 11 disulfide bonds
between the heavy chains, which ensures stabilization of
the IgG structure due to intra� and inter�molecular
crosslinking of the heavy and light chain. Two inter�chain
disulfide bonds stabilize each light chain, and four stabi�
lize each heavy chain. All of this provides stability and
impacts the duration of antibody half�life. Another factor
determining the relatively long duration of the human
blood half�life of the antibody (Table 1) is the ability of the
antibody Fc�region to bind specific receptors [17, 18].
Antibody effector functions. The modern notion on
antibody catabolism in the human body is based on the
idea Brambell suggested more than 50 years ago [19]. To
explain long survival of IgG relative to other plasma pro�
teins, Brambell postulated the availability of specific pro�
tection receptors (FcRp) that would bind IgG in pinocyt�
ic vacuoles and redirect them to the circulation. When the
FcRp is saturated, the excess of unbound IgG is subject�
ed to proteolysis in lysosomes. Brambell’s idea was cor�
roborated experimentally [20]. According to present
views, the neonatal receptor FcRn, which transfers IgG
from mother to fetus across the placenta and through the
proximal region of its small intestine during feeding with
breast milk, plays an important role in antibody metabo�
lism [21]. It must be noted that the FcRn represents a
variant of the major histocompatibility complex class I
and is accumulated not only in endothelium in adults, but
also in monocytes, macrophages, and dendrite cells. The
FcRn binding site in the IgG is located on the interface
between domains CH2–CH3 and is responsible for: (i) its
long half�life; (ii) transport across the placenta, and (iii)
reciprocal movement of IgG along the surface of mucous
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 837
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
membranes. Following synthesis the IgG molecules, they
are pinocyted by endothelial cells that bind with high
affinity (KD ≈ 10 nM) to histidine residues of the binding
site, forming the IgG–FcRn complex at lower pH (6.0�
6.5), thus avoiding degradation in lysosomes. After
returning to the cell surface, the IgG is released at physio�
logical pH (7.0�7.5). As a result, this system ensures long
circulation of IgG [20, 21], for 1�3 weeks depending on
the subclass (Table 1).
The Fc�region of the antibody also defines other
effector properties of the antibody (Fig. 2), such as anti�
body�dependent cell cytotoxicity (ADCC) [22], comple�
ment�dependent cytotoxicity (CDC) [23], antibody�
dependent cellular phagocytosis (ADCP) [24], and anti�
inflammatory activity [18]. The receptors or ligands that
initiate effector functions of IgG include: (i) three struc�
turally homologous receptors FcγR (Table 1); (ii) compo�
nent complement C1q; and (iii) neonatal FcRn described
above. The receptors FcγR, which differ structurally from
the neonatal FcRn, are classified into three types: FcγRI
(CD64), FcγRII (CD32), and FcγRIII (CD16) [17]. The
receptor FcγRI, which plays an important role in protec�
IgG3
κ2γ2
λ2γ2
γ3
170
3
7�21b
++/+++b
+++
61
0.89�0.91
0.017
7.7�9.8
1.1
–
–
++/+++b
–
+++
+++
IgG2
κ2γ2
λ2γ2
γ2
146
3
21
++
+
–
0.1�0.45
0.02
0.02�0.03
–
–
–
+++
++++
++++
++++
IgG1
κ2γ2
λ2γ2
γ1
146
3
21
++++
+
65
3.5�5.2
0.12
1.2�2.0
0.2
–
–
+++
+++
++++
++++
Molecular formula [10]
Subclasses [10]
Molecular mass, kDa [10]
Carbohydrate content, % [10]
Half�life, days [9]
Transport across placenta [9]
Binding to C1q [9]
Binding toFcγR [9]
Binding to FcαIR [11]
Binding to FcεR [12]
Binding to FcRn [9]
Binding to protein Аc [13]
Binding to protein Gc [14]
Binding to protein Ld [15]
Table 1. Properties of human immunoglobulins
a For dimeric form of secretory IgA.b Depending on allotype.c Staphylococcus aureus.d Peptostreptococcus magnus.
IgE
κ2ε2
λ2ε2
190
12
2.5
–
–
–
–
+
–
+
–
+++
IgD
κ2δ2
λ2δ2
–
185
12
2.8
–
–
–
–
–
–
++
–
+++
IgM
κ2μ2
λ2μ2
–
950
12
5
–
+
–
–
–
–
–
–
+++
IgA
κ2α2
λ2α2
α1�2
385а
7.5
5�6
–
–
–
+
–
–
+
–
+++
IgG4
κ2α2
λ2α2
γ4
146
3
21
+++
–
34
0.17�0.24
0.02
0.2�0.25
–
–
–
+++
++++
++++
++++
IgG
FcγRI (CD64)
FcγRIIA (CD32)
FcγRIIB (CD32)
FcγRIIIA (CD16a)
FcγRIIIB (CD16b)
Association constant for monovalent ligand(×10–6 M)
838 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
tion against bacterial infections, demonstrates the highest
affinity to both monomeric IgG and immune complexes
due to formation of additional hydrogen bonds and salt
bridges with the lower part of the Fc hinge region [19]. On
the other hand, FcγRIIA and FcγRIIIA bind effectively
immune complexes, but demonstrate weak binding of
monomeric IgG due to low affinity [18]. The ADCC
reaction is initiated by interaction between the Fc�region
of the antibody bound to the target cell and FcγRIIIA
(CD16a), which is present on the surface of natural killer
(NK) cells (Fig. 2). This causes the release of cytotoxic
granules from the activated NK cells, which contain per�
forin and granzymes. FcγRIIIA binds to the human IgG
subclasses with different affinities (Table 1), and only the
Fc�regions of IgG1 and IgG3 are capable of induction of
ADCC in humans [9]. It is important for the discussed
topic that the interaction of FcγR with antibodies defines
the efficiency of TMAs [25]. It is known that the anti�
cancer effect of rituximab and trastuzumab is significant�
ly lower when tested in mice deficient in FcγR receptors
[26] or having a single nucleotide replacement in the
FcγRIIA gene at position 131 (rs1801274) and in the
FcγRIIIA gene at position 158 (rs396991) [27].
Some modifications of IgG could directly affect its
binding to FcγR and following activation of cells. For
example, a triple mutant of the Fc�region of IgG1
(S298A/E333A/L334A) increases efficiency of antibody
binding to FcγRIIIA and following activation of ADCC
[28]. The Fc mutant variants S239D/I332E and
S238D/I332E/A330L stimulate manifestation of ADCC
via increased ability to bind FcγRIIIA with simultaneous
decrease in the ability to bind FcγRIIB and activate CDC
[29]. These observations are in agreement with the results
of testing of recombinant anti�HER2 monoclonal antibod�
Fig. 1. Schematic representation of main structural and functional elements of immunoglobulin G. The light chain of the antibody consists of
variable (VL) and constant (CL) domains, and the heavy chain contains variable (VH) and three constant domains (CH1, CH2, CH3). The anti�
gen binding fragment (Fab) is bound to the Fc�domain via a flexible hinge region (HR). Variable domains VL and VH contain hypervariable
regions (HVR) defining antibody specificity and forming paratopes. The antibody structure is stabilized by intra� and inter�molecular disul�
fide bonds.
HR
HVRParatope
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 839
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
Fig. 2. Major effector functions of antibodies. The Fc�region in antibodies is responsible for induction of antibody�dependent cell cytotoxic�
ity (ADCC) via interaction with the FcγIIIA receptor on the surface of immune cells (for example, NK cells) that initiates production of cyto�
toxic granules, which results in degradation of the target cell (for example, a cancer cell) membrane. Binding to the FcγIIA receptor in mono�
cytes (M) occurs during induction of antibody�dependent cellular phagocytosis (ADCP), which initiates phagocytosis. The Fc�region is also
an inducer of complement�dependent cytotoxicity (CDC): interaction of the complement C1q system with the antibody Fc fragment initi�
ates a reaction cascade causing the destruction of the target cell membrane.
