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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5366_Библиотеки_им_академика_М_И_Перельмана.pdf
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R. Pranomphon etal.
complex N-glycans that occur as nal glycan products. Biosynthesis of hybrid
and complex N-glycans begins in the medial-Golgi with the action of an
N-acetylglucosaminyltransferase (GnT-1 or mgat1), which adds a GlcNAc to
Man5GlcNAc2 [34]. The majority of N-glycans are then trimmed by Golgi
α-mannosidase II, removing two mannoses (Man) from GlcNAcMan5GlcNAc2 to
yield GlcNAcMan3GlcNAc2. Hybrid N-glycans result when a structure such as
GlcNAcMan3GlcNAc2 undergoes no further extension or trimming to remove
exposed mannose residues, resulting in structures with one or two terminal mannose residues. In addition, another GlcNAc can be added to the innermost Man
group by the enzyme β1,4-N-acetylglucosaminyltransferase III (GnT-III or mgat3)
in the medial Golgi, resulting in bisecting GlcNAc structures, which can also alter
the capacity for other downstream enzymes to act on the glycan structure. Next, the
enzyme β-1,2-N-acetylglucosaminyltransferase II (GnT-II or mgat2) adds a GlcNAc
to the GlcNAcMan3GlcNAc2 structure to generate the glycan product
GlcNAc2Man3GlcNAc2, which is the precursor for all multiantennary complex
N-glycans. Tri- and tetra-antennary branches can be achieved by adding GlcNAc at
the α(1,3)-mannose site by N-acetylglucosaminyltransferase IV (GnT-IV or mgat 4)
and/or at the α(1,6)-mannose site by N-acetylglucosaminyltransferase V (GnT-V or
mgat 5). Additional modications of complex and hybrid N-glycans can occur in
the trans-Golgi including the addition of core α(1,6)-fucose (Fuc) to the GlcNAc
adjacent to Asn at the N-glycan sites by α-(1,6)-fucosyltransferase and branch elongation by the addition of a β-linked galactose residue to GlcNAc by galactosyltransferase to produce Galβ1-4GlcNAc, or poly-acetyllactosamine (poly-LacNAc)
sequences. Finally, these terminal Gal residues can serve as acceptors for several
sialyltransferases, leading to even more complexity.
6.2.2 O-Linked Glycosylation
O-linked glycosylation occurs in either the ER or Golgi apparatus of animal cells.
O-glycosylation begins with the attachment of a single monosaccharide
(N-acetylgalactosamine, GalNAc) onto the hydroxyl group of the amino acids Ser
or Thr on the polypeptide [35]. As O-glycosylation lacks consensus sequences, glycosylated Ser/Thr residues are often located in the “proline-glutamate-serinethreonine (PEST)” region of the protein [36]. This processing step is further
modied by the addition of other monosaccharides including GlcNAc, GalNAc,
sialic acid, fucose, galactose, xylose, and/or polylactosamine [14, 35].
O-glycosylation does not share a common core structure; it is believed to be primarily involved in protein stability and degradation [36].
6 Metabolic and Process Engineering to Control Glycan Structures…
141
6.2.3 Glycosaminoglycan Synthesis
Glycosaminoglycans are generally synthesized as proteoglycans attached to core
proteins such as serglycin, syndecan, and glypican. Biosynthesis occurs in the ER
or Golgi apparatus. GAGs are attached to the core proteins through a β-linkage
between xylose and either serine or threonine on the core protein. GAGs are long
(up to ~1Mda) linear chains of repeating disaccharide units, consisting of either
GlcNAc or GalNAc alternating with glucuronic acid and/or iduronic acid or Gal.
Extensive chain modications including N-deacetylation and N-sulfonation,
O-sulfonations, and epimerization then occur under the actions of specic enzymes,
many of which have a variety of isozymes whose expression is cell-type dependent.
6.3 Effects ofGlycosylation onBiological Activity
The importance of glycosylation and the question of how glycosylation patterns
affect different properties of recombinant proteins have attracted much attention
over the last decade. For potential therapeutic use, large, complex proteins need to
have human-like posttranslational modications to be functional and nonimmunogenic. Proper glycosylation proles promote biological activity and stability,
increase half-life, and reduce the immunogenicity of protein therapeutics [37, 38].
Obtaining a consistent glycoform prole in production is desired due to regulatory
concerns because a molecule can be dened, in part, by its carbohydrate structures.
