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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5886_Библиотеки_им_академика_М_И_Перельмана.pdf
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R. Pranomphon etal.
Increasing sialic acid content in the cytoplasm or CMP-SA in the nucleus by different strategies increases sialylation by varying amounts. Overexpression of CMPSAT in CHO cells expressing recombinant human interferon gamma (IFN-γ)
increased IFN-γ sialylation by 4–16% [88]. Similarly, a modest increase in
sialylation (10–20%) was observed upon supplementation with ManNAc, which led
to a 12-fold increase in the intracellular pool of CMP-sialic acid [89]. In another
study, supplementation with 1,3,4-O-Bu3ManNAc, a chemical analog of the sialic
acid precursor ManNAc increased the nal sialic acid content of rhEPO >40% in
CHO cells at a 100-fold lower concentration than natural ManNAc [90].
Co-overexpressing several genes together enhanced sialylation more signicantly than overexpressing single genes individually. CHO cells expressing CMPSAS in combination with CMP-SAT and human α-2,3-SiaT exhibited greater
rhEPO sialylation compared with CHO cells overexpressing α-2,3-SiaT or CMPSAS individually [91]. GNE, catalyzing the rst two steps in the synthesis of sialic
acid, is a rate-limiting enzyme, and its activity is regulated by feedback inhibition
from free cytoplasmic CMP-Neu5Ac [71, 87]. Coexpressing a mutant version of
GNE lacking feedback regulation with CMP-SAS enhanced the sialylation modestly, but when the mutant version of GNE was coexpressed with CMP-SAT and
human α-2,3-SiaT, CHO cells produced rhEPO having 43% increased sialylation.
Coexpression experiments involving CMP-SAT clearly indicated that endogenous
CMP-SAT is insufcient, and its overexpression was essential for increasing the
sialylation [92]. Recently, epigenetic modulators have been employed to turn on the
silenced α-2,6-SiaT in CHO cells by demethylating its promoter using a CRISPRbased approach [93].
Sialidases are enzymes that catalyze the removal of sialic acid residues from
glycoproteins and glycolipids. CHO cells contain four different sialidases (Neu1-4)
distributed in the lysosome (Neu1 and Neu4), cytosol (Neu2), and plasma membrane (Neu3) [94]. During glycoprotein biologic manufacture, lowering the activity
of these enzymes is desirable, but not completely, due to their important roles in
crucial biological functions [95–97]. The cytosolic sialidase, Neu2, is released into
the supernatant during cell lysis and preferentially removes α-2,3-linked sialic acids
from the glycoprotein products [98]. RNA-mediated suppression of Neu2 [99] and
plasma membrane-bound Neu3 [74] activities by 40 and 98%, respectively,
increased sialic acid content (up to 33% in model proteins), but the effect of Neu2
suppression was observed in the death phase only.
6.5.3 Manipulating Fucosylation
Core Fuc residues on IgG antibodies have a negative effect on their effector function
[51]. Effector function is essential in IgGs designed for use in tumor therapy; after
binding to antigens on cancer cells, the IgG Fc region binds strongly to FcγRIIIa
receptors present on natural killer cells, causing cancer cell death by lysis via the
ADCC mechanism [48, 51]. Fucose-containing N-glycans present at Asn-297in the
6 Metabolic and Process Engineering to Control Glycan Structures…
151
IgG adversely affect the Fc-FcɣRIII interaction [100]. Multiple studies have successfully demonstrated that removal of the core Fuc residue from the N-glycan in
human IgG1increases the binding afnity of Fc toward FcɣRIII, which, in turn,
enhances the invivo ADCC signicantly [48, 51]. Such fucose-free antibodies can
be benecial to patients as their higher potency enables lower dosage administration
[101, 102].
In mammals, fut8 is the only gene encoding for a fucosyltransferase capable of
adding Fuc to N-glycans [103]. Inhibition or knockout of fut8 and interference with
transport and synthesis of donor substrate GDP-Fuc are two approaches for reducing or inhibiting Fuc addition to N-glycans in CHO cells [33]. Knockout of fut8in
CHO cells using zinc nger nucleases and homologous recombination produced
fully afucosylated antibodies, showing enhanced ADCC [104, 105]. Further, Chan
etal. showed that inactivating Slc35c1, encoding for GDP-fucose transporter, also
generated CHO cells producing fucose-free glycans [106].