Membrane
destruction
Activation
of complement
system
Lysis
Target cell
840 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
ies with replacements L235V, F243L, R292P, Y300L, and
P396L: enhanced ability to bind the FcγRIIIA receptor
and kill cancer cells was observed for these antibodies [30].
The Fc�region of IgG1 and IgG3 are also potent
inducers of phagocytosis and CDC (Fig. 2). The CDC
mechanism uses the complement protein system, when
the C1q component of the complement in conjunction
with the Fc�region of antibody bound to the cell initiate
the reaction cascade, destroying the target cell membrane
[17]. Bindings of the Fc�region of IgG1 or IgG3 with C1q
or FcγR are two mutually exclusive processes. The anti�
body molecules bind to C1q or FcR�carrying cells, but
not with both targets simultaneously [31].
SHORT ESSAY ON N� AND O�GLYCOSYLATION
OF PROTEINS IN EUKARYOTES
Antibodies synthesized in an animal cell are typical
glycoproteins containing more than 10 monosaccharides
and belonging to two major types: N�glycoproteins with
carbohydrate linked to an asparagine (Asn) nitrogen
atom, and O�glycoproteins that are bound via oxygen
atom of serine (Ser) or threonine (Thr) [5]. The N�gly�
cans of antibodies are most extensively studied [32].
Animal glycoproteins predominantly contain 10 types of
monosaccharides with seven of them found in the human
body: glucose (Glc), galactose (Gal), fucose (Fuc), man�
nose (Man), N�acetylglucosamine (GlcNAc), N�acetyl�
galactosamine (GalNAc), and N�acetylneuraminic acid
(Neu5Ac).
Biosynthesis of polysaccharides (glycans) in mam�
mals occurs in the endoplasmic reticulum (ER) and
Golgi apparatus and involves actions of multiple trans�
membrane enzymes such as glycosyltransferases and gly�
cosidases [33].
The process of protein N�glycosylation occurs with
participation of dolichol, which consists of a chain of
five�carbon isoprene units with linear “head�to�tail”
linkage. The process starts with the synthesis of a
dolichol�glycan precursor, which is a dolichol�phosphate
(Dol�P) bound to the pre�assembled oligosaccharide
containing 14 monosaccharides (Glc3Man9GlcNAc2)
conserved across all eukaryotes [5].
The N�glycosylation is initiated in ER by the
oligosaccharyltransferase protein complex (OST) and the
transfer of the pre�assembled glycan (Glc3Man9GlcNAc2)
(Fig. 3) to asparagine (located in the amino acid sequence
with Asn�X�Ser/Thr motif, where X represent any amino
acid except proline) of the synthesized polypeptide chain
[34]. Next, a series of trimming and arrangement reac�
tions common for all eukaryote cells occurs in the lumen
of the ER following the attachment of the precursor to Asn
residues (Fig. 3).
The initial maturation steps involve sequential
removal of three Glc residues by glycosidase I and II
(GCS I, II) and production of the Man9GlcNAc2 glycan,
from which the Man8GlcNAc2 polysaccharide is generat�
ed by endomannosidases (MNS) that eventually becomes
a substrate for diversification and maturation in the Golgi
apparatus. The Man8GlcNAc2 diversification in humans
starts with removal of three mannoses by MNS I, which is
followed by addition of β1�2�GlcNAc catalyzed by
GlcNAc�transferase I (GNT I) and removal of the α1�3�
and α1�6�Man by MNS II. The resulting hybrid N�glycan
GlcNAc1Man3GlcNAc2 is a specific substrate for the
Fig. 3. Major stages of formation of N�linked glycans in humans. The process of protein N�glycosylation starts in the ER, where the “glycan�
precursor” (Glc3Man9GlcNAc2) is assembled on dolichol phosphate, from which the oligosaccharide is transferred next to the asparagine of
the protein target catalyzed by oligosaccharyltransferase (OST). Next, cleaving of Glc residues occurs in ER catalyzed by glycosidase, which
is followed by further modification of the attached glycan in the Golgi apparatus: cleavage of Man from high�mannose forms by mannosidas�
es (MNS I, II) with further addition of GlcNAc, Fuc, Gal, and Neu5Ac. As a result, either hybrid glycans can be formed or the complex type
of glycans. GNT I and II, GlcNAc�transferase I and II; FUT8, α1,6�fucosyltransferase; GALT, β�1,4�galactosyltransferase; SIAT, α�2,3/6�
sialyltransferase.
ER Golgi apparatusCis� Trans�
High
mannose
glycans
Hybrid
glycans
Complex glycans
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 841
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
GNT II enzyme, which catalyzes transition from hybrid
to complex N�glycans [35].
Hence, the N�glycan structures are subclassified into
three categories: with high mannose content, hybrid, and
complex types. The high�mannose type of N�glycans can
have from two to six additional mannose residues linked to
the trimannosyl core of the glycan. The complex type does
not have additional Man residues, but it has additional
polysaccharide branches containing GlcNAc, Gal, and
sialic acid residues. The hybrid type has one branch of the
high�mannose type and another branch of the complex
type. The hybrid and complex N�glycan can be bi� (such
as immunoglobulins), tri�, and tetra antennary complexes
(such as FcγR) according to the number of branches [34].
Unlike the N�linked glycosylation, O�glycosylation
of proteins has been investigated to a much lesser extent
[36]. Nevertheless, O�glycosylation is in many ways sim�
pler and starts in humans in the Golgi apparatus, typical�
ly via attachment of one of either GalNAc, Man, or Fuc
residues to serine or threonine of the polypeptide chain.
There is no known consensus sequence found for O�gly�
cosylation, although glycosylated residues of serine and
threonine have been observed that are flanked by proline.
The next step might be the addition of a sialic acid
residue, which completes the chain, or step�wise addition
of a large number of monosaccharides to form longer lin�
ear or branched chains. The O�glycans are usually shorter
than the N�linked ones.
SYSTEMS FOR TMA PRODUCTION
The fundamentals of TMA production technology
were suggested by Köhler and Milstein [37], who devel�
oped a technique for converting B�lymphocytes into an
immortal form, which, in turn, allowed synthesizing mono�
clonal antibodies in cell culture. The pharmaceutical
industry immediately accepted the hybridoma technology
because on one hand the technology could not be patent�
ed, being already published in 1975, and on the other hand
antibodies produced using this technology became the
subjects of separate patents, which was very profitable for
pharmaceutical companies producing TMAs. These cir�
cumstances allowed designing and testing on short notice
mouse antibodies such as orthoclone OKT3
(Muromonab�CD3), which were approved by the FDA
already in 1985. Hence, OKT3, which prevented rejection
of a donor organ during kidney transplantation, became
the first TMA [38]. In the next steps of development of this
technology, the problem of hybridoma instability was
solved and techniques were developed for humanizing
mouse antibodies, which alleviate the problem of induc�
tion of anti�mouse antibodies in patients [39].
Biosynthesis of antibodies or their fragments using
genetic engineering and heterologous expression systems
in cells of such organisms as bacteria, yeasts, insects,
plants, and mammals became a promising alternative to
hybridoma technology. Now TMAs are produced pre�
dominantly in animal cells, because these cells ensure the
synthesis of antibodies that are the closest to human anti�
bodies in carbohydrate composition. The TMAs are pro�
duced in one of three cell lines: Chinese hamster ovary
cells (CHO), murine NS0 (non�secreting mouse myeloma
cells), or SP2/0 (murine myeloma cells), and less often in
human cells lines HEK293 or PER.C6 [8, 39�42].
The CHO cell line continues to be a “work horse”
for production of more than 70% of therapeutic proteins
(Table 2) since its first successful application for produc�
tion of plasminogen activator (Activase®) in 1986. The
reason for this lies in the fact that CHO cells ensure the
highest level of antibody production, reaching the titer of
1 g/liter in a batch�system (production in a limited vol�
ume) and from 1 to 10 g/liter in a fed�batch�system (batch
process with addition of nutrients) [43]. The CHO cells
can be genetically modified and cultivated either as adhe�
sive cells or as suspensions. The procedures of cell trans�
fection, gene amplification, and clone selection in CHO
cells are well developed and widely used. At present,
CHO cells are the most popular choice for posttransla�
tional modification of TMAs, in particular their glyco�
modification including fucosylation and sialylation. The
fact that these cells are not susceptible to human viruses is
an important feature of this cell line. It must be taken into
account that viral infections could interfere with the
process of cultivation of cell producers, affecting quality
and yield of TMA. Hence, it follows that that the resist�
ance of CHO cells to human viruses makes them the cells
of choice as host cells for TMA production.
