Functional Role of Carbohydrate Residues in Human ......Subdivision of IgG into four subclasses is determined by their relative concentration in normal human serum, where IgG1, IgG2,

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


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.


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



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.


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



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.


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 chimeric(mouse/human),monomethylauristatin E conjugate

IgG1 (human)

biospecific, rat IgG2b/mouse IgG2a

IgG1 chimeric(mouse/human)

IgG1 (mouse) humanized

IgG1 (human)

IgG2 (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®)



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


IgG1 (human)

IgG1 (mouse)


IgG1 (human)



IgG1 (mouse)humanized, with afucosylatedFc�fragment


IgG4 (human)

(mouse) completelyhumanized, with afucosylatedFc�fragment

IgG1 (human)



IgG2 (human)



IgG1 (human)

IgG1 (human)


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.


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)



IgG2aλ (mouse)


IgG1 (human)


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.



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.


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.



(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.


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



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.


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)



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.


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



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).

REFERENCES

1. Kunert, R., and Casanova, E. (2013) Recent advances in

recombinant protein production: BAC�based expression

vectors, the bigger the better, Bioengineered, 4, 258�261.

2. Walsh, G. (2014) Biopharmaceutical benchmarks 2014,

Nat. Biotechnol., 32, 992�1000.

3. Niwa, R., and Satoh, M. (2015) The current status and

prospects of antibody engineering for therapeutic use: focus

on glycoengineering technology, J. Pharm. Sci., 104, 930�

941.

4. Higel, F., Seidl, A., Sorgel, F., and Friess, W. (2016) N�gly�

cosylation heterogeneity and the influence on structure,

function and pharmacokinetics of monoclonal antibodies

and Fc fusion proteins, Eur. J. Pharm. Biopharm., 100, 94�

100.

5. Costa, A. R., Rodrigues, M. E., Henriques, M., Oliveira,

R., and Azeredo, J. (2014) Glycosylation: impact, control

and improvement during therapeutic protein production,

Crit. Rev. Biotechnol., 34, 281�299.

6. Jefferis, R. (2009) Glycosylation as a strategy to improve

antibody�based therapeutics, Nat. Rev. Drug Discov., 8,

226�234.

7. Reusch, D., and Tejada, M. L. (2015) Fc glycans of thera�

peutic antibodies as critical quality attributes, Glycobiology,

25, 1325�1334.

8. Altshuler, E. P., Serebryanaya, D. V., and Katrukha, A. G.

(2010) Generation of recombinant antibodies and means

for increasing their affinity, Biochemistry (Moscow), 75,

1584�1605.

9. Vidarsson, G., Dekkers, G., and Rispens, T. (2014) IgG

subclasses and allotypes: from structure to effector func�

tions, Front. Immunol., 5, 520.

10. Nezlin, R. (1998) in Chapter 1. General Characteristics of

Immunoglobulin Molecules, Academic Press, New York, pp.

3�73.

11. Otten, M. A., and Van Egmond, M. (2004) The Fc recep�

tor for IgA (FcalphaRI, CD89), Immunol. Lett., 92, 23�31.

12. Fridman, W. H. (1991) Fc receptors and immunoglobulin

binding factors, FASEB J., 5, 2684�2690.

13. Roque, A. C. A., Silva, C. S. O., and Taipa, M. A. (2007)

Affinity�based methodologies and ligands for antibody

purification: advances and perspectives, J. Chromatogr. A,

1160, 44�55.

14. Cheng, Y., and Patel, D. J. (2004) An efficient system for

small protein expression and refolding, Biochem. Biophys.

Res. Commun., 317, 401�405.

15. Nilson, B. H., Logdberg, L., Kastern, W., Bjorck, L., and

Akerstrom, B. (1993) Purification of antibodies using pro�

tein L�binding framework structures in the light chain vari�

able domain, J. Immunol. Methods, 164, 33�40.

16. Irani, V., Guy, A. J., Andrew, D., Beeson, J. G., Ramsland,

P. A., and Richards, J. S. (2015) Molecular properties of

human IgG subclasses and their implications for designing

therapeutic monoclonal antibodies against infectious dis�

eases, Mol. Immunol., 67, 171�182.

17. Chan, K. R., Ong, E. Z., Mok, D. Z. L., and Ooi, E. E.

(2015) Fc receptors and their influence on efficacy of ther�

apeutic antibodies for treatment of viral diseases, Expert

Rev. Anti Infect. Ther., 13, 1351�1360.