Alternatively, antibodies devoid of core Fuc can also be produced by CHO cells
overexpressing the GnT-III enzyme, which catalyzes bisecting GlcNAc addition
onto the common core structure (Man3GlcNAc2) in N-glycans [107]. Upon addition of bisecting GlcNAc, the oligosaccharide cannot act as a suitable substrate for
subsequent glycosylation enzymes, especially Golgi-mannosidase II (Man-II),
GalT, and FucT [108], leading to the production of fucose-free mAbs showing
increased ADCC, but decreased complement-dependent cytotoxicity. The decreased
CDC is due to hybrid N-linked oligosaccharide structures, resulting from incomplete mannose cleavage [109].
6.5.4 Manipulating Branching
In mammals, branching in N-glycans occurs in the medial Golgi and is carried out
by GnT-I, -II, -IV, and –V enzymes, which control GlcNAc addition at the branch
point in a stepwise manner [108]. Branching produces bi-, tri-, and tetra-antennary
structures, which can be extended by enzymes in the trans-Golgi capable of adding
Gal, Fuc, and sialic acids (Fig.6.2) [110]. GnT-I and GnT-II control the formation
of bi-antennary structures; tri-antennary structure formation is controlled by GnT-IV
or GnT-V, and the tetra-antennary structure formation is controlled by the combined
action of GnT-IV and GnT-V (Fig.6.2). Higher branching (tri and tetra) provides
additional sites for the attachment of sialic acid residues, which enhance biological
activity and circulatory lifetime. Increased branching of therapeutic glycoproteins is
thus of signicant clinical as well as commercial interest.
Multiple studies have shown that branching can be increased by overexpression
of GnT-IV and/or GnT-V enzymes. Tri-antennary structures were signicantly
increased (over 50%) by overexpression of GnT-IV or GnT-V individually in CHO
cells producing IFN-γ with predominantly bi-antennary sugar chains [111]. In the
same study, tetra-antennary structures were increased up to 56% of the total sugar
chains when GnT-IV and GnT-V enzymes were coexpressed. However, compared
152
to observed increases in tri- and tetra-antennary structures, the corresponding
increase in sialylation was insignicant. Inadequate sialylation was attributed to
insufcient intracellular sialyltransferase or CMP-SAS activity. The reduced sialic
acid capping on tri- and tetra-antennary structures in rhEPO was solved by Yin and
coworkers by additionally expressing human α-2,6-SiaT [112]. They showed that
coexpression of α-2,6-SiaT with GnT-IV and GnT-V produced rhEPO containing
approximately 92% tri- and tetra-antennary N-glycans with a 45% increase in the
sialic acid content compared with rhEPO obtained from wild-type CHO-K1 cells.
R. Pranomphon etal.
6.6 Effects ofBioprocess Conditions
A variety of bioprocess strategies have been employed to alter glycosylation proles, sometimes individually, but often in combination. Here, we group the studies
based on the predominant manipulation while recognizing that more than one
parameter may have been varied in these studies.
6.6.1 Temperature
Mellahi and colleagues investigated the effect of various feeding regimes and temperature shift strategies to improve antibody productivity and ensure product quality
of an inducible CHO cell line (cumate gene switch) expressing rituximab [113,
114]. Experiments were performed in both shake asks and bioreactors, with induc-
tion performed at cell densities ranging from 1×106 to 10×106 cells/mL.While the
cell density at induction had a signicant impact on product titer, it had little effect
on the glycan distribution. In contrast, the culture duration signicantly affected
glycosylation with a signicant decrease in galactosylation observed as the culture
proceeded. They further explored temperature shifts from 37°C to either 34°C or
30°C and from 34°C to 30°C, coinciding with induction of production at either
4×106 or 10×106 cells/mL.Induction at high cell density combined with a temperature shift from 37 °C to 30 °C led to the highest antibody concentrations.
Growth at 37°C before induction yielded slightly higher percentages of galactosylation, sialylation, and fucosylation, compared to cultures started at 34°C, but the
differences were not statistically signicant. Slightly lower galactosylation was
seen later in culture, an effect that was less pronounced for cultures grown at 34°C
before induction.