The murine cell lines NS0 and SP2/0 demonstrate
10�fold lower yield of antibodies as compared with CHO
[44]. The human cells HEK293 are more often used for
transient expression of genes encoding chains of antibod�
ies, and the antibody yield in these cells is 2�3�fold lower
in comparison with CHO. Good parameters were
demonstrated for the human embryonic retinoblast cell
line PER.C6: the antibody yield was comparable with
CHO and reached 0.5 and 8 g/liter for the batch� and fed�
batch�systems, respectively. In the case of perfusion culti�
vation mode, the PER.C6 cells (http://www.lonza.com)
can produce up to 27 g/liter [45]. It must be noted that
the realized parameters of antibody yields in animal cell
systems can be overridden [39] as a result of: (i) improve�
ment of vector systems [1]; (ii) genetic modification of
cells aimed to enhance biosynthetic activity via decreas�
ing probability of apoptosis; (iii) increasing efficiency of
mRNA translation of target genes [39], and (iv) improv�
ing antibody secretion by cells [46].
Hence, the production system based on mammalian
cells provides the complete spectrum of posttranslational
modifications including N�glycosylation and therefore is
widely used for production of various recombinant pro�
teins, including TMAs.
842 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
No.
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Area of applicationand treatment
6
rheumatoid arthritis, ankylosing spondylitis,Crohn’s disease, ulcerativecolitis
chronic lymphoid leukemia,skin T�cell lymphoma, multiple sclerosis
high cholesterol level not controlled by diet and statins
to prevent organ rejectionduring transplantation
systemic lupus erythematosus
inhibits growth of new bloodvessels in tumors
classical Hodgkin’s lymphoma, systemic anaplastic large cell lymphoma
cryopyrin�associated periodic syndrome
malignant ascites, conditions developing in metastatic cancer
colorectal cancer with wild�type KRAS homolog (Kirsten rat sarcoma viraloncogene)
prophylaxis of kidney transplant rejection
multiple myeloma
osteoporosis
neuroblastoma
treatment for paroxysmalnocturnal hemoglobinuria
high cholesterol level not controlled by diet and statins
Table 2. List of TMAs approved for clinical use
Target
5
TNF alpha
CD52
PCSK9
IL2R
BLyS
VEGF�А
CD30
IL�1β
EpCAM/CD3
EGFR
receptor IL�2
CD38
RANK�L
GD2
complementcomponent C5
PCSK9
Antibody format
4
IgG1 (human)
IgG1 (rat) humanized
IgG1 (mouse)humanized
IgG1, chimeric (mouse/human)
IgG1 (human)
IgG1 (mouse)humanized
IgG1 chimeric(mouse/human),monomethylauristatin E conjugate
IgG1 (human)
biospecific, rat IgG2b/mouse IgG2a
IgG1 chimeric(mouse/human)
IgG1 (mouse) humanized
IgG1 (human)
IgG2 (human)
IgG1 chimeric(mouse/human)
IgG2/4 (mouse)humanized
IgG1 (human)
Cell line –antibody
producers
3
CHO
CHO
CHO
Sp2/0
NS0
CHO
CHO
Sp2/0
hybridoma
Sp2/0
CHO
CHO
CHO
SP2/0
NS0
CHO
Name according to USANC*(commercial name)
2
Adalimumab (Humira®)
Alemtuzumab (MabCampath, Campath�1H®)
Alirocumab (Praluent®)
Basiliximab (Simulect®)
Belimumab (Benlysta®)
Bevacizumab (Avastin®)
Brentuximab vedotin(Adcentris®)
Canakinumab (Ilaris®)
Catumaxomab (Removab®)
Cetuximab (Erbitux®)
Daclizumab (Zenapax®)
Daratumumab (Darzalex®)
Denosumab (Prolia®)
Dinutuximab (Unituxin®)
Eculizumab (Soliris®)
Evolocumab (Repatha®)
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 843
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
1
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
6
acute myeloid leukemia
rheumatoid and psoriaticarthritis, ankylosingspondylitis
radioimmunotherapy of resistant lymphomas
Crohn’s disease, ulcerative colitis psoriasis,rheumatoid arthritis
melanoma
psoriasis
asthma, atopic dermatitis
T�cell leukemia and adultleukemia
multiple sclerosis andCrohn's disease
melanoma
chronic lymphoid leukemia,follicular non�Hodgkin’slymphoma
chronic lymphoid leukemia,follicular non�Hodgkin’slymphoma
prevention of allergic asthma
prevention of RSV infection,for example, in prematurebabies
rectal cancer
melanoma
HER2�positive breast cancer
stomach cancer
anthrax
asthma
Table 2 (Contd.)
5
CD33
TNF alpha
CD20
TNF alpha
CTLA�4
IL�17a
IL�5
CCR4 (C–Cchemokinereceptor 4)
α4�integrin
PD1
CD20
CD20
Fc�fragment of IgE
RSV, protein F
EGFR
PD1
HER2
receptorVEGF
Bacillus anthracis
IL�5
4
IgG4 (mouse) humanized
IgG1 (human)
IgG1 (mouse)
IgG1 chimeric(mouse/human)
IgG1 (human)
IgG1 (mouse)humanized
IgG1 (mouse)humanized
IgG1 (mouse)humanized, with afucosylatedFc�fragment
IgG4 (mouse)humanized
IgG4 (human)
(mouse) completelyhumanized, with afucosylatedFc�fragment
IgG1 (human)
IgG1 (mouse)humanized
IgG1 (mouse)humanized
IgG2 (human)
IgG4 (mouse)humanized
IgG1 (mouse)humanized
IgG1 (human)
IgG1 (human)
IgG1 (mouse) humanized
3
NS0
Sp2/0
CHO
Sp2/0
CHO
–***
CHO
CHO
NS0
CHO
CHO
NS0
CHO
NS0
CHO
CHO
CHO
NS0
NS0
NS0
2
Gemtuzumab ozogamicin(Mylotarg®)
Golimumab (Simponi®)
Ibritumomab tiuxetan(Zevalin®)
Infliximab (Remicade®)
Ipilimumab (Yervoy®)
Ixekizumab** (Taltz)
Mepolizumab (Nucala®)
Mogamulizumab (Poteligeo®)
Natalizumab (Tysabri®)
Nivolumab (Opdivo®)
Obinutuzumab (Gazyva®)
Ofatumumab (Arzerra®)
Omalizumab (Xolair®)
Palivizumab (Synagis®)
Panitumumab (Perjeta®)
Pembrolizumab (Keytruda®)
Pertuzumab (Perjeta®)
Ramucirumab (Cyramza®)
Raxibacumab
Reslizumab** (Cinqair)
844 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
THERAPEUTIC MONOCLONAL ANTIBODIES
The TMA nomenclature is suggested by the govern�
ing body of the American Medical Association together
with the US Adopted Name Council (USANC) and
approved by the World Health Organization (http://www.
who.int/medicines/services/inn/). The list of prepara�
tions approved for clinical studies is changed annually as
a results of inclusion or removal of TMA [47]. Moreover,
the USANC does not revise the name of an antibody after
that so as not to confuse doctors and patients. The full
name of a TMA includes three main elements signifying
sequentially – [target] – [source] – [abbreviated designa�
tion of monoclonal antibody, suffix �mab] (for polyclonal
antibody suffix �pab is used). In this manner the letter
indicating the source (�u� human, �o� mouse, �a� rat,
�zu� humanized, �e� hamster, �i� primates, �xi� chimera,
�axo� chimeric chain rat/mouse, and �xizu�, indicating
combination of humanized and chimeric chains) pre�
cedes the suffix �mab. The source designation is preceded
by the TMA target designation or the disease that is an
encrypted indication of the disease, organ, tumor or its
subclass against which the antibody has been raised: �vir�
viral, �bac� bacterial, �lim� immune system, �les� inflam�
matory lesions, �cir� cardiovascular system, �fung� anti�
fungal, �ner� nervous system, �kin� interleukin, �mul�
musculoskeletal system, �os� bones, �toxa� target toxin,
�col� colon, �mel� melanoma, �mar� mammals, �got� tes�
ticle, �gov� ovary, �pr(o)� prostate, and �tum� various
other tumors. The company producing TMA must add a
prefix to this triad (from two or more syllables) to the
beginning of the name in order not to repeat or be in con�
flict with the existing list of known TMAs. Let us select a
well�known antibody – trastuzumab – to illustrate this
principle and break the name into elements: [tras] –
[tu] – [zu] – [mab], which correspond to [syllable(s) dif�
fering from the previously used TMAs] – [type of disease
or target, the last consonant is left out to simplify pro�
nunciation] – [source of antibody] – [suffix].