An optimal prole may involve a spectrum of product glycans that confers a desired
therapeutic efcacy, or a homogeneous glycoform prole that can be systemically
monitored [1]. As monoclonal antibodies have dominated protein therapeutics,
much work has focused on glycosylation effects on mAbs, though certainly other
proteins, particularly those containing sialic acids can be substantially affected.
N-linked glycosylation is the most prevalent glycan form seen in recombinant
mAbs, and IgG antibodies contain one conserved glycosylation site on each of the
two Asn–297in the constant heavy 2 (CH2) domains. This site has gained recent
importance as it has been demonstrated that the Fc domain of the IgG can be engineered for improved functionality [39].
6.3.1 Mannosylation
In general, human IgG contains low levels (<5%) of high mannose (HM) glycoforms (Man5-9) [5, 40]. The HM glycans increase antibody-dependent cellular
cytotoxicity (ADCC), which is likely due to the lack of core fucose, but signicantly
increase serum clearance, thereby impacting the therapeutic antibody efcacy
[41–43]. For this reason, antibodies with HM glycans must be controlled. Cell type
and cell culture parameters modulate the level of high mannose glycans [44].
142
R. Pranomphon etal.
6.3.2 Fucosylation
Fucosylation involves the addition of fucose from GDP-Fuc to the GlcNAc residue
of Fc oligosaccharides via an α1,6-linkage catalyzed by fucosyltransferase 8 (fut8)
[45]. Almost all therapeutic antibodies produced in CHO cells, mouse myeloma
NS0, and SP2/0 are fucosylated [46, 47]. Removal of the core fucose residue from
Fc oligosaccharides enhances ADCC by improving the binding afnity of the IgG
Fc domain to Fcγ receptor IIIa [48–50]. For example, non-fucosylated therapeutic
antibodies show up to 100-fold increased ADCC [51].
6.3.3 Galactosylation
Galactosylation involves the addition of Gal residues from UDP-Gal to the nonreducing terminal of the Man α1-3 and Man α1-6 arms of the bi-antennary core glycan structure [52, 53]. Terminal Gal residues of Fc glycans affect
complement-dependent cytotoxicity (CDC) [54, 55], protein folding, stability, and
aggregation [56, 57].
6.3.4 Sialylation
Terminal sialic acids in N-linked complex glycans play a major role in the circulatory half-life of glycoproteins as sialic acid masks the penultimate sugar, Gal, and
prevents recognition and uptake of the glycoprotein by asialoglycoprotein receptors
(ASGPR) on hepatocytes, resulting in a longer serum half-life of the therapeutic
protein [58–60].
A variety of factors, both extrinsic and intrinsic, limit our abilities to produce the
desired glycans in cultured mammalian cells. A summary of commonly understood
cellular bottlenecks inuencing glycosylation in CHO cells is illustrated in Fig.6.2.
The remaining sections in the chapter address strategies to overcome these limitations, including choice of host cell line, glycoengineering, and bioprocess
optimization.
6.4 Choice ofHost Cell Line
While CHO cell lines are the dominant mammalian cell host for producing therapeutic proteins, other rodent and human cell lines have been employed, with varying
success at obtaining desired glycan proles. Enzymatic differences between
different host cell types and the implications are shown in Table6.1 (adapted from
6 Metabolic and Process Engineering to Control Glycan Structures…
Fig. 6.2 Various experimentally observed bottlenecks in the glycosylation pathway in cells.
(Adapted from [61] with permission from Springer-Nature)
143
[62, 63]). In addition, different CHO cell hosts (e.g., CHO-K1, CHO DG44) exhibit
different glycosylation patterns, an important consideration when developing a biosimilar product in a different host cell line.