18. Nimmerjahn, F., and Ravetch, J. V. (2008) Fc�gamma

receptors as regulators of immune responses, Nat. Rev.

Immunol., 8, 34�47.

19. Brambell, F. W., Hemmings, W. A., and Morris, I. G.

(1964) A theoretical model of gamma�globulin catabolism,

Nature, 203, 1352�1354.

20. Junghans, R. P., and Anderson, C. L. (1996) The protec�

tion receptor for IgG catabolism is the beta2�microglobu�

lin�containing neonatal intestinal transport receptor, Proc.

Natl. Acad. Sci. USA, 93, 5512�5516.

21. Roopenian, D. C., and Akilesh, S. (2007) FcRn: the

neonatal Fc receptor comes of age, Nat. Rev. Immunol., 7,

715�725.

22. Roman, V. R. G., Murray, J. C., and Weiner, L. M. (2013)

in Antibody Fc: Chapter 1. Antibody�Dependent Cellular

Cytotoxicity, Academic Press.

23. Lindorfer, M. A., Kohl, J., and Taylor, R. P. (2014) in

Chapter 3. Interactions between the Complement System and

854 DOROKHOV et al.


Fcγ Receptors A2 (Nimmerjahn, F., and Ackerman, M. E.,

eds.) Academic Press, Boston, pp. 49�74.

24. Gul, N., and Van Egmond, M. (2015) Antibody�dependent

phagocytosis of tumor cells by macrophages: a potent effec�

tor mechanism of monoclonal antibody therapy of cancer,

Cancer Res., 75, 5008�5013.

25. Lu, J., Chu, J., Zou, Z., Hamacher, N. B., Rixon, M. W.,

and Sun, P. D. (2015) Structure of FcγRI in complex with

Fc reveals the importance of glycan recognition for high�

affinity IgG binding, Proc. Natl. Acad. Sci. USA, 112, 833�

838.

26. Bulliard, Y., Jolicoeur, R., Windman, M., Rue, S. M.,

Ettenberg, S., Knee, D. A., Wilson, N. S., Dranoff, G., and

Brogdon, J. L. (2013) Activating Fcγ receptors contribute

to the antitumor activities of immunoregulatory receptor�

targeting antibodies, J. Exp. Med., 210, 1685�1693.

27. Bruhns, P., Iannascoli, B., England, P., Mancardi, D. A.,

Fernandez, N., Jorieux, S., and Daeron, M. (2009)

Specificity and affinity of human Fc�gamma receptors and

their polymorphic variants for human IgG subclasses,

Blood, 113, 3716�3725.

28. Shields, R. L., Namenuk, A. K., Hong, K., Meng, Y. G.,

Rae, J., Briggs, J., Xie, D., Lai, J., Stadlen, A., Li, B., Fox,

J. A., and Presta, L. G. (2001) High resolution mapping of

the binding site on human IgG1 for Fc�gamma RI, Fc�

gamma RII, Fc�gamma RIII, and FcRn and design of IgG1

variants with improved binding to the Fc�gamma R, J. Biol.

Chem., 276, 6591�6604.

29. Lazar, G. A., Dang, W., Karki, S., Vafa, O., Peng, J. S.,

Hyun, L., Chan, C., Chung, H. S., Eivazi, A., Yoder, S. C.,

Vielmetter, J., Carmichael, D. F., Hayes, R. J., and

Dahiyat, B. I. (2006) Engineered antibody Fc variants with

enhanced effector function, Proc. Natl. Acad. Sci. USA,

103, 4005�4010.

30. Nordstrom, J. L., Gorlatov, S., Zhang, W., Yang, Y.,

Huang, L., Burke, S., Li, H., Ciccarone, V., Zhang, T.,

Stavenhagen, J., Koenig, S., Stewart, S. J., Moore, P. A.,

Johnson, S., and Bonvini, E. (2011) Anti�tumor activity

and toxicokinetics analysis of MGAH22, an anti�HER2

monoclonal antibody with enhanced Fcγ receptor binding

properties, Breast Cancer Res., 13, R123.

31. Nezlin, R., and Ghetie, V. (2004) Interactions of

immunoglobulins outside the antigen�combining site, Adv.

Immunol., 82, 155�215.

32. Lin, Z., Lo, A., Simeone, D. M., Ruffin, M. T., and

Lubman, D. M. (2012) An N�glycosylation analysis of

human alpha�2�macroglobulin using an integrated

approach, J. Proteom. Bioinform., 5, 127�134.