Numerous TMAs have been created since the late
1990s [47]. The majority of licensed antibodies are full�
size TMAs (Table 2) rather than Fab�fragments, because
full�size antibodies demonstrate longer lifetime in the
blood stream of patients as a result of interaction with
FcRn. Most TMAs are either humanized or fully human
because the use of murine or chimeric antibodies is
accompanied with increased risk of unfavorable immuno�
logical reactions in patients.
The TMAs are classified into various types in accor�
dance with the target of their activity: either they are the
1
37
38
39
40
41
42
43
44
6
lymphomas, leukemia, transplant rejection, and autoimmune diseases
immunosuppression, transplant medicine
lymphoproliferative disorder:Castleman’s disease
immunosuppressive drug to treat rheumatoid arthritisand its other forms
rituximab�resistant lymphomas
HER2�positive breast cancer
psoriasis, multiple sclerosis
ulcerative colitis, Crohn’s disease
Table 2 (Contd.)
5
CD20
IL�17a
IL�6
receptor IL�6
CD20
HER2
IL�12 and IL�23
Integrin α4β7
4
IgG1κ chimeric(mouse/human)
IgG1 (human)
IgG1 chimeric(mouse/human)
IgG1 (mouse)humanized
IgG2aλ (mouse)
IgG1 (mouse)humanized
IgG1 (human)
IgG1 (mouse)humanized
3
CHO
CHO
CHO
CHO
Hybridoma
CHO
Sp2/0
CHO
2
Rituximab (MabThera®,Rituxan®)
Secukinumab (Cosentyx®)
Siltuximab (Sylvant®)
Tocilizumab (RoActemra,Actemra®)
Tositumomab and 131I�labeledTositumomab (Bexxar®)
Trastuzumab (Herceptin®)
Ustekinumab (Stelara®)
Vedolizumab (Entyvio®)
* USANC, US Adopted Name Council.
** Approved by FDA in 2016.
*** No information.
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 845
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
agents neutralizing pathogens or the agents targeting
molecules causing diseases in humans (Table 2). From 44
antibodies licensed at the beginning of 2016, only two –
palivizumab (Synagis®) and raxibacumab – are intend�
ed for treatment of infectious diseases. This is signifi�
cantly lower than the number of antibodies for treatment
of cancer or autoimmune diseases. In this category the
leaders are the antibodies directed against tumor necro�
sis factor α (TNFα). According to the data from
Pharmacompass (http://www.pharmacompass.com),
adalimumab (Humira®) intended for treatment of
rheumatoid arthritis and Crohn’s disease was the best�
seller in 2015. Rituximab (MabThera®, Rituxan®)
intended predominantly for treatment of lymphomas and
leukemia is the bestseller among the anticancer prepara�
tions. This is followed by bevacizumab (Avastin®) and
trastuzumab (Herceptin®) (Table 2).
The high profit from sales of anticancer TMAs is due
to the high price of immunotherapy in the United States:
the cost per patient in the USA is from 20,000 to 100,000
dollars annually [3]. The fact is that for immunotherapy
to be efficient, it is necessary to maintain a high concen�
tration of the preparation in the patient’s blood serum,
which is achieved via repeated introduction of antibodies
(from 1 to 10 mg/kg). Multiple administrations of the
preparation require several grams of TMA per patient.
The necessity of introduction of such amounts of TMA is
explained by the facts that: (i) there is competition
between the therapeutic antibody and endogenous IgG
for the FcγR that inhibits ADCC [48], and (ii) only small
part of the total amount of antibodies introduced to the
patient finds its way into the solid tumor due to the high
molecular mass (≈150 kDa) of the full�size TMA. For
example, it was shown that the fraction of antibodies in
the bulk of the rectal cancer tumor is no more than 0.1%
of the total introduced amount [49]. On the other hand,
the fact that the serum TMA concentration following its
administration could be at the level of several hun�
dred μg/ml for more than two weeks contributes to the
high efficiency of immunotherapy. It is related to the fact
that full�size TMAs approved for use in clinical practice
(Table 2) have usually been created on the basis of IgG1
(less often IgG2 and IgG4), which demonstrate long half�
life (Table 1), this being explained by the mechanism of
antibody recirculation and the presence of FcRn in
endothelial cells of blood vessels. The lack of approved
TMAs based on IgG3 is explained [16] by the increased
probability of their proteolysis due to the extensive hinge
region of the antibody [50] and inability of IgG3 to bind
the protein (Table 1).
In addition to the original licensed TMAs (Table 2),
so�called biosimilar antibodies (or biosimilars) started to
enter market after 2014 when patents expired, which were
usually produced by other companies. The term “biosim�
ilar” is commonly used for high molecular weight com�
pounds including TMAs; it should not be confused with
the term “generic” used for relatively low molecular
weight pharmaceutical preparations. The process of com�
mercialization of biosimilars is controlled by the FDA (in
United States) and by EMA (European Medicines
Agency, EU) [51]. The main requirement is that biosimi�
lars must be characterized thoroughly to confirm suffi�
cient comparability with the respective original standard
TMA.
In the Russian Federation, the pharmaceutical com�
pany Biocad (http://biocad.ru/) began production of a
rituximab biosimilar (trade name Acellbia™) in 2014,
bevacizumab in 2015, and trastuzumab (trade name
HERtiCAD™) in 2016.
The TMAs, both original and biosimilars, must com�
ply with the critical quality attributes [52] determining
safety, efficiency, pharmacokinetics, and clearance of
antibodies [7]. It must be considered in the process that
the antibodies produced in human and mammalian cells
display molecular heterogeneity, which must be con�
trolled.
GLYCOSYLATION OF HUMAN SERUM
IMMUNOGLOBULIN IgG
As in the case of majority of glycoproteins, the
immunoglobulins from a healthy human are subjected to
glycosylation in the ER and Golgi apparatus [9]. The
degree of glycosylation of different classes of
immunoglobulins varies: glycans comprise ∼3% of the
total mass of the IgG antibody, which is a relatively low
value in comparison with IgM, IgD, and IgE (Table 1).
The N�linked glycans are found in both the Fc� and Fab�
fragments of all types of immunoglobulins. The O�glycans
have been found in the hinge region of IgD, IgA, and
IgG3 [53]. Polyclonal human IgG serum contains mainly
N�glycans of the complex type (Fig. 3). The glycans of
Fc� and Fab�fragments differ significantly in the content
of specific residues, although they have common bianten�
nal structure consisting of branching heptasaccharide and
two disaccharide units (antennas). The availability of
large multiantennal glycans such as tri�, and tetra�anten�
nal glycans in the IgG composition of healthy humans has
not been confirmed. The structure of biantennal N�gly�
cans of Fab� and Fc�fragments of the antibody are shown
in Fig. 4, where the heptasaccharide core consists of two
GlcNAc residues, three Man residues, as well as two
GlcNAc residues. The Fuc residue branching from the
GlcNAc is also added together with Gal and negatively
charged sialic acid. The glycan Fab�fragments of IgG
contain a higher percentage of the branching GlcNAc
residue in the mannose branch, and of Gal and sialic
acid, but lower percentage of Fuc in comparison with the
Fc�fragment.