McHugh and colleagues examined the effects of a temperature shift on therapeutic protein production, charge variants, N-linked glycosylation, and protein aggregation [115]. In this study, two CHO-K1 GS cell lines expressing different human
mAbs (IgG1 mAb1 and IgG4 mAb2, respectively) exhibited differential responses
to a temperature shift. They observed that the nal mAb1 titer with a temperature
shift to 32°C was about 25% greater than unshifted cultures. Shift temperatures of
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153
Δ1.5°C increments signicantly affected nal titer and charge variants. Furthermore,
lower shift temperature decreased acidic charge variants for both cell lines.
Signicant differences in G0 and Man5 of about 1% and a slight decrease in galactosylation were observed between shifted and unshifted conditions for mAb1 production. In addition, an early shift temperature on Day 3 signicantly reduced
mAb2 galactosylation similarly to mAb1 such that G1F was reduced by 5% and
G2F was reduced by 0.8 with a 3% increase in G0F.
6.6.2 pH
Ivarsson and colleagues investigated the impact of chemical stress parameters (pH,
dissolved oxygen tension (DOT) and osmolarity) and mechanical stress parameters
(sparging) on cell growth, productivity, and N-linked glycosylation of a murine
hybridoma cell line producing an IgG1 antibody, using a shift-experiment methodology in batch cultures [116]. A shift in one or more of the process parameters was
performed during the early exponential growth phase. They observed that cell
growth was dependent on pH, followed by osmolarity. mAb productivity was
dependent on DOT, followed by pH. In contrast, sparging had little effect on the
specic growth rate or production rate in a range of 0.05–0.2vvm. Increasing pH
between 6.8 and 8.0 monotonically decreased galactosylation and sialylation indices, with a maximum decrease of 50% between pH6.8 and 8.0. The reduction in
galactosylation was due to decreases in both G1F and G2F glycans with an increase
in G0F glycans from 16 to 35% of total glycans. A slight decrease in fucosylation
(from 99% to 94%) was also observed over that pH range. Compared with 50% of
air saturation, DOT of 10% and 90% air saturation showed small increases in both
galactosylation and sialylation. Increases in osmolarity from 320 to 420mOsm/kg
at the control pH of 7.2 did not affect glycosylation; however, at elevated pH (>7.2),
osmotically stressed cultures showed increased galactosylation and sialylation, offsetting the decreases caused by increased pH.
Seo and coworkers evaluated the effects of different cultivation pH (6.8, 7.0, 7.2,
7.4, and 7.6) and temperature (33.0°C and 37.0°C) on a novel human cell line,
F2N78, established by somatic fusion of HEK293 and Namalwa lymphoma cells,
producing an antibody against rabies virus. The maximum antibody concentration
was observed at 37°C and pH6.8. Regardless of temperature, the highest specic
growth rate occurred at a pH range of 7.0–7.4. The dominant glycan form was G1F,
followed by G2F and then G0F, a signicant increase in galactosylation compared
with antibody production in CHO cells. Increasing pH decreased G2F glycans and
increased G0F glycans while culture temperature had little effect on the glycan
distribution. Notably, a signicant fraction of the antibody obtained from the stationary phase and later points in culture was aglycosylated, particularly at pH values
between 7.0 and 7.4, which they attributed to glucose depletion [117].
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R. Pranomphon etal.
Jiang and colleagues investigated the impact of pH excursions on cell culture
performance and N-linked glycosylation for three mAb-producing Chinese hamster
ovary cell lines [118]. Antibody galactosylation (G1F+G2F) was greater at higher
pH values in the range of 6.90–7.10 while cell-specic productivity decreased with
the increased pH for one cell line, suggesting that the impact of pH excursions on
antibody N-linked glycosylation is cell-line specic.