33. Sha, S., Agarabi, C., Brorson, K., Lee, D.�Y., and Yoon, S.

(2016) N�Glycosylation design and control of therapeutic

monoclonal antibodies, Trends Biotechnol., March 22, pii:

S0167�7799(16)00047�0; doi: 10.1016/j.tibtech.2016.02.013

[Epub ahead of print].

34. Butters, T. D. (2002) Control in the N�linked glycoprotein

biosynthesis pathway, Chem. Biol., 9, 1266�1268.

35. Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H.,

Stanley, P., Bertozzi, C. R., Hart, G. W., and Etzler, M. E.

(2009) in Essentials of Glycobiology, Cold Spring Harbor

Laboratory Press, N. Y.

36. Dicker, M., and Strasser, R. (2015) Using glyco�engineer�

ing to produce therapeutic proteins, Expert Opin. Biol.

Ther., 15, 1501�1516.

37. Köhler, G., and Milstein, C. (1975) Continuous cultures of

fused cells secreting antibody of predefined specificity,

Nature, 256, 495�497.

38. Smith, S. L. (1996) Ten years of Orthoclone OKT3

(muromonab�CD3): a review, J. Transpl. Coord., 6, 109�

119, 120�121.

39. Kunert, R., and Reinhart, D. (2016) Advances in recombi�

nant antibody manufacturing, Appl. Microbiol. Biotechnol.,

pii: S0167�7799(16)00047�0.

40. Dumont, J., Euwart, D., Mei, B., Estes, S., and Kshirsagar,

R. (2015) Human cell lines for biopharmaceutical manu�

facturing: history, status, and future perspectives, Crit. Rev.

Biotechnol., 1�13.

41. Ghaderi, D., Zhang, M., Hurtado�Ziola, N., and Varki, A.

(2012) Production platforms for biotherapeutic glyco�

proteins. Occurrence, impact, and challenges of non�

human sialylation, Biotechnol. Genet. Eng. Rev., 28, 147�

175.

42. Hossler, P. (2012) Protein glycosylation control in mam�

malian cell culture: past precedents and contemporary

prospects, Adv. Biochem. Eng. Biotechnol., 127, 187�219.

43. Zboray, K., Sommeregger, W., Bogner, E., Gili, A.,

Sterovsky, T., Fauland, K., Grabner, B., Stiedl, P., Moll, H.

P., Bauer, A., Kunert, R., and Casanova, E. (2015)

Heterologous protein production using euchromatin�con�

taining expression vectors in mammalian cells, Nucleic

Acids Res., 43, e102.

44. Ho, Y., Kiparissides, A., Pistikopoulos, E. N., and

Mantalaris, A. (2012) Computational approach for under�

standing and improving GS�NS0 antibody production under

hyperosmotic conditions, J. Biosci. Bioeng., 113, 88�98.

45. Kuczewski, M., Schirmer, E., Lain, B., and Zarbis�

Papastoitsis, G. (2011) A single�use purification process for

the production of a monoclonal antibody produced in a

PER.C6 human cell line, Biotechnol. J., 6, 56�65.

46. Le Fourn, V., Girod, P.�A., Buceta, M., Regamey, A., and

Mermod, N. (2014) CHO cell engineering to prevent

polypeptide aggregation and improve therapeutic protein

secretion, Metab. Eng., 21, 91�102.

47. Reichert, J. M. (2016) Antibodies to watch in 2016, mAbs,

8, 197�204.

48. Preithner, S., Elm, S., Lippold, S., Locher, M., Wolf, A.,

Da Silva, A. J., Baeuerle, P. A., and Prang, N. S. (2006)

High concentrations of therapeutic IgG1 antibodies are

needed to compensate for inhibition of antibody�depend�

ent cellular cytotoxicity by excess endogenous immuno�

globulin G, Mol. Immunol., 43, 1183�1193.

49. Gallinger, S., Reilly, R. M., Kirsh, J. C., Odze, R. D.,

Schmocker, B. J., Hay, K., Polihronis, J., Damani, M. T.,

Shpitz, B., and Stern, H. S. (1993) Comparative dual label

study of first and second generation antitumor�associated

glycoprotein�72 monoclonal antibodies in colorectal can�

cer patients, Cancer Res., 53, 271�278.

50. Carter, P. J. (2006) Potent antibody therapeutics by design,

Nat. Rev. Immunol., 6, 343�357.

51. Ornes, S. (2015) Core concepts: biosimilars, Proc. Natl.

Acad. Sci. USA, 112, 15261�15262.