Glycosylation of Fab�region of IgG. Approximately
15�25% of Fab in the IgG from healthy humans is glyco�
846 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
sylated [54]. Such dispersion is related with both the used
testing methods and the physiological state of the human.
It was found that the sialic acid content increases in preg�
nant women, and the fraction of branching GlcNAc
residues in the glycans of Fab�fragments decreases [54].
Interestingly, the fraction of monosialylated glycans
increases during pregnancy from 37 to 41% (p = 0.0001)
with simultaneous decrease in the disialylated glycans
from 56 to 51% (p = 0.0001) [55]. It was suggested that
the increased glycosylation of Fab�fragments during preg�
nancy provides protective functions minimizing elimina�
tion of genes inherited from the father from the fetus [54].
The change in glycosylation degree of IgG Fab�fragments
has been observed in human pathological states. For
example, increased up to 80% carbohydrate content in
the Fab�fragment of autoantibodies was recorded in
patients with rheumatoid arthritis, with simultaneous
decrease in the ability of autoantibodies to bind antigen
[56]. The emergence of new N�glycosylation sites in the
Fab�fragment was observed in B�cell lymphoma [57].
Tumor�specific N�glycans demonstrate high�mannose
structure, while normal N�glycans of the complex type
have been found in the Fc�fragment [58].
N�glycosylation of Fab�fragments can both increase
and decrease the following: (i) ability of IgG to bind anti�
gen [59, 60]; (ii) half�life of antibody [61�63]; (iii) aggre�
gation of antibody and formation of immune complex
[64, 65]. The mechanism of the effect of glycans on these
properties of antibody is unclear, but a hypothesis on the
effect of a carbohydrate residue on conformation of the
variable region of the antibody has been suggested as an
explanation for these effects [54].
Glycosylation of Fc�fragment of IgG. Significantly
more studies have been devoted to glycosylation of the
Fc�fragment of IgG in comparison with the Fab�fragment
because the possibility for modification of the functional
activity of therapeutic antibodies attracts significant
interest. The N�linked glycan is located in the CH2
domain at position Asn297 (in accordance with the
nomenclature based on numbering of the heavy γ�chain
sequence of IgG) or Asn84.4 (according to the nomen�
clature of the international ImMunoGeneTics system
(www.imgt.org)).
The model of crystal structure of the human IgG Fc�
fragment contains a horseshoe�like structure that, as sug�
gested, mediates effector functions of the antibody
Fig. 4. N�linked Fab and Fc glycans. Schematic representation of IgG with indication of the glycan fraction containing fucose, galactose, sial�
ic acid, and branched GlcNAc shown by arrows. Curly brackets show percent of glycans containing two residues of sialic acid.
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 847
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
(ADCC, CDC, and ADCP) and antiinflammatory activ�
ity [66]. The pair of N�linked glycans attached to Asn297
in the CH2 domain forms a hydrophobic core. No direct
contact between the FcγR and glycan attached to Asn297
was found, but the presence of the latter affected the
degree of accessibility of the horseshoe�like structure and,
as a result, affected the binding of the Fc�fragment with
FcγR and C1q. The hydrophobic residues (Phe241,
Phe243, Val262, and Val264) in the Fc�fragment jointly
limit flexibility of the Fc�fragment glycan via simultane�
ous interaction with the hexose ring of the pentasaccha�
ride core of the glycan and the GlcNAc, which causes as
suggested the decrease in the level of terminal galactosy�
lation and sialylation of the Asn297�linked glycan [67].
The complete removal of the Asn297�linked glycan fol�
lowing treatment of antibodies with glycosidases results in
formation of the closed conformation of the Fc�frag�
ment, which decreases significantly the efficiency of IgG
binding with the majority of FcγR [68]. If only one
GlcNAc residue remains attached to Asn297 as a result of
the enzyme treatment, the binding of the Fc�fragment
with the FcγRIIIA receptor is completely canceled [69].
If the terminal monosaccharide residues of the Asn297�
linked glycan are removed by exoglycosidases, the posi�
tion of the CH3 domains does not change, but the dis�
tance between the two CH2 domains decreases in tandem
[70] and the ADCC function decreases [71]. The used
experimental approach allowed determining the mini�
mum size of the Asn297�linked glycan (three�sugar
nucleus) that still guarantees structural stability of the Fc�
fragment and its effector function [72].
Another important structural feature of the Asn297�
linked glycan defining properties and function of the
human IgG is fucosylation of the glycan core. Fucose
attached to the glycan nucleus via the α1,6�bond decreas�
es the efficiency of interaction of the Fc�fragment with
FcγRIIIA and activation of ADCC [73]. Defucosylation
of the Asn297�linked glycan decreases the efficiency of Fc
binding with the FcγRI manifold, but at the same time it
increases the degree of its binding to the FcγRIIIA and,
respectively, increases the effector ADCC function.
Comparison of crystal structures of the fucosylated and
non�fucosylated Fc�fragment–FcγRIIIA complexes pro�
vided an explanation to these results: the Fc�fragment
with defucosylated Asn297�glycan demonstrates stronger
binding with the FcγRIIIA receptor via interaction with
the Asn162�glycan of the receptor [73]. Understanding of
the role of fucose in the effector function of antibody pro�
vides a new possibility for developing the method of afu�
cosylation to increase TMA activity.
Increase in the ADCC�function of an antibody can
also be achieved via attachment of a GlcNAc residue to
the mannose fork via enhanced expression of β1,4�N�
acetylglucosoaminyltransferase III (GNT III) [74]. The
presence of GlcNAc in the mannose fork inhibits further
addition of fucose during biosynthesis of the Asn297�gly�
can, and that is probably why the efficiency of the
ADCC�function of the antibody increases.
The terminal galactose of the Asn297�glycan (Fig. 4)
also participates in binding of the Fc�fragment with
FcγRIIIA. However, unlike the core fucose, not the
removal of galactose, but rather hypergalactosylation
increases the affinity of the Fc�fragment due to, as sug�
gested, an increase in the structural rigidity of the CH2
domains [75].
Negatively charged sialic acid and the neutral man�
nose in the terminal region of the Asn297�glycan also
have an impact on the effector function of an antibody. It
is suggested that the presence of the bulky and negatively
charged sialic acid residue on the biantennal terminal of
the glycan influences the structure of the hinge region and
decreases the binding efficiency of the Fc�fragment to
FcγRIIIA [76]. As for mannose, despite the fact that the
fraction of high�mannose glycoforms of the Asn297�gly�
can in the total amount of Fc�fragment glycoforms is not
large, artificial increase in mannose content in the
Asn297�glycan increases the ability of the Fc�fragment to
bind FcγRIIIA in comparison with the major biantennal
complex glycan. It was suggested for explanation of this
phenomenon that it could be caused by: (i) the lack of
fucose that decreases affinity of the Fc�fragment, and (ii)
the ability of the large number of mannose residues to dis�
turb the tertiary structure of the Fc�fragment.
Glycosylation of the Fc�fragment is also important for its
efficient binding with C1q and complement activation.
Different glycoforms demonstrate different affinities
towards C1q and, respectively, different efficiency of
CDC activation [77]. In particular, the mature complex
type of the Asn297�glycan activates complement more
efficiently than a hybrid of high�mannose form even if it
is fucosylated. On the other hand, the terminal, negative�
ly charged sialic acid inhibits and the galactose activates
the binding of the Fc�fragment with the C1q.