Goh and Ng [63] provided a detailed comparison of the glycosylation proles of
four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII
(FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced from different sources including human urinary- or plasma-derived proteins, different CHO
cell hosts, NS0 and J558L murine myeloma cells, baby hamster kidney (BHK)
cells, and the human cell lines HEK293/T (human embryonic kidney with or without Epstein Barr transformation), PER.C6 (retinoblastoma), HT-1080 (brosarcoma), and AGE1.HN (human neural tissue derived). They noted that for IgG the
NS0-derived structures had signicantly lower galactose levels than the plasma- or
CHO-derived structures, with increased high mannose structures in the NS0-derived
IgG.Interestingly, while terminal sialylation was under 5% on IgG derived from
human plasma, CHO-K1, J558L, and HEK293 cells, 11.8% of glycans on IgG produced in NS0 cells were sialylated. For coagulation factor VII (FVII), which has
two N glycosylation sites, N145 and N322, glycans on plasma-derived FVII were
predominantly complex-type bi- and tri-antennary structures without core fucosylation. CHO- and BHK-derived FVII showed primarily complex-type biantennary
di-sialylated core-fucosylated structures, while HEK293-derived FVII demonstrated the most heterogeneous N-linked glycans with about 20 different glycostructures. Native EPO is N-glycosylated at sites N24, N38, and N83 and is primarily
144
Table 6.1 Enzymatic differences between host species
Glycosylation enzyme Function
β-1,4N-acetylglucosaminyl-
transferase III (GnT- III)
α1,3/4 fucosyltransferase Catalyzes the transfer of
α1,3 galactosyltransferase
(α1,3-GT)
β-galactoside α2,6sialyltransferase (Gal
α2,6-ST)
CMP-Neu5Ac hydroxylase
(CMAH)
Catalyzes the transfer of
GlcNAc from UDPGlcNAc to a core-β-Man
of an N-glycan via
β1,4-linkage to form a
bisecting GlcNAc residue
L-fucose from GDP-Fuc
to N-acetyllactosamine,
generating Lewisx, sialyl
Lewisx structures and
their combinations
Catalyzes the transfer of
Gal onto terminal
lactosaminide residues
via an α1,3 linkage, to
synthesize a Gal-α
1,3-Gal group (alpha-Gal)
Attaches sialic acid with
an alpha-2,6 linkage onto
Gal or GalNAc residues
Catalyzes the conversion
of the precursor molecule
CMP-N-acetylneuraminic
acid (Neu5Ac) to
CMP-N-
glycolylneuraminic acid
(Neu5Gc)
Expression in
mammalian
cells
Expressed in
human cells;
absent in
CHO cells
Expressed in
human cells;
absent in
CHO cells
Inactive in
human and
CHO cells;
functional in
BHK and
murine cells
Expressed in
human and
murine cells;
absent in
CHO and
BHK cells
Inactive in
humans;
functional in
animal cells
R. Pranomphon etal.
Impact on
recombinant proteins
Absence of bisecting
GlcNAc in CHO
derived proteins,
leading to reduced
biological activity of
mAbs
May perturb proper
biodistribution and
pharmacokinetics
Possibility of
immunogenic
glycoforms in
BHK- and murinederived proteins
Undersialylation,
reduced serum
half-life of CHOand BHK-derived
proteins
Presence of
immunogenic form
of sialic acid
particularly in
murine-derived
proteins
decorated with tetra-antennary complex-type glycostructures. Recombinant EPO
expressed in BHK-21, CHO, and HT-1080 cells demonstrated predominantly tetraantennary structures for ~60–80% of the total glycoforms observed. The structures
were largely sialylated in both the native and recombinant forms although the fraction of antennae that were sialylated varied between host cells, leading to charge
variation in the proteins depending on host. Alpha-1 antitrypsin (A1AT) has three
main N-linked glycosylation sites at positions N46, N83, and N247. In the native
A1AT, the glycans are typically disialylated biantennary complex-type structures.
The predominant glycan in CHO-derived, recombinant A1AT was also a disialylated, complex-type biantennary structure but with a lower fraction of 47.9%
compared with the 74–77% found in two plasma studies. Studies in human cell lines
6 Metabolic and Process Engineering to Control Glycan Structures…
145
AGE1.HN, HEK293, HEK293T, and PER.C6 also observed that the predominant
glycan in recombinant A1AT was a biantennary complex-type structure; however,
the terminal sialylation on the predominant glycan in the AGE1.HN, HEK293, and
HEK293T cell lines was signicantly lower than in the native or CHO-derived glycans. In addition, while minimal fucosylation was observed in the plasma-derived
A1AT, recombinant A1AT produced in CHO and human cell lines predominantly
contained core α(1,6) fucose and to a certain degree, Lewisx fucose.
Even among different host CHO cell lines (e.g., CHO-K1 and CHO DG44) and
individual clones derived from the same host, different glycosylation patterns occur.