52. Rathore, A. S., Weiskopf, A., and Reason, A. J. (2016) Defining

critical quality attributes for monoclonal antibody therapeutic

products (http://www.biopharminternational.com/defining�

critical�quality�attributes�monoclonal�antibody�therapeutic�

products).



53. Plomp, R., Dekkers, G., Rombouts, Y., Visser, R.,

Koeleman, C. A. M., Kammeijer, G. S. M., Jansen, B. C.,

Rispens, T., Hensbergen, P. J., Vidarsson, G., and Wuhrer,

M. (2015) Hinge�region O�glycosylation of human

immunoglobulin G3 (IgG3), Mol. Cell. Proteom., 14, 1373�

1384.

54. Van de Bovenkamp, F. S., Hafkenscheid, L., Rispens, T.,

and Rombouts, Y. (2016) The emerging importance of IgG

Fab glycosylation in immunity, J. Immunol., 196, 1435�

1441.

55. Bondt, A., Rombouts, Y., Selman, M. H. J., Hensbergen,

P. J., Reiding, K. R., Hazes, J. M. W., Dolhain, R. J. E. M.,

and Wuhrer, M. (2014) Immunoglobulin G (IgG) Fab gly�

cosylation analysis using a new mass spectrometric high�

throughput profiling method reveals pregnancy�associated

changes, Mol. Cell. Proteom., 13, 3029�3039.

56. Rombouts, Y., Willemze, A., Van Beers, J. J. B. C., Shi, J.,

Kerkman, P. F., Van Toorn, L., Janssen, G. M. C.,

Zaldumbide, A., Hoeben, R. C., Pruijn, G. J. M., Deelder,

A. M., Wolbink, G., Rispens, T., Van Veelen, P. A.,

Huizinga, T. W. J., Wuhrer, M., Trouw, L. A., Scherer, H.

U., and Toes, R. E. M. (2016) Extensive glycosylation of

ACPA�IgG variable domains modulates binding to citrulli�

nated antigens in rheumatoid arthritis, Ann. Rheum. Dis.,

75, 578�585.

57. Zhu, D., Ottensmeier, C. H., Du, M.�Q., McCarthy, H.,

and Stevenson, F. K. (2003) Incidence of potential glycosy�

lation sites in immunoglobulin variable regions distinguish�

es between subsets of Burkitt’s lymphoma and mucosa�

associated lymphoid tissue lymphoma, Br. J. Haematol.,

120, 217�222.

58. Radcliffe, C. M., Arnold, J. N., Suter, D. M., Wormald, M.

R., Harvey, D. J., Royle, L., Mimura, Y., Kimura, Y., Sim,

R. B., Inoges, S., Rodriguez�Calvillo, M., Zabalegui, N.,

De Cerio, A. L.�D., Potter, K. N., Mockridge, C. I., Dwek,

R. A., Bendandi, M., Rudd, P. M., and Stevenson, F. K.

(2007) Human follicular lymphoma cells contain oligo�

mannose glycans in the antigen�binding site of the B�cell

receptor, J. Biol. Chem., 282, 7405�7415.

59. Leibiger, H., Wustner, D., Stigler, R. D., and Marx, U.

(1999) Variable domain�linked oligosaccharides of a human

monoclonal IgG: structure and influence on antigen bind�

ing, Biochem. J., 338 (Pt. 2), 529�538.

60. Schneider, D., Duhren�von Minden, M., Alkhatib, A., Setz,

C., Van Bergen, C. A. M., Benkisser�Petersen, M., Wilhelm,

I., Villringer, S., Krysov, S., Packham, G., Zirlik, K., Romer,

W., Buske, C., Stevenson, F. K., Veelken, H., and Jumaa, H.

(2015) Lectins from opportunistic bacteria interact with

acquired variable�region glycans of surface immunoglobulin

in follicular lymphoma, Blood, 125, 3287�3296.

61. Alessandri, L., Ouellette, D., Acquah, A., Rieser, M.,

Leblond, D., Saltarelli, M., Radziejewski, C., Fujimori, T.,

and Correia, I. (2012) Increased serum clearance of oligo�

mannose species present on a human IgG1 molecule,

mAbs, 4, 509�520.

62. Bork, K., Horstkorte, R., and Weidemann, W. (2009)

Increasing the sialylation of therapeutic glycoproteins: the

potential of the sialic acid biosynthetic pathway, J. Pharm.