Much like the Fab�fragment, glycosylation of the
IgG Fc�fragment in human blood serum changes with age
and during pregnancy [78]. The content of agalactosylat�
ed forms increases, while the fraction of sialylated forms
decreases [79]. It is interesting that galactosylation and
sialylation of the IgG caused by pregnancy improves the
course of rheumatoid arthritis [80]. Decrease in galactose
content is also observed during systemic vasculitis such as
Wegener’s granulomatosis and microscopic polyangiitis
[81]. Whereas the normal content of glycoforms in the
Fc�fragment with zero galactose is around 26%, the con�
tent of such glycoforms increases to 55% during systemic
vasculitis. Emergence of the agalactosylated Asn297�gly�
can is characteristic for autoimmune diseases such as
rheumatoid arthritis [81]. It was shown that the content of
agalactosylated structures increased with simultaneous
decrease in digalactosylated N�glycans [82].
Terminal sialylation of the IgG Asn297�glycan can
be considered as a protective reaction during autoim�
848 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
mune diseases [83]. For example, introduction of IgG
with sialylated Asn297�glycan produces a strong antiin�
flammatory effect, which can be used as a basis for devel�
opment of glycomodified TMAs [84].
PECULIARITIES OF TMA GLYCOSYLATION
General characteristic. In addition to relative sim�
plicity of cultivation, cell lines were selected for TMA
production because the antibodies produced in them are
characterized by a carbohydrate profile that is the closest
to the IgG from human blood serum [42, 85]. Now all the
TMAs used in clinics have been produced in Chinese
hamster ovary tumor cells (CHO) or murine myeloma
cells (NS0 or SP2/0) (Table 2). Despite the fact that both
production systems have been developed on the basis of
cells of organisms belonging to the same class of mam�
mals, comparison of the TMA glycosylation profiles
reveals noticeable differences important for clinical appli�
cations. These differences are explained by different con�
tent of intracellular glycosylation factors in the cells
including the pool of synthetases, glycosyltransferases,
and glycosidases, as well as by the differences in the pool
of protein transporters of nucleotide derivatives of sugars
[85].
Moreover, heterogeneity of the carbohydrate com�
position in a TMA preparation produced in the same cul�
ture was observed. For example, the rituximab prepara�
tion (Table 2) is highly heterogeneous with respect to gly�
cosylation of the Fc�fragment [86]. In addition to meta�
bolic features of each individual cell, the cleavage of car�
bohydrate residues by extracellular glycosidases released
from the disrupted producing cells, which is especially
characteristic for the batch� and fed�batch production
systems, contributes to TMA heterogeneity.
Interestingly, high productivity, which is the goal of a
manufacturing company, can facilitate emergence of
immature high�mannose forms of antibodies that are
formed in an early stage of glycosylation prior to addition
of GlcNAc or fucose. This could happen in the case of
high production of antibodies in the Golgi apparatus of
the cell line because of formation of a deficit of the com�
ponents of the system due to insufficient rate of the sub�
strate transfer through cellular and intracellular mem�
brane barrier or insufficient activity of glycosylation
enzymes [87]. The emergence of high�mannose forms of
antibodies has detrimental effect for therapy, because the
high�mannose forms exhibit high rate of clearance of the
preparation from the organism [7, 33].
Immunogenic glycans. One of the features of TMA
production systems in mammalian cells that are different
from human ones is the synthesis of immunogenic glyco�
proteins. Glycan epitopes are known in TMAs that could
cause adverse immunological reactions if administered to
patients. Among these are residues of galactose�α1,3�
galactose (α�Gal) and N�glycolylneuraminic acid
(Neu5Gc) located in the terminal region of the glycan.
Mouse cells contain the enzyme α1,3�galactosyltrans�
ferase, which produces glycans containing α�Gal [39].
This enzyme is inactive in humans but is present in the
majority of other mammals. The negatively charged ter�
minal Neu5Gc residue, which is characteristic for mam�
malian cells (but not for human ones), is formed in a
hydroxylation reaction catalyzed by CMP�Neu5Ac
hydroxylase [41]. The α�Gal and Neu5Gc epitopes are
more characteristic for mouse cells rather than for CHO
and, consequently, the likelihood of a TMA being
immunogenic for humans is higher for the TMAs pro�
duced in mouse cells [85]. This was corroborated by the
medical fact that administration of the cetuximab TMA
to patients, which was produced in murine cell culture
SP2/0 (Table 2) and contained α�Gal and Neu5Gc,
could cause severe anaphylactic shock accompanied by
induction of IgE specific for α�Gal [88]. It must be men�
tioned that the IgE specific for α�Gal and Neu5Gc epi�
topes is present even in healthy humans [89]. Thus far
there is no documented evidence on unfavorable
immunological reactions due to administrations to
patient of TMAs synthesized in CHO. Nevertheless, it
must be taken into account that the enzymes required for
formation of similar immunogenic epitopes are available
in CHO cells [90].
Sialylation. In addition to immunogenic glycans,
other differences in glycosylation pattern of TMA in pro�
ducing cells were observed [39]. For example, a higher
level of sialylation of glycoproteins is characteristic for
CHO cells. It must be noted that the level of sialylation of
the IgG1 from healthy human blood plasma varies in the
range 2�5%, but if the TMAs are produced in CHO cells,
murine myeloma cells J558L, and human HEK293 cells
their content increases to 31, 10, and 33% [83]. The com�
parison of HEK293 or PER.C6 cell cultures with CHO
reveals that both α2,3� and α2,6�sialyltransferase are syn�
thesized in human cells, while the α2,6�sialyltransferase
is absent in the CHO cells. Hence, the terminal sialyla�
tion occurs in the CHO cells exclusively via the α2,3�
bond [83].
Although increased terminal sialylation of the Fc�
fragment of TMA decreases the ADCC effector function
of the TMA [76], it can produce a beneficial effect in
patients. First, all these preparation can have an antiin�
flammatory effect [83, 84]. Second, it is likely that the
negatively charged sialic acid increases stability of TMA
against proteolysis in the patient’s bloodstream, which is
explained by shielding of the terminal galactose residues
(Fig. 4) and thus preventing their binding to galactose�
specific receptors on hepatocytes and following endocy�
tosis [5]. Finally, despite the fact that TMA sialylation
decreases the efficiency of binding to FcγRIII and conse�
quently inhibits the ADCC effector function of TMA,
more favorable conditions are created for interaction with
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 849
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
FcγRIIA and FcγRIIB, and the probability of phagocyto�
sis increases [91].
Fucosylation. More than 95% of the TMAs produced
in CHO cells are fucosylated and do not contain the
branching GlcNAc residue in the mannose fork, while only
70�90% of the TMAs produced in mouse myeloma cells
are fucosylated and they contain ∼10% of glycans with a
branching GlcNAc residue in the mannose fork [92].
Galactosylation. The Asn297�glycans of the Fc�frag�
ment of TMA contain 0, 1, or 2 (G0, G1, and G2, respec�
tively) terminal galactose residues [93] (Fig. 5). The
TMAs synthesized in CHO cells are usually less galacto�
sylated as compared with the TMAs isolated from murine
myeloma cells. Despite the large diversity in galactose
content, so far no adverse consequence was observed in
the practice of TMA application. Nevertheless, galacto�
sylation impacts mechanisms of action of some TMAs, in
particular their effector functions. For example, change
in binding with C1q complement and manifestation of
CDC was observed for rituximab [94].
Summing up the differences in the ability of the pro�
ducing cells to glycosylate the TMAs used in practice, we
note (Fig. 6) that the synthesis in CHO cells results in the
absence of α2,6�sialic acid and of branching GlcNAc
residue in the mannose fork as well as increased sialylation
of antibodies (up to 31%). TMA production in mouse
myeloma cells results in emergence of α1,3�galactose and
increased sialylation of the antibodies (up to 10%).
Role of culture medium. The particular profile of
TMA glycosylation depends not only on the cell line, but
also on the culture medium [42]. For example, terminal
sialylation and glycosylation of IgG1 increases in serum�
free medium [5]. The glucose content in the medium
influences the TMA glycosylation. For example, reduc�
tion of glucose content up to its complete absence results
in increase in non�glycosylated forms by 45% [95]. The
TMA glycosylation profile is affected by the method of
cell cultivation including pH of the medium [96], cultiva�
tion temperature [97], and content of dissolved oxygen
[98]. Change in medium components aimed at change in
TMA glycosylation profile could inhibit growth of cells,
decrease their titer, and, eventually decrease productivity
of the culture [33]. For example, the use of GlcNAc as a
medium component has a detrimental effect on the cul�
ture growth by suppressing glucose transport into the cell
[99].