Zheng and coworkers investigated the effect of a pH shift during the expression
phase on a CHO cell line producing anti-CD52 mAbs in perfusion culture and compared the production process, CQAs and the biological potency invitro [119]. In
this study, they shifted the culture pH from 7.15±0.05 to 6.85±0.05 at day 9 and
maintained a control group at 7.15±0.05in a 15-L bioreactor. Under pH shift conditions, the cell density and viability were stable and high throughout the perfusion
culture. In addition, the qGlc and qLac were higher in the pH-shifted conditions
than in the pH control conditions. Even though the specic productivity was lower
under pH shift, the volumetric productivity remained high. Conversely, the charge
and size variants of mAbs were similar in both pH conditions. Interestingly, six
major glycoforms (G0, G0F, G1, G1F, G2F, and Man5) were observed under the
two pH conditions. All forms were signicantly different except Man5 and G1. The
galactosylation (G1+G1F+G2F) increased signicantly upon pH shift, due primarily to a decrease in G0F and increases in G1F and G2F.However, a signicant
increase in G0 was also observed, leading to an overall decrease in fucosylation.
CDC and ADCC efcacy were improved signicantly in the antibodies derived
from the pH-shifted cultures, which can be attributed to the increased galactosylation and decreased fucosylation, respectively.
6.6.3 Feeding Strategies andOther Bioprocess Manipulations
Reinhart and coworkers examined cell growth and product formation in batch, fedbatch, and semicontinuous perfusion cultures of three CHO cell lines (CHO-K1,
CHO-S, and CHO DG44) producing the same monoclonal antibody in an isogenic
format by transfection with the same bacterial articial chromosome (BAC) constructs [120]. Additionally, two different cell culture media were used to investigate
the effects on the bioprocess and mAb quality. Noticeably, fucosylated, mannosylated and aglycosylated glycans were less inuenced by the media but were primarily inuenced by the host cell line. Generally, there were core-fucosylated, complex
biantennary Fc glycans with predominantly G0F, G1F, and G2F glycoforms present
in all three CHO cell lines. Mannosylation was highest in CHO DG44 (11–13%)
followed by CHO-K1 (5–9%), and then CHO-S (2–3%). Fucosylation was lowest
in CHO DG44 (71–83%) followed by CHO-K1 (82–84%) and CHO-S (94–96%).
The light chain region also had a glycosylation site containing sialic acid residues.
CHO-K1 cells had the highest amount of sialylated mAb (21–36%) followed by
CHO DG44 (15–19%) and CHO-S cultures (6–15%). Generally, CHO-S cells
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155
showed greater biomass synthesis while CHO-K1 showed the highest mAb
production.
Wang and coworkers investigated the effects of different media compositions,
EX-CELL (EX) medium and immediate advantage (IA) medium from MilliporeSigma- Aldrich, on the glycan prole of a recombinant protein, EPO-Fc, secreted
from a CHO-GS cell line using an intact glycopeptide analysis method [121]. In this
study, the culture supernatant containing recombinant EPO-Fc protein was harvested on day 5 and day 8 of a batch culture. The three EPO N-glycosylation sites
exhibited bi-, tri-, and tetra-branched glycan structures with various fucosylation
and sialylation levels at each site, while the Fc N-glycosylation site exhibited mainly
fully fucosylated, asialylated bi-antennary N-glycans as expected. Noticeably,
EPO-Fc protein in the EX medium produced a more complex tetra- antennary
N-glycan prole on the three EPO N-glycosylation sites than IA medium, while IA
medium produced more bi- and tri-antennary structures at these same sites. The
EPO-Fc exhibited about 25% sialylation, predominantly monosialylation on the
EPO N-glycosylation sites. No sialylation was seen on the Fc site, as expected.
While sialylation remained relativelyconstant between day 5 and day 8in the EX
medium, it decreased somewhat in the IA medium, particularly at the rst
N-glycosylation site. Furthermore, small amounts of the immunogenic Neu5Gc
(2–6%) were detected in this study, and the content signicantly increased on day 8
compared to cultures on day 5in both media.
Wang and coworkers investigated the effect of ultralow carbon dioxide partial
pressure (pCO2) on mAb N-glycosylation in CHO cell culture [122]. In fed-batch
bioreactor cultures, they decreased the medium pCO2 to an ultralow value
(<25mmHg) by increasing the headspace aeration rate and observed that cell viability and mAb production decreased. In addition, low pCO2 increased the percentage
of galactosylation to 27.45 ± 2.13% compared to the normal pCO2 condition
(21.36 ± 1.66%), while the fucosylation percentage was not affected. They suggested that the increased mAb galactosylation under low pCO2 conditions may be
caused by the upregulated transcription levels of relevant glycosyltransferases
(GnT-1 and B4galt1) and nucleotide sugar transporters (UDP-GlcNAcT and
UDP-GalT).