Sci., 98, 3499�3508.

63. Naso, M. F., Tam, S. H., Scallon, B. J., and Raju, T. S.

(2010) Engineering host cell lines to reduce terminal sialy�

lation of secreted antibodies, mAbs, 2, 519�527.

64. Courtois, F., Agrawal, N. J., Lauer, T. M., and Trout, B. L.

(2016) Rational design of therapeutic mAbs against aggre�

gation through protein engineering and incorporation of

glycosylation motifs applied to bevacizumab, mAbs, 8, 99�

112.

65. Retamozo, S., Brito�Zeron, P., Bosch, X., Stone, J. H.,

and Ramos�Casals, M. (2013) Cryoglobulinemic disease,

Oncology (Williston Park), 27, 1098�1105, 1110�1116.

66. Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U.

(2000) The 3.2�Å crystal structure of the human IgG1 Fc

fragment–Fc�gammaRIII complex, Nature, 406, 267�273.

67. Jefferis, R., Lund, J., and Pound, J. D. (1998) IgG�Fc�

mediated effector functions: molecular definition of inter�

action sites for effector ligands and the role of glycosyla�

tion, Immunol. Rev., 163, 59�76.

68. Sazinsky, S. L., Ott, R. G., Silver, N. W., Tidor, B.,

Ravetch, J. V., and Wittrup, K. D. (2008) Aglycosylated

immunoglobulin G1 variants productively engage activat�

ing Fc receptors, Proc. Natl. Acad. Sci. USA, 105, 20167�

20172.

69. Baruah, K., Bowden, T. A., Krishna, B. A., Dwek, R. A.,

Crispin, M., and Scanlan, C. N. (2012) Selective deactiva�

tion of serum IgG: a general strategy for the enhancement

of monoclonal antibody receptor interactions, J. Mol. Biol.,

420, 1�7.

70. Barb, A. W., and Prestegard, J. H. (2011) NMR analysis

demonstrates immunoglobulin G N�glycans are accessible

and dynamic, Nat. Chem. Biol., 7, 147�153.

71. Stewart, R., Thom, G., Levens, M., Guler�Gane, G.,

Holgate, R., Rudd, P. M., Webster, C., Jermutus, L., and

Lund, J. (2011) A variant human IgG1�Fc mediates

improved ADCC, Protein Eng. Des. Sel., 24, 671�678.

72. Mimura, Y., Ashton, P. R., Takahashi, N., Harvey, D. J.,

and Jefferis, R. (2007) Contrasting glycosylation profiles

between Fab and Fc of a human IgG protein studied by

electrospray ionization mass spectrometry, J. Immunol.

Methods, 326, 116�126.

73. Ferrara, C., Grau, S., Jager, C., Sondermann, P., Brunker,

P., Waldhauer, I., Hennig, M., Ruf, A., Rufer, A. C., Stihle,

M., Umana, P., and Benz, J. (2011) Unique carbohy�

drate–carbohydrate interactions are required for high

affinity binding between Fc�gammaRIII and antibodies

lacking core fucose, Proc. Natl. Acad. Sci. USA, 108, 12669�

12674.

74. Ferrara, C., Brunker, P., Suter, T., Moser, S., Puntener, U.,

and Umana, P. (2006) Modulation of therapeutic antibody

effector functions by glycosylation engineering: influence

of Golgi enzyme localization domain and co�expression of

heterologous beta1,4�N�acetylglucosaminyltransferase III

and Golgi alpha�mannosidase II, Biotechnol. Bioeng., 93,

851�861.

75. Houde, D., Peng, Y., Berkowitz, S. A., and Engen, J. R.

(2010) Post�translational modifications differentially affect

IgG1 conformation and receptor binding, Mol. Cell.

Proteom., 9, 1716�1728.

76. Scallon, B. J., Tam, S. H., McCarthy, S. G., Cai, A. N.,

and Raju, T. S. (2007) Higher levels of sialylated Fc glycans

in immunoglobulin G molecules can adversely impact

functionality, Mol. Immunol., 44, 1524�1534.

77. Kanda, Y., Yamada, T., Mori, K., Okazaki, A., Inoue, M.,

Kitajima�Miyama, K., Kuni�Kamochi, R., Nakano, R.,

Yano, K., Kakita, S., Shitara, K., and Satoh, M. (2007)

856 DOROKHOV et al.


Comparison of biological activity among nonfucosylated

therapeutic IgG1 antibodies with three different N�linked

Fc oligosaccharides: the high�mannose, hybrid, and com�

plex types, Glycobiology, 17, 104�118.