A specific glycosylation pattern is characteristic for
each of the three most used regimes of TMA production:
batch�system (production in a limited volume), fed�
batch�system (process with nutrient addition during cul�
tivation), and perfusion (continuous renewal of the cul�
ture medium). A large number of studies have been devot�
ed to this topic, and they have been reviewed in a recent
publication of Kunert and Reinhart [39]. It is worth men�
tioning here that periodic cultivation in the batch�system
results in exhaustion of nutrients as well as accumulation
of products of metabolism and cell degradation, causing
changes in glycosylation during cultivation. The fed�
batch�system that increases lifetime of the culture and cell
density due to periodic addition of nutrients could result
in degradation of glycans by extracellular enzymes
released by cells or via cell lysis, which also could be
responsible for heterogeneity of glycoforms [85]. The per�
fusion system does not suffer from these drawbacks.
Continuous renewal of the culture medium provides more
homogenous composition of glycoforms and, as an exam�
ple, increases the degree of sialylation [100].
The procedure for production of biosimilars
approved by FDA and EMA recommends using the same
type of producing cells as for the original TMAs, because
the glycosylation profile is significantly different for dif�
ferent types of cell cultures. Moreover, the EMA and
Fig. 5. Variants of N�linked glycans of Fc of TMAs produced in CHO or murine cell line with different galactose content.
850 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
FDA require detailed comparison of carbohydrate struc�
tures including general profile and site�specific glycosyla�
tion of TMAs.
Chemical modifications of TMAs. Consideration of
the role of chemical modifications of TMAs that play an
important part in their practical application is out of
scope of this review. We will mention here only briefly that
in addition to structural differences in oligosaccharides,
multiple chemical modifications have been detected in
TMA preparations including: (i) N�terminal modifica�
tions, which result from alternative cleavage of the signal
peptide that does not affect binding to antigen and FcRn
[101]; (ii) C�terminal heavy chain proline amidation, also
not affecting binding to antigen and FcRn [102]; (iii) Asn
deamidation in Fab�fragment that decreases affinity of
the therapeutic antibody to antigen [103]; (iv) oxidation
of recombinant monoclonal antibodies observed for
methionine residues and less often for tryptophan, histi�
dine, and other residues (oxidation usually decreases the
activity of antibodies and their stability) [104]; (v) degra�
dation of Asn and Asp, which is considered the most
important change because it significantly affects the
structure, stability, and functions of the therapeutic anti�
body. The regulation bodies of the United States and
European Union also control isomerization of Asp with
formation of isoAsp, which results in a small change in
charge as well as formation of trisulfide bonds, thioesters,
and other chemical modifications. In addition to the
effect of the culture itself on the emergence of chemical
modification in the preparation of an antibody as
described above, the emergence of modifications (glyca�
tion, deamidation, and N�terminal cyclization) caused by
the purification procedure and storage of preparation also
has been mentioned. These modifications can be prevent�
ed or avoided by decreasing temperature and by pH opti�
mization.
Human
Fig. 6. Major differences in glycosylation of TMAs produced in commercial producer cell cultures from human IgG.
Hamster(CHO)
Mouse(SP2/0�NSO)
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 851
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
GLYCOMODIFICATION AS A MEANS
TO ENHANCE THERAPEUTIC ACTIVITY OF A TMA
Recognizing the important role of carbohydrate
residues in therapeutic activity of TMAs motivates
researcher to search for methods to optimize their carbo�
hydrate profile. Pharmaceutical companies encourage
this, because the biosimilar TMAs (biosimilars) enter
markets in recent years as patent protection expires.
These preparations are significantly cheaper than the
original TMAs because the producing companies do not
invest money in the development of the original TMAs,
and this, in turn, could significantly decrease profits of
the companies that developed the original TMAs.
Glycomodification of potential TMAs can be per�
formed using several approaches. The first approach com�
prises point mutation in the TMA amino acid sequence.
For example, replacement of the phenylalanine residue at
position 243 of the human IgG1 Fc by alanine increased 5�
15�fold the yield of sialylated glycoform [83]. The second
approach involves the use of genetically modified cell lines.
As for now, the greatest success achieved was in using
genetically modified cell lines for production of non�fuco�
sylated TMAs [85]. This is mainly explained by the fact
that researchers are convinced that this is exactly the right
modification for increasing therapeutic effect. This opin�
ion is based on three facts. First, the existing producer cell
lines usually produce hyperfucosylated forms of TMAs
[105]. Second, published studies indicate the ability of
fucose located in the core region of the biantennal Asn297�
glycan to suppress the ADCC effector function, and that
fucose removal increases the ability of the antibody to
induce ADCC 100�fold [106] without affecting pharmaco�
kinetics in the process, i.e. the fate of the introduced anti�
body, at least in the organism of macaques [107].
The Asn297�glycan is fucosylated in mammalian
cells by α1,6�fucosyltransferase (FUT8), which catalyzes
transfer of fucose from GTP�fucose to the inner GlcNAc
residue via an α1,6�bond (Fig. 3). The CHO FUT8(–/–)
knockout cell line constructed by common methods pro�
duces non�fucosylated antibody, but it suffers from insta�
bility [108]. It must be noted that construction of mam�
malian somatic cell knockouts producing antibodies as a
means for fucosylation control remains a difficult prob�
lem. Several alternative methods have been suggested.
One of the methods allowed production of the complete�
ly non�fucosylated IgG1 with enhanced ADCC effector
function in CHO cells using small interfering RNA
(siRNA) directed against the genes involved in the fuco�
sylation process, such as FUT8, GTP�mannose 4,6�
dehydratase, and GTP�fucose transporter [109]. Another
method suggests the use of the zinc finger nuclease
(ZFN), which cleaves the FUT8 gene in CHO cells at the
site encoding the catalytic center of the FUT8 [110]. It
was shown possible to construct stable cell lines express�
ing model non�fucosylated antibodies using ZFN.
Another promising approach is based on the use of
the CRISPR/Cas9 system (Clustered Regularly
Interspaced Short Palindromic Repeats/CRISPR�associ�
ated protein 9) that provides for targeted destruction of
the FUT8 gene in the genome of CHO cells [111]. The
CHO FUT8(–/–) clone constructed in such a manner
was able to produce non�fucosylated TMAs without dis�
turbing the growth and viability of the producing cells.
Another example of the possibility of glycomodification
of antibody is CMP�Neu5Ac hydroxylase gene knockout
in CHO cells, which decreased the content of potentially
immunogenic sialic acid Neu5Gc in the TMA prepara�
tion [112].
Comparative modeling of the antibody that binds
TNFα reveals that the fucose in the Asn297�glycan does
not affect the interaction of Fab with TNFα, but it signif�
icantly influences the interaction with FcγRIIIA. The
removal of fucose causes more intimate interaction of the
Fc�fragment with FcγRIIIA and establishes new strong
interactions between G129 of the FcγRIIIA receptor and
S301 of the Fc�fragment. Moreover, analysis of the 3D�
model indicates emergence of new polar interactions
between the Fc�fragment (residues Y299, N300, and
S301) and FcγRIIIA (residues K128, G129, R130, and
R155) [113].
The greatest success for the use of the genetic engi�
neering approach was demonstrated in the construction
of non�fucosylated TMAs obinutuzumab (Gazyva®)
[114] and mogamulizumab (Poteligeo®) [115] (Table 2).
Obinutuzumab was approved on February 26, 2016 by
FDA for application in United States clinics primarily for
treatment of lymphomas and leukemia [114]. Its Fab
interacts with the extracellular loop of the transmem�
brane antigen CD20 on the surface of benign and malig�
nant B�lymphocytes, but it does not interact with
hematopoietic stem cells and normal plasma cells. The
non�fucosylated Fc�fragment of obinutuzumab efficient�
ly binds to FcγRIII on the surface of immune effector
cells such as NK�cells, macrophages, and monocytes,
and it activates ADCC.