6.6.3.1 Glucose andOther Glycosylation Precursors
Zhang and coworkers investigated the effect of glycosylation precursors on the glycosylation proles of IgG expressed in CHO cells [123]. In this study, when glucose
was depleted, leaving lactate as a complementary carbon source, different sugar
combinations of fructose, fucose, mannose, and galactose were fed into the culture
to study their effects on glycosylation. They observed that feeding with mannose or
glucose lowered HM glycans by 3–7% compared to feeding without mannose or
glucose. Feeding galactose increased G1 glycans by 8–20% with a 2–6% increase
in G2 glycans compared to feeding without galactose or glucose. Furthermore,
feeding fucose signicantly increased concentrations of intracellular GDP-Fuc.
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R. Pranomphon etal.
However, as the afucosylated fraction (other than high mannose glycans) was very
small, fucose feeding had no effect on fucosylation.
6.6.3.2 Amino Acids
Ghaffari and coworkers investigated the effect of limiting cysteine, asparagine, or
glutamine on batch and fed-batch cultures for three antibody-producing Chinese
hamster ovary cell lines (CHO-DXB11, CHO-K1SV, and CHO-S) [124]. They
found that cysteine limitation had a negative impact on CHO cell growth, mAb
productivity, and the glycosylation pattern of all three cell lines. Glutamine limitation reduced cell growth but not specic productivity. In contrast to glutamine,
asparagine limitation did not signicantly affect either specic productivity or cell
growth. Under the limiting conditions, there were minor differences in the glycosylation proles of the EG2-hFc antibody produced in the CHO-DXB11 cell line. In
the case of cysteine limitation in the CHO-K1SV cells, the fucosylated glycans
were reduced by 16% and the antennarity index by 15%. For the CHO-S cells, the
fucosylated glycans were reduced by 12% and in the antennarity index by 16%.
Restoring the cysteine levels after 1day of cysteine limitation resulted in partial
recovery of cell growth and productivity in CHO-DXB11 culture. Interestingly,
they observed that supplementing cultures with cysteine resulted in similar cellspecic productivity as using commercial feeds.
6.6.3.3 Glycosaminoglycan Production
In an effort to improve the glycan structures for the bioengineered heparin described
above, Baik etal. evaluated the effects of bioprocess conditions on productivity,
structure, and anticoagulant activity. Supplementing the cultures with a chemically
dened feed medium increased IVCD approximately twofold and product titer
nearly threefold. However, the glycan compositions were largely unchanged.
Adding a bolus of cysteine increased the anticoagulant activity approximately twofold, but this was not sufcient to obtain pharmacological anticoagulant activity [125].
6.6.4 Culture Additives
Dang and coworkers investigated the effects of carbon dioxide partial pressure,
media hold duration (at 37 °C), manganese, and high-temperature short-time
(HTST) treatment of culture media on afucosylation of mAb (IgG1) produced in
CHO cells [126]. Afucosylation level was highest (~7.1%) in the low pCO2 model
employing the other process parameters (i.e., media HTST treatment, Mn, and
media hold), while the lowest afucosylation level (~2.5%) was observed for the
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157
condition without these three factors. Moreover, they observed that HTST treatment
may not be associated with the higher afucosylation in the condition with Mn supplementation and media HTST.Noticeably, G0F was reduced in conditions with Mn
supplementation, whereas Man5 was ~1% lower in all the conditions tested.
Interestingly, there was a synergistic effect of these three independent parameters
(pCO2, Mn, and media hold) on afucosylation. Afucosylation increased (~4%) with
increasing these process parameters. In addition, they investigated the effects of
high pCO2, Na+, and osmolality on afucosylation. They found that afucosylation
increased with increasing Na+ resulting from Na2CO3 addition to maintain pH in the
high pCO2 model.