78. Rook, G. A., Steele, J., Brealey, R., Whyte, A., Isenberg,

D., Sumar, N., Nelson, J. L., Bodman, K. B., Young, A.,

and Roitt, I. M. (1991) Changes in IgG glycoform levels are

associated with remission of arthritis during pregnancy, J.

Autoimmun., 4, 779�794.

79. Knezevic, A., Gornik, O., Polasek, O., Pucic, M., Redzic,

I., Novokmet, M., Rudd, P. M., Wright, A. F., Campbell,

H., Rudan, I., and Lauc, G. (2010) Effects of aging, body

mass index, plasma lipid profiles, and smoking on human

plasma N�glycans, Glycobiology, 20, 959�969.

80. Van de Geijn, F. E., Wuhrer, M., Selman, M. H.,

Willemsen, S. P., De Man, Y. A., Deelder, A. M., Hazes, J.

M., and Dolhain, R. J. (2009) Immunoglobulin G galacto�

sylation and sialylation are associated with pregnancy�

induced improvement of rheumatoid arthritis and the post�

partum flare: results from a large prospective cohort study,

Arthritis Res. Ther., 11, R193.

81. Holland, M., Yagi, H., Takahashi, N., Kato, K., Savage, C.

O. S., Goodall, D. M., and Jefferis, R. (2006) Differential

glycosylation of polyclonal IgG, IgG–Fc and IgG–Fab iso�

lated from the sera of patients with ANCA�associated sys�

temic vasculitis, Biochim. Biophys. Acta, 1760, 669�677.

82. Axford, J. S., Cunnane, G., Fitzgerald, O., Bland, J. M.,

Bresnihan, B., and Frears, E. R. (2003) Rheumatic disease

differentiation using immunoglobulin G sugar printing by

high density electrophoresis, J. Rheumatol., 30, 2540�2546.

83. Mimura, Y., Kelly, R. M., Unwin, L., Albrecht, S., Jefferis,

R., Goodall, M., Mizukami, Y., Mimura�Kimura, Y.,

Matsumoto, T., Ueoka, H., and Rudd, P. M. (2016)

Enhanced sialylation of a human chimeric IgG1 variant

produced in human and rodent cell lines, J. Immunol.

Methods, 428, 30�36.

84. Anthony, R. M., Nimmerjahn, F., Ashline, D. J., Reinhold,

V. N., Paulson, J. C., and Ravetch, J. V. (2008)

Recapitulation of IVIG anti�inflammatory activity with a

recombinant IgG Fc, Science, 320, 373�376.

85. Butler, M., and Spearman, M. (2014) The choice of mam�

malian cell host and possibilities for glycosylation engineer�

ing, Curr. Opin. Biotechnol., 30, 107�112.

86. Schiestl, M., Stangler, T., Torella, C., Cepeljnik, T., Toll,

H., and Grau, R. (2011) Acceptable changes in quality

attributes of glycosylated biopharmaceuticals, Nat.

Biotechnol., 29, 310�312.

87. Zalai, D., Hever, H., Lovasz, K., Molnar, D.,

Wechselberger, P., Hofer, A., Parta, L., Putics, A., and

Herwig, C. (2016) A control strategy to investigate the rela�

tionship between specific productivity and high�mannose

glycoforms in CHO cells, Appl. Microbiol. Biotechnol.,

[Epub ahead of print].

88. Chung, C. H., Mirakhur, B., Chan, E., Le, Q.�T., Berlin, J.,

Morse, M., Murphy, B. A., Satinover, S. M., Hosen, J.,

Mauro, D., Slebos, R. J., Zhou, Q., Gold, D., Hatley, T.,

Hicklin, D. J., and Platts�Mills, T. A. E. (2008) Cetuximab�

induced anaphylaxis and IgE specific for galactose�alpha�

1,3�galactose, N. Engl. J. Med., 358, 1109�1117.

89. Padler�Karavani, V., and Varki, A. (2011) Potential impact

of the non�human sialic acid N�glycolylneuraminic acid on

transplant rejection risk, Xenotransplantation, 18, 1�5.

90. Bosques, C. J., Collins, B. E., Meador, J. W., Sarvaiya, H.,

Murphy, J. L., Dellorusso, G., Bulik, D. A., Hsu, I.�H.,

Washburn, N., Sipsey, S. F., Myette, J. R., Raman, R.,

Shriver, Z., Sasisekharan, R., and Venkataraman, G.