Another glycomodified TMA, mogamulizumab
(Poteligeo®) [115], comprises humanized mouse anti�
body constructed on the basis of IgG1 with non�fucosy�
lated Fc�fragment and is directed for treatment of the
rather rare and aggressive disease adult T�cell leukemia/
lymphoma (ATLL) caused by infection with the human
T�lymphotropic virus 1 (HTLV�1). This virus is endemic
in southwest regions of Japan as well as in Caribbean
countries and some parts of Africa. This disease is resist�
ant to known chemotherapeutic agents and requires a
search for targets for efficient immunotherapy. The C�C
chemokine receptor type 4 (CCR4) located on the sur�
face of tumor cells in patients with ATLL was found to be
such a target. CCR4 is one of the chemokine receptors
participating in lymphocyte migration. It is present in T�
helper type 2 cells and regulatory T�cells. Regulation
852 DOROKHOV et al.
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
bodies have approved mogamulizumab for treating
patients in clinics of Japan, and for the phase III clinical
trials in the United States and the European Union by the
FDA and EMA, respectively. It is known that moga�
mulizumab exhibits clearly pronounced ability to activate
ADCC (but not CDC) [115].
The third approach for construction of cell lines pro�
ducing glycomodified antibodies comprises transient
expression of foreign genes that control antibody glycosy�
lation in these lines [33]. For example, it is possible to
increase the effector function of antibody related to the
suppression of fucosylation of the Asn297�glycan by the
expression of heterologous GNT III. GNT III catalyzes
addition of the branching GlcNAc residue in the man�
nose fork of the core part of biantennal Asn297�glycan
(Fig. 4). If the GlcNAc is present in the mannose fork, it
creates steric hindrance for addition of a fucose residue,
which eventually enhances ADCC�function [74].
Transient expression could be also used for production of
antibodies with increased content of sialic acid residues at
the terminals of the biantennal structure of the Asn297�
glycan (Figs. 3 and 4). In general, hypersialylation has an
adverse effect on the binding of Fc�fragment with
FcγRIII and, consequently, decreases the ability of an
antibody to activate ADCC. The approach developed for
production of human erythropoietin in CHO cells opens
the possibility of influencing the sialylation process. The
simultaneous transfection of cells with constructs con�
taining genes encoding CMP�sialic acid synthetase,
CMP�sialic acid transporter, and α2,3�sialyltransferase
increases sialic acid content in the erythropoietin by 43%
[116]. The amount of sialic acid in a glycoprotein can also
be increased if the sialidase is inhibited in CHO cells by
RNA interference [117].
Finally, control over growth conditions of the cell
culture and selection of optimum composition of the
medium also is an efficient way for targeted glycomodifi�
cation of antibodies [33]. It is known that a cocktail of
medium components including uridine, manganese, and
galactose increases the degree of TMA galactosylation
[95]. To decrease the level of high�mannose forms in the
population of antibodies, it is necessary to exclude man�
nose as a carbon source [118]. The point is that abnor�
mally high mannose concentrations in the medium inhib�
it activity of the α�mannosidase, which facilitates pro�
duction of TMAs with high mannose content. The con�
tent of amino acids in the medium also affects the carbo�
hydrate composition produced in CHO cells. For exam�
ple, glutamine as a carbon source facilitates ammonia
formation, and this negatively affects sialylation and
galactosylation of TMAs. Replacement of glutamine with
gluten hydrolysate or addition of threonine, proline, and
glycine into the medium overcomes the toxic effect of
ammonia.
Introduction of glycosylation inhibitors into the
medium is another method for the control of glycosyla�
tion. For example, MNS I inhibitors (deoxymannojiri�
mycin and kifunensine) facilitate emergence of high�
mannose glycans, and an MNS II inhibitor (swainsonine)
provides hybrid glycans [85]. The GCS and MNS I
inhibitors in turn decrease IgG fucosylation and, hence,
increase ADCC.
Since the discovery of the connection between the
removal of fucose from the core part of the biantennal
Asn297�glycan of the Fc�fragment and ADCC activation
numerous non�fucosylated antibodies have been con�
structed, with more than 20 of them recommended for
clinical trials [3]. However, only two (obinutuzumab and
mogamulizumab) were approved as TMAs and intro�
duced into clinical practice. It is interesting that these
TMAs target blood cells. The question arises as to why
there are no efficient glycomodified antibodies against
solid tumors such as breast or rectal cancer. Different
bioavailability of antibodies against malignant blood cells
and the cells in solid tumors and the presence of NK�cells
in blood are suggested as the main explanations [3]. A
possible connection of this phenomenon with FcγRIII
polymorphism has also been considered. Further studies
are needed to understand the reasons for this situation
and to develop relevant and efficient glycomodified TMA
for solid tumors. This possibility is very likely because
positive results of clinical trials of the non�fucosylated
variant of trastuzumab have been reported [105].
CONCLUSIONS
Antibodies play an important role in human humoral
immunity and have been used for a long time as an effi�
cient means for treatment of such human diseases as can�
cer, viral infections, and immune diseases.
As in the case of majority of glycoproteins,
immunoglobulins in healthy humans are subjected to gly�
cosylation in the ER and Golgi apparatus. The N�glycans
are found in all classes of immunoglobulins, both in Fc�
and Fab�fragments.
The Asn297�linked glycan of the human IgG1 Fc�
fragment has a biantennal structure with the core consist�
ing of four GlcNAc residues and three mannose residues.
The fucose is added to the heptasaccharide core that
branches from the GlcNAc residue together with the
galactose and negatively charged Neu5Ac.
The fucose attached to the glycan core via α1,6�
bond decreases efficiency of Fc�fragment binding to
FcγRIIIA and ADCC activation. Defucosylation of the
Asn297�glycan increases the degree of its binding to the
FcγRIIIA manifold and, respectively, increases the
ADCC effector function.
Despite the fact that efficient methods were devel�
oped for production of antibodies in bacteria, yeasts, and
plants, the commercially available TMAs are produced in
animal cells, predominantly in one of three mammalian
ROLE OF CARBOHYDRATE RESIDUES IN IMMUNOGLOBULIN 853
BIOCHEMISTRY (Moscow) Vol. 81 No. 8 2016
cell lines: Chinese hamster ovary cells and murine NS0 or
SP2/0. The vast majority of more than 40 commercial
TMAs are either chimeric (mouse/human) or humanized
mouse antibodies belonging to the IgG1 class.
The biological activity of TMAs can vary depending
on the particular oligosaccharide attached to it. The TMA
glycosylation profile is determined by the cell line and
procedures and conditions of cultivation. Synthesis in
CHO cells results in: (i) the absence of α2,6�sialic acid
and of branching GlcNAc residue in the mannose fork,
and (ii) increased degree of antibody sialylation. TMA
production in mouse myeloma cells results in emergence
of α�Gal and enhanced sialylation.
Methods for glycomodification were developed as
means to increase therapeutic and biological activities of
TMAs, including: (i) genetic methods directed to either
optimization of amino acid sequence of the TMA Fc�
fragment or genetic modification of cell line producers of
TMAs; (ii) methods of transient expression of foreign
genes in producer cells that control glycosylation of anti�
bodies, and (iii) methods for control of cultivation condi�
tions and selection of the optimum medium composition
for the growth of producer cells.
The possibility for production of glycomodified
TMAs demonstrating the highest therapeutic effects has
been established. Further development of this line of
investigation will likely involve increase in: (i) the TMA
stability in the bloodstream; (ii) the fraction of antibodies
delivered to a solid tumor, and (iii) competitiveness of
TMAs for binding to FcγR.
Acknowledgements
This work was financially supported by the Russian
Science Foundation (project No. 16�14�00002; section
devoted to production and glycomodification of TMAs)
and by the Russian Foundation for Basic Research (proj�
ect No. 16�34�60002_mol_a_dk; section devoted to
mechanisms of protein glycosylation).
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