Yeo and colleagues compared the glycan proles for a recombinant mAb produced
in CHO-K1 and CHO DG44 cell lines [64]. The mAb produced in CHO-K1 cells
had predominantly G0F glycans (~60%) with approximately 25% G1F glycans and
a much smaller fraction of G2F glycans. In contrast, the DG44-derived mAbs had a
much lower fraction of G0F glycans (typically 20–30%) with 40–50% G1F glycans
and ~20% G2F glycans. In both cell lines, the fraction of sialylated and high mannose glycans was relatively small, but it was signicantly greater in the DG44derived mAbs than in the mAb produced in CHO-K1. A similar study was performed
by Könitzer and coworkers at Boehringer Ingelheim in which they compared mAbs
produced in two DG44-derived cell lines and one CHO-K1-derived cell line. In
addition to comparing the glycan patterns, they performed RNA-Seq to compare the
gene expression patterns between the different host cell lines. The CHO-K1-derived
cell line produced a higher fraction of nonfucosylated antibodies compared to either
of the DG44 cell lines. When looking at high mannose sugar structures, antibodies
produced in DG44 cell line 2 demonstrated the lowest amount (median: 3.7%;
range: 0.1–35.0%), followed by DG44 cell line 1 (median: 7.9%; range: 1.1–37.0%)
and the CHO-K1 cell line (median: 9.7%; range:1.7–24.4%). Terminal sialylation
was also increased in DG44 cell line 2 compared to the other two cell lines. Lastly,
antibody galactosylation was consistently lower in the DG44 cell line 1 and CHOK1 cell line than in DG44 cell line 2. In general, the glycan proles from DG44 cell
line 1 and the CHO-K1 cell line show more similarity to each other than to DG44
cell line 2. Using RNA-Seq, they compared the transcriptome of the three cell lines
and identied 188 glycosylation-related genes that were expressed in all 3 lines, 4
genes that were unique to the CHO-K1 cell line, 8 genes that were only expressed
in the DG44 cell lines, and 2 genes expressed uniquely in either of the 2 DG44 cell
lines. They further characterized relative expression levels of several key enzymes
and noted that expression of a recombinant mAb led to differential expression of
several glycosylation-related genes.
For production of recombinant or bioengineered glycosaminoglycans such as
heparin, the choice of host cell lines appears even more critical. Baik and coworkers
attempted to produce a bioengineered heparin in CHO cells by engineering the
GAG biosynthesis pathways [65]. They overexpressed two critical enzymes in the
pathway, NDST2, which removes the acetyl group from GlcNAc replacing it with a
sulfate group, and 3OST, which sulfonates the critical 3-hydroxyl position on
146
GlcNAc responsible for anticoagulant activity. While they were able to dramatically
increase sulfation and signicantly increase anticoagulant activity, the resulting
product was very different from pharmaceutical heparin both in terms of structure
and anticoagulant activity. In contrast, Thacker and colleagues performed similar
metabolic engineering manipulations on murine mastocytoma cells (a tumorigenic
form of mast cells, which are the source of heparin in vivo). They were able to
obtain heparin-like GAGs with an anticoagulant potency that exceeds porcinederived heparin [66].
R. Pranomphon etal.
6.5 Glycoengineering
Genetic approaches for enhancing N-glycosylation are the most common strategies
and employ gene editing or transient expression techniques to change the activity of
glycosyltransferases and increase or decrease the precursors involved in the
N-glycosylation process. Genetic approaches altering heterogeneity, sialylation,
fucosylation, and branching in N-glycan structures are described below and shown
in Fig.6.3.
6.5.1 Manipulating Heterogeneity
Although glycoprotein biologics produced by CHO cells containing heterogeneous
mixtures of N-glycans are considered safe as human therapeutics, excess heterogeneity can be an issue. This excess heterogeneity arises due to the variability of
N-glycan processing and can compromise the safety and activity of such glycotherapeutics. Having homogenous glycoforms allows comparative studies of their
biological effects, which can be advantageous in the development of therapeutic
candidates [67]. Yang etal. showed that CHO cells can be genetically engineered to
produce glycoproteins in a nearly homogenous form without any deleterious effect
on their growth or other compensatory changes [68]. To achieve this goal, the invivo
function of each of the 19 glycosyltransferases potentially participating in N-glycan
formation and processing was determined in CHO cells by individual and/or multiple glycosyltransferase gene knockouts. The effects of knocking out each of the 19
genes involved in N-glycan branching (mgat1/2/3/4A/4B/4C/5/5B), galactosylation
(B4galt1/2/3/4), N-acetyllactosamine (LacNAc) elongation (B3gnt1/2/8), terminal
capping by sialylation (st3gal3/4/6), and core α-6-fucosylation (fut8) were determined using a stably expressed model protein, a recombinant human erythropoietin
(rhEPO) containing in its structure three N-glycans with heterogeneous tetraantennary structures, low poly-LacNAc and terminal α-2,3-linked sialic acid.