Zhou and coworkers investigated the effects of different components, in particular bovine serum albumin (BSA), on IgG N-glycoforms in hybridoma culture media
[15]. They found that small molecule additives including glucose, glutamine, pyruvate, and sodium bicarbonate caused minor changes in glycan species. Noticeably,
glutamine addition decreased galactosylation and sialylation, presumably due to
ammonia accumulation. On the other hand, BSA addition increased galactosylation
and sialylation levels, with a decrease in fucosylation level. Furthermore, two new
species of IgG N-glycans in the BSA+ conditions were discovered, afucosylated
sialylated N-glycans and Neu5Ac-containing N-glycans, rather than the Neu5Gccontaining glycans commonly seen in rodent species.
The effects of deoxymannojirimycin (DMJ), an α-D-mannosidase inhibitor, on
high mannose and core fucose levels were studied during IgG1 production in two
different CHO cell lines (CHO-DXB-11 and CHO-K1SV) by Schwarz etal. (2020)
[127]. DMJ at concentrations of 500 μM signicantly decreased the levels of
complex- type glycans and core-fucosylated complex glycans by 15- and 14-fold,
respectively, and signicantly increased the HM glycan level by 21-fold. However,
they observed that the relative fucosylated complex and fucosylated bi-antennary
levels were not affected by DMJ, demonstrating that DMJ had no effect on fucosyltransferase activity. Therefore, they suggest that the decrease in core-fucosylated
glycans upon DMJ feeding resulted from increased HM glycans rather than an
increase in afucosylated complex glycoforms.
The effect of rafnose supplementation on high mannose species was studied in
high-throughput systems using two cell lines (CHO-K1 and CHO-S) expressing
humanized and human IgG1 antibodies (cell line 1 and cell line 2) by Bruhlmann
etal. [128]. Cultures supplemented with rafnose at constant medium osmolality
(315mOsm/kg) exhibited up to sixfold increased HM species with increasing rafnose. Interestingly, cell line 1 predominantly expressed Man5 and Man6 glycans,
while Man7 was not detected, and small amounts of Man8 were observed. In addition, cell line 2 exhibited mainly Man5 glycans. They highlighted that rafnose
mainly favored Man5 glycans. Supplementing the culture with 30mM rafnose at
three different osmolalities signicantly increased HM glycans. Notably, culture
supplementation with rafnose inuenced the expression levels of glycosylationrelated genes. In particular, sialyltransferase gene expression was highly upregulated, whereas galactosyltransferase gene expression was downregulated.
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Ehret and coworkers investigated the impact of cell culture media additives on
glycosylation proles of recombinant IgG1 produced in Chinese hamster ovary
cells (CHO-K1and CHO DG44) [129]{Prabhu, 2019 #157}. Addition of 15μM
kifunensine into the feed medium increased HM species by 85.8%. Moreover, supplementing the culture with 800μM 2-F-peracetyl fucose lowered fucosylation by
76.1%. Notably, the combination of 24μM uridine, 48μM manganese, and 120mM
galactose increased total galactosylation by 40.9%. In addition, the presence of
30 μM dexamethasone in combination with galactose (120 mM), manganese
(48μM), and uridine (24μM) increased sialylation by 6.9%.
Yin and coworkers examined the impact of the butyrated N-acetylmannosamine
analog, 1,3,4-O-Bu3ManNAc on recombinant protein expression in CHO cell culture [90, 130]. Addition of 1,3,4-O-Bu3ManNAc increased protein expression of
EPO- and human IgG-expressing CHO cell lines with negligible impact on cell
growth, viability, glucose consumption, and lactate production. Conversely, sodium
butyrate (NaBu) addition decreased viable cell density and viability by ∼20 and
∼30%, respectively, compared to untreated or 1,3,4-O-Bu3ManNAc treated cells for
both products. NaBu-treated cells exhibited lower sialylation levels on puried EPO
compared to the untreated control while the addition of 1,3,4-O-Bu3ManNAc
increased sialylation levels compared to both the NaBu-treated and untreated control cultures. They concluded that, at the same butyrate concentration, supplementation with 1,3,4-O-Bu3ManNAc has a negligible impact on cell viability, cytotoxicity,
and apoptosis while also enhancing protein sialylation, compared to NaBu in CHO
cell culture.
Wells and colleagues investigated the effects of galactose, uridine, and
2-F-peracetyl fucose (2FP) on cell growth, titer, and gene expression of a CHO cell
line producing an IgG1 antibody in a batch culture [131]. Supplementation with
200μM uridine increased galactosylation by 2% and decreased fucosylation by 6%
compared with unsupplemented controls. Furthermore, the mRNA levels of
mgat1–2, B4galt1–6, and the sugar transporter Slc35a3 were upregulated compared
with controls on day 4. Cells grown in the presence of 100mM galactose exhibited
up to 21% decreased G0 glycans with increased G1 or G2 glycans compared to
unsupplemented cultures on day 7. In addition, this supplementation increased
fucosylation by about 5%. Interestingly, galactose addition upregulated mgat2,
mgat5, and B4galt3–4 by Day 4 of the cultures. Supplementing cultures with 50μM
2FP decreased relative fucosylation by 48%. Moreover, 2FP supplementation signicantly decreased the expression of fut8 and nucleotide sugar transporter gene
expression (Slc35a3, Slc35a1, and Slc35c1). They observed that galactose and 2FP
had no negative effects on cell growth, metabolism, or titer. In addition, the uridinesupplemented cells demonstrated ∼23% increased viable cell density and ∼30%
decreased mAb titer.
Prabhu and coworkers investigated the effects of nickel and cobalt on glycosylation of a recombinant IgG produced in CHO cells [132]. Nickel concentrations
greater than 500 μM signicantly reduced galactosylation, while concentrations
lower than 500μM had no signicant impact on glycosylation. In addition, fucosylation was not impacted at any nickel concentration tested. 50μM cobalt slightly
6 Metabolic and Process Engineering to Control Glycan Structures…
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increased G1F glycans, whereas higher concentrations increased G0F glycans.
Interestingly, uridine and galactose supplementation into cultures containing either
Ni2+ or Co2+ may alleviate the effect of Ni2+ and Co2+ on galactosylation due to the
increased availability of UDP-Gal.
Pande and colleagues investigated the effect of a small molecule ionophore,
monensin, on high mannose glycans in different IgG-producing CHO cell lines
[133]. Noticeably, monensin addition increased HM glycans, particularly Man5.
Furthermore, no negative impacts on HM or cell culture parameters were observed
at monensin concentrations between 0.1 to 10 nM. However, 50 nM monensin
greatly increased HM glycans with negative effects on cell growth, viability, and
titer. Interestingly, when monensin was added as a 25 nM bolus into the culture,
total HM glycans on IgG increased without negative impacts on cell culture parameters. They next tested the effect of monensin on different production cell lines in a
10-day mock perfusion assay designed to mimic the conditions in a perfusion bioreactor. Three production cell lines producing mAbs with low levels of HM glycans
(less than 10%) and one production cell line that consistently produced mAbs with
high levels of HM glycans (more than 10%) were used. Monensin was added at
25nM to all cultures on day 3. After that, cultures were subjected to daily partial
medium exchanges with perfusion medium containing either 25nM monensin or
increasing monensin concentrations. They observed that monensin increased HM
glycans on all four antibodies in a dose-dependent manner though the magnitude of
the response was cell-line specic.
6.7 Perspectives andFuture Directions
CHO cells have been successfully used to express complex and efcacious biotherapeutics at signicant scales. One cannot overstate the progress in the eld
made possible by complex cellular engineering [134] and process engineering [1],
which has been vital to large-scale manufacturing. Indeed, the examples of the
highly complicated glycoproteins erythropoietin [135] and abatacept (Orencia®)
[136] produced and marketed as therapeutics are an indication of how CHO cells
can achieve reproducible CQAs without immunogenic glycans. While signicant
process development is required for successful production of a biosimilar, including
cell line screening, manipulation of process conditions, and possibly cell line glycoengineering, the regulatory approval of over 60 biosimilars in Europe and over 30in
the United States demonstrates that matching CQAs without the innovator cell line
or knowledge of proprietary bioprocess conditions is achievable. This success is a
testament to the plasticity of CHO cells and a growing understanding of the effects
of process conditions on glycan distributions.
Still, monoclonal antibodies, the dominant class of recombinant therapeutic proteins, have relatively straightforward glycosylation patterns with typically one glycosylation site on each heavy chain and biantennary, asialylated glycans [137]. As
more challenging structures such as heparin, bispecic antibodies, complicated