(2010) Chinese hamster ovary cells can produce galactose�

α�1,3�galactose antigens on proteins, Nat. Biotechnol., 28,

1153�1156.

91. Dashivets, T., Thomann, M., Rueger, P., Knaupp, A.,

Buchner, J., and Schlothauer, T. (2015) Multi�angle effec�

tor function analysis of human monoclonal IgG glycovari�

ants, PLoS One, 10, e0143520.

92. Raju, T. S. (2008) Terminal sugars of Fc glycans influence

antibody effector functions of IgGs, Curr. Opin. Immunol.,

20, 471�478.

93. Raju, T. S., and Jordan, R. E. (2012) Galactosylation vari�

ations in marketed therapeutic antibodies, mAbs, 4, 385�

391.

94. Hodoniczky, J., Zheng, Y. Z., and James, D. C. (2005)

Control of recombinant monoclonal antibody effector

functions by Fc N�glycan remodeling in vitro, Biotechnol.

Prog., 21, 1644�1652.

95. Liu, B., Spearman, M., Doering, J., Lattova, E.,

Perreault, H., and Butler, M. (2014) The availability of

glucose to CHO cells affects the intracellular lipid�linked

oligosaccharide distribution, site occupancy and the N�

glycosylation profile of a monoclonal antibody, J.

Biotechnol., 170, 17�27.

96. Muthing, J., Kemminer, S. E., Conradt, H. S., Sagi, D.,

Nimtz, M., Karst, U., and Peter�Katalinic, J. (2003)

Effects of buffering conditions and culture pH on produc�

tion rates and glycosylation of clinical phase I anti�

melanoma mouse IgG3 monoclonal antibody R24,

Biotechnol. Bioeng., 83, 321�334.

97. Yoon, S. K., Song, J. Y., and Lee, G. M. (2003) Effect of

low culture temperature on specific productivity, tran�

scription level, and heterogeneity of erythropoietin in

Chinese hamster ovary cells, Biotechnol. Bioeng., 82, 289�

298.

98. Kunkel, J. P., Jan, D. C., Jamieson, J. C., and Butler, M.

(1998) Dissolved oxygen concentration in serum�free con�

tinuous culture affects N�linked glycosylation of a mono�

clonal antibody, J. Biotechnol., 62, 55�71.

99. Grainger, R. K., and James, D. C. (2013) CHO cell line

specific prediction and control of recombinant monoclon�

al antibody N�glycosylation, Biotechnol. Bioeng., 110,

2970�2983.

100. Lipscomb, M. L., Palomares, L. A., Hernandez, V.,

Ramirez, O. T., and Kompala, D. S. (2005) Effect of pro�

duction method and gene amplification on the glycosyla�

tion pattern of a secreted reporter protein in CHO cells,

Biotechnol. Prog., 21, 40�49.

101. Kotia, R. B., and Raghani, A. R. (2010) Analysis of mono�

clonal antibody product heterogeneity resulting from

alternate cleavage sites of signal peptide, Anal. Biochem.,

399, 190�195.

102. Johnson, K. A., Paisley�Flango, K., Tangarone, B. S.,

Porter, T. J., and Rouse, J. C. (2007) Cation exchange�

HPLC and mass spectrometry reveal C�terminal amid�

ation of an IgG1 heavy chain, Anal. Biochem., 360, 75�83.

103. Vlasak, J., Bussat, M. C., Wang, S., Wagner�Rousset, E.,

Schaefer, M., Klinguer�Hamour, C., Kirchmeier, M.,

Corvaia, N., Ionescu, R., and Beck, A. (2009)



Identification and characterization of asparagine deami�

dation in the light chain CDR1 of a humanized IgG1 anti�

body, Anal. Biochem., 392, 145�154.

104. Liu, D., Ren, D., Huang, H., Dankberg, J., Rosenfeld, R.,

Cocco, M. J., Li, L., Brems, D. N., and Remmele, R. L.

(2008) Structure and stability changes of human IgG1 Fc

as a consequence of methionine oxidation, Biochemistry,

47, 5088�5100.

105. Suzuki, E., Niwa, R., Saji, S., Muta, M., Hirose, M., Iida,

S., Shiotsu, Y., Satoh, M., Shitara, K., Kondo, M., and

Toi, M. (2007) A nonfucosylated anti�HER2 antibody

augments antibody�dependent cellular cytotoxicity in

breast cancer patients, Clin. Cancer Res., 13, 1875�1882.