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6 Metabolic and Process Engineering to Control Glycan Structures…
147
Eliminates b4-branched
tetra-antennary N-glycans
Eliminates a-2,3 linked sialic acids
Eliminates a-2,3 linked sialic acids
( bi-antennary N-glycans but
with increased poly-LacNAc)
Eliminates poly-LacNAc
Increases tri-antennary N-glycans
over bi-antennary forms
Increases tetra-antennary N-
a-glycans over bi-antennary forms
Adds α-2,6 linked sialic acids
Eliminates a-2,3 linked sialic acids
Increases a-2,3 linked sialic acids
23supression
o
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N-glycosylation genes
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mutant GNE gene
&
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i
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Asn
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k
-
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knockout of
CMP-SAT gene,
CMP-SAS gene
&
G
n
,
i
a
l
Increases a-2,3 linked sialic acids
Eliminates b6-branched
tetra-antennary N-glycans
Eliminates galactose (>90%)
Eliminates immunoreactivity for
LacNAc in cells containing
knockout
of
Eliminates fucose
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Increases complex
e
bisected glycans
Introduces additional N-glycan
attachment sites in the protein
Gene alteraton
Increases galactose
Adds & increases a-2,6 linked
sialic acids & monosialylation
Adds & increases a-2,6 linked
sialic acids. Increases branching
Increases a-2,3 linked sialic acids
Bisects
N-glycans
Fig. 6.3 Summary of genetic approaches for manipulating glycosylation in CHO cells described
in this chapter. Gene knockouts are represented by yellow segments and gene knock-ins, overexpression and alteration are represented by green segments. For convenience, tetra-antennary
N-glycans and genes participating in N-glycosylation are also shown
6.5.2 Manipulating Sialylation
Sialylation refers to the glycosidic addition of a negatively charged monosaccharide, a sialic acid, by sialyltransferases, generally to terminal Gal or GalNAc, though
occasionally to GlcNAc or sialic acid itself in complex N-glycans [69]. CHO cells
148
R. Pranomphon etal.
contain α-2,3-sialyltransferases (α-2,3-SiaT), whereas human cells have α-2,6-SiaT
in addition to α-2,3-SiaT [70, 71]. Due to this difference, N-glycans produced by
CHO cells only contain sialic acid residues linked by α-2,3-glycosidic linkages,
whereas human glycans contain both α-2,3- and α-2,6-linked sialic acid residues
[72]. Sialic acid at the termini in complex N-glycans masks terminal Gal from recognition by hepatocyte asialoglycoprotein receptors that lead to rapid clearance
from the circulation [73]. Insufcient or lack of sialylation in glycoprotein biologics
can lead to inconsistency in the pharmacodynamics and cause challenges in formulating reproducible dosages. Thus, correct and generally, maximal sialylation is necessary to ensure longer plasma half-lives and maximum in vivo activity and
therapeutic efcacy [74].
Of the numerous (>50) sialic acids occurring in nature, N-acetylneuraminic acid
(Neu5Ac) is the most abundant [69]. Its derivative, N-glycolylneuraminic acid
(Neu5Gc), is also a major sialic acid found on mammalian cell surfaces, formed by
Neu5Ac hydroxylation by cytidine monophosphate (CMP)-Neu5Ac hydroxylase.
Humans lack CMP-Neu5Ac hydroxylase and are unable to synthesize Neu5Gc.
Glycoproteins synthesized by CHO cells occasionally contain N-glycans capped
with Neu5Gc. Neu5Gc in glycans can be a cause for concern as Neu5Gc-capped
glycans act as “xeno-autoantigens” in humans and are a cause of “xenosialitis,” an
inammatory process initiated by binding of naturally occurring antibodies against
Neu5Gc in the human body [75–77]. The Neu5Gc-dependent antigenicity of glycoproteins obtained from CHO cells depends on the amount and locations of Neu5Gc
in the glycan structure. For example, mAbs and rhEPO produced by CHO cells
containing 1–2% Neu5Gc did not elicit an immune response, whereas, in the same
study, fetuin with high levels of Neu5Gc (7% of total sialic acid residues) elicited an
immune response in chickens [78]. A recent study by Yu etal. on different clinical
mAbs containing Neu5Gc residues concluded that mAbs containing a single
Neu5Gc residue do not bind to anti-Neu5Gc antibodies, while only a minor fraction
of mAbs containing two or more Neu5Gc showed binding to anti-Neu5Gc antibodies [79]. Further, this study suggested that the binding of anti-Neu5Gc antibodies to
mAbs containing multiple Neu5Gc depends on Neu5Gc location in the mAb structure. A recent study has implicated a newly discovered miRNA, cgr-miR-111, in
controlling the expression of CMP-Neu5Ac hydroxylase, suggesting another strategy for control of Neu5Gc on recombinant proteins produced in CHO cells [80].
