Current Pharmaceutical Biotechnology, 2010, 11, 233-240

233

Cell Engineering and Cultivation of Chinese Hamster Ovary (CHO) Cells

Takeshi Omasa*, Masayoshi Onitsuka, and Wook-Dong Kim
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 5650871, Japan
Abstract: Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to
their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary
(CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for
therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The
CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost
of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development.
Recent developments in omics technologies have been essential for the development of rational methods of constructing a
cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of
therapeutic proteins using a CHO cell expression system.

Keywords: Chinese hamster ovary (CHO) cell, cell line development, cultivation process, post-translational process, bacterial
artificial chromosome (BAC) library, glycosylation control, omics technology, mammalian cell.
INTRODUCTION
In vitro animal cell cultivation was first performed by
R.G. Harrison about one hundred years ago. Harrison,
known as the father of tissue culture, cultivated tissues
from frog embryos into frog lymph clots and grew nerve
fibers from the tissue [1]. Despite the shorter history of animal cell cultivation than that of microbial cultivation, it is
now a promising, indispensable and fundamental technology
in biotechnology and bioindustry. In 1986, human tissue
plasminogen activator (tPA) became the first approved
therapeutic protein produced in Chinese hamster ovary
(CHO) cells [2]. Nowadays, more than 40 products produced
by mammalian cell cultivation are launched in the market
and large-scale (more than 20,000 L) cultivation is performed all over the world. According to Walshs report, 165
biopharmaceutical products (recombinant proteins, monoclonal antibodies and nucleic-acid-based drugs) gained approval in 2006 for the EU and USA markets [3]. We classified the host cells of 107 recombinant protein pharmaceuticals in accordance with this report and other data. Forty-two
percent of products are produced by Escherichia coli, 21%
are produced by Saccharomyces cerevisiae and 29% are produced by CHO cells. Taken together, more than 90% of
products are produced by these three host cells. Among the
therapeutic antibodies launched in the EU, USA and Japanese markets. CHO cells produce about 50% of these.
At present, CHO cells are the most important industrial
mammalian cell line, similarly to Escherichia coli or Sac*Address correspondence to this author at the Department of Biotechnology,
Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita,
Osaka 565-0871, Japan; Tel: +81-6-6879-7437; Fax: +81-6-6879-7439;
E-mail: omasa@bio.eng.osaka-u.ac.jp

1389-2010/10 $55.00+.00

charomyces cerevisiae cells, among various mammalian cell

lines. The reasons for the wide usage of CHO cells are considered to be as follows: (1) The de facto standard of good
manufacturing practice (GMP)-certified production and
safety issues for industrial applications are clearly defined.
(2) The industrial serum-free media are developed and serum-free adaptation is relatively easy. (3) The construction
of highly productive cell lines is well known and more than
10 g/L production of antibody has been reported. (4) Highdensity large-scale fed-batch cultivations are developed and
scale-up technology is well established.
1. CURRENT STRATEGY FOR CONSTRUCTION OF
CULTIVATION PROCESS
Fig. (1) shows a typical upstream process flow of industrial therapeutic antibody production by CHO cell cultivation. The CHO-based technologies are now well established
and are general platforms of therapeutic antibody production.
As a first step, a master cell bank (MCB) is established after
the construction of a productive cell line. Serum-containing
media have been used for past industrial production processes, but recent regulation requires the use of serum-free
media in industrial processes. The serum-free adaptation is a
common process before the construction of an MCB. Various types of serum-free medium are commercially available
now, but the development of serum-free media is a continuous challenge in cell culture engineering. Shibuya et al. developed serum substitutes for fed-batch cultivation, which
consist of 12 components [4]. Nonanimal serum substitutes
have been actively developed recently. Sericin, which is a
protein derived from silkworms, promotes cell growth and
antibody production and can be autoclaved [5]. Phosphatidic
acid is also a promising constituent of low-protein serumfree media [6].
2010 Bentham Science Publishers Ltd.

234 Current Pharmaceutical Biotechnology, 2010, Vol. 11, No. 3

After establishment of an MCB, a working cell bank

(WCB) is constructed from the MCB. Each expansion culture and hence an inoculation culture starts with WCB in a
small flask, and culture volume is increased by 5-8 times
until large production-scale cultivation (Fig. 1). The development of scale-up technology is an important key factor for
successful industrial production. The oxygen supply and
dissolved carbon dioxide stripping are the most important
factors. Matsunaga et al. evaluated the volumetric dissolved
carbon dioxide transfer coefficient kLaCO2 of an 80-10,000 L
reactor and constructed a model medium similar to cell culture medium [7,8]. Takuma et al. reported the relationship
between glucose limitation and CO2 partial pressure in antibody production using a recombinant CHO cell line [9].
Other important factors are pH, temperature and static pressure. Oguchi et al. reported that a temperature shift improved
mRNA stability and prolonged the cell culture time [10].
Fujiwara et al. suggested that static pressure affected intracellular pH and enhanced the recombinant protein production
of recombinant CHO cells [11]. Generation time (doubling
time or population doubling level (PDL)) is an important
parameter for evaluation of culture expansion. Each cell culture expansion is managed on the basis of the PDL from the
MCB. Recently, more than 10 g/L titer has been reported in
the production of recombinant antibodies using CHO cells.
This high titer is achieved by selection and using both the
highly productive cell lines (for example, more than 20 pcd

Omasa et al.

(pg cell-1 day-1) specific production rate is reported) and the

long-term fed-batch operation to increase the integral value
of viable cell concentration (IVC).
It has been mentioned in many reports that the production
cost of mammalian cell cultivation is high and that this expense is a disadvantage of the mammalian cell system. Hood
et al. compared the cost of production of therapeutic proteins
in various production systems [12]. According to the report,
the production cost of CHO cells is 300$/g-product, which is
3,000 times higher than that of transgenic plants. They assumed that the cost of processing and purification in transgenic plants is the same as that in mammalian cell culture.
However, CHO cells can secrete recombinant proteins into a
medium and more than 15% of supernatant proteins is the
target product even though the supernatant protein concentration is as low as 10-20 mg/L. It is likely that the purification cost of CHO cell cultivation is inexpensive compared
with that of other production systems including recombinant
plant culture. The accurate and realistic cell culture cost of
the industrial production of a therapeutic antibody by recombinant CHO cells was estimated to be only 2$/g-product in
the case of a 5g/L expression level and a 10,000L production
[13].
The advantage of using the mammalian cells for recombinant production system is that they could secrete the properly folded and glycosylated form of a protein. The disad-

Fig. (1). Typical upstream process of mammalian cell culture. Mammalian cell culture is scaled up from a working cell bank (WCB) to
production scale.

Cell Engineering and Cultivation of Chinese Hamster Ovary (CHO) Cells

vantage of the mammalian cell production system is not the

high cost; the most significant disadvantage is the low
growth rate. The typical specific growth rate is from 0.03 to
0.05h-1 (doubling time, 14-23h), which is less than one-20th
of the microbial growth rate. The slow specific growth rate
causes the long time required for cultivation, selection and
construction of a cell line. The other disadvantage is the low
cell concentration. The typical cell concentration is from 1 to
5
106cells mL-1. The dry cell weight of mammalian cells is
about 3
10-10g cell-1. Consequently, the typical cell concentration based on the dry cell weight is from 0.3 to 1.5 g-drycell L-1 in mammalian cell cultivation. In contrast, the typical
microbial cell concentration is about 10 g-dry-cell L-