106. Zhang, P., Woen, S., Wang, T., Liau, B., Zhao, S., Chen,

C., Yang, Y., Song, Z., Wormald, M. R., Yu, C., and Rudd,

P. M. (2016) Challenges of glycosylation analysis and con�

trol: an integrated approach to producing optimal and

consistent therapeutic drugs, Drug Discov. Today, 21, 740�

765.

107. Leabman, M. K., Meng, Y. G., Kelley, R. F., DeForge, L.

E., Cowan, K. J., and Iyer, S. (2013) Effects of altered

FcγR binding on antibody pharmacokinetics in cynomol�

gus monkeys, mAbs, 5, 896�903.

108. Yamane�Ohnuki, N., Kinoshita, S., Inoue�Urakubo, M.,

Kusunoki, M., Iida, S., Nakano, R., Wakitani, M., Niwa,

R., Sakurada, M., Uchida, K., Shitara, K., and Satoh, M.

(2004) Establishment of FUT8 knockout Chinese hamster

ovary cells: an ideal host cell line for producing complete�

ly defucosylated antibodies with enhanced antibody�

dependent cellular cytotoxicity, Biotechnol. Bioeng., 87,

614�622.

109. Imai�Nishiya, H., Mori, K., Inoue, M., Wakitani, M.,

Iida, S., Shitara, K., and Satoh, M. (2007) Double knock�

down of alpha1,6�fucosyltransferase (FUT8) and GDP�

mannose 4,6�dehydratase (GMD) in antibody�producing

cells: a new strategy for generating fully non�fucosylated

therapeutic antibodies with enhanced ADCC, BMC

Biotechnol., 7, 84.

110. Malphettes, L., Freyvert, Y., Chang, J., Liu, P.�Q., Chan,

E., Miller, J. C., Zhou, Z., Nguyen, T., Tsai, C., Snowden,

A. W., Collingwood, T. N., Gregory, P. D., and Cost, G. J.

(2010) Highly efficient deletion of FUT8 in CHO cell lines

using zinc�finger nucleases yields cells that produce com�

pletely nonfucosylated antibodies, Biotechnol. Bioeng.,

106, 774�783.

111. Sun, T., Li, C., Han, L., Jiang, H., Xie, Y., Zhang, B.,

Qian, X., Lu, H., and Zhu, J. (2015) Functional knockout

of FUT8 in Chinese hamster ovary cells using

CRISPR/Cas9 to produce a defucosylated antibody, Eng.

Life Sci., 15, 660�666.

112. Chenu, S., Gregoire, A., Malykh, Y., Visvikis, A.,

Monaco, L., Shaw, L., Schauer, R., Marc, A., and

Goergen, J.�L. (2003) Reduction of CMP�N�acetylneur�

aminic acid hydroxylase activity in engineered Chinese

hamster ovary cells using an antisense�RNA strategy,

Biochim. Biophys. Acta, 1622, 133�144.

113. Pierri, C. L., Bossis, F., Punzi, G., De Grassi, A., Cetrone,

M., Parisi, G., and Tricarico, D. (2016) Molecular model�

ing of antibodies for the treatment of TNFα�related

immunological diseases, Pharmacol. Res. Perspect., 4,

e00197.

114. Evans, S. S., and Clemmons, A. B. (2015) Obinutuzumab:

a novel anti�CD20 monoclonal antibody for chronic lym�

phocytic leukemia, J. Adv. Pract. Oncol., 6, 370�374.

115. Subramaniam, J. M., Whiteside, G., McKeage, K., and

Croxtall, J. C. (2012) Mogamulizumab: first global

approval, Drugs, 72, 1293�1298.

116. Son, Y.�D., Jeong, Y. T., Park, S.�Y., and Kim, J. H.

(2011) Enhanced sialylation of recombinant human ery�

thropoietin in Chinese hamster ovary cells by combinator�

ial engineering of selected genes, Glycobiology, 21, 1019�

1028.

117. Ngantung, F. A., Miller, P. G., Brushett, F. R., Tang, G.

L., and Wang, D. I. C. (2006) RNA interference of siali�

dase improves glycoprotein sialic acid content consistency,

Biotechnol. Bioeng., 95, 106�119.

118. Slade, P. G., Caspary, R. G., Nargund, S., and Huang, C.�

J. (2016) Mannose metabolism in recombinant CHO cells

and its effect on IgG glycosylation, Biotechnol. Bioeng.,

113, 468�480.

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