6.5.2.1 Increasing α-2,6 Sialylation
Glycoprotein biologics that contain α-2,6-linked sialic acid residues are suggested
to be more “human-like” and perform better invivo [81, 82]. In two recent studies,
chemo-enzymatic modication (in vitro glycosylation) of two IgGs (anti-Her2 antibody and rituximab) creating homogenous glycans containing α-2,6-sialic acid residues enhanced ADCC due to stronger interaction of α-2,6-sialylated glycans with
FcγRIIIa receptors on natural killer cells [81, 83]. Similarly, in two previous studies
involving in vitro glycosylation, α-2,6-sialylated versions of IgGs produced
6 Metabolic and Process Engineering to Control Glycan Structures…
149
superior anti-inammatory responses compared to asialylated or α-2,3-sialylated
versions [82, 84].
CHO cells were successfully engineered to produce rhEPO containing almost
exclusively α-2,6-sialylation by knockout of st3gal4/6 genes (encoding α-2,3SiaTs) and knock-in of st6gal-I gene (encoding an α-2,6-SiaT) [68]. Furthermore,
in the same study, homogeneous bi-antennary N-glycans capped by α-2,6-NeuA
were produced by additional knockout of mgat4A/4B/5 genes.
6.5.2.2 Increasing theSialic Acid Content
Besides knockout and knock-in of sialyltransferase genes, increases in sialic acid
content can be obtained by overexpressing genes participating in steps prior to
sialylation or inhibiting genes encoding for sialidases that remove the sialic acid
after the sialylation step. The addition of sialic acid residues to a growing N-glycan
chain can be limited by the absence of Gal, which acts as an acceptor substrate for
sialyltransferases. Increasing the Gal content in N-glycans by overexpressing GalT
produces a corresponding increase in the sialic acid content in CHO cells overexpressing sialyltransferase enzymes. Raymond etal. transiently coexpressed genes
encoding for GalT, α-2,6-SiaT, and an IgG1 antibody (trastuzumab/Herceptin®) and
produced efcient α-2,6-sialylation in trastuzumab’s Fc region. The glycans under
investigation were monosialylated, a physiologically relevant form found in circulating human IgGs. Over 85% of sialic acids on trastuzumab showed α-2,6- sialylation
due to overexpression of human GalT, which inserts Gal residues in the glycan that
are preferentially used by α-2,6-SiaT rather than α-2,3- SiaT. Overexpression of
GalT alone increased the Gal content of the Fc glycans, but had no effect on increasing the sialylation, whereas overexpression of α-2,6-SiaT only increased the
sialylation moderately [85]. In a similar study, coexpression of GalT was benecial
in CHO cells overexpressing α-2,3-SiaT to increase the sialylation. Trisialylated
glycans on rhEPO increased from 17.3 to 35.5% when expressed in CHO EC1 cells
co-overexpressing both human α-2,3-SiaT and GalT [86].
Sialic acid content can also be enhanced by increasing the concentration and
availability of the donor sugar nucleotide CMP-sialic acid (CMP-SA) present in the
Golgi apparatus. Inside the nucleus, CMP-SA is generated from sialic acid by CMP
sialic acid synthetase (CMP-SAS) and later transported to the Golgi by CMP-sialic
acid transporter (CMP-SAT). In eukaryotes, sialic acid is synthesized in the cytoplasm by three enzymes in a four-step process. The two rst steps are catalyzed by
a bifunctional enzyme, GNE (UDP-N-acetylglucosamine-2-epimerase/N- acetylmannosaminekinase), having kinase and epimerase activity. The epimerase
activity of GNE converts UDP-GlcNAc to N-acetylmannosamine (ManNAc), which
is then converted to ManNAc-6-phosphate by the kinase activity of GNE.The last
two steps are catalyzed by two enzymes, Neu5Ac-9-phosphate synthase and
Neu5Ac-9-phosphate phosphatase, which produce Neu5Ac from ManNAc-6phosphate by condensation and dephosphorylation reactions, respectively. GNE is a
rate-limiting enzyme for the synthesis of sialic acid in the cytoplasm [71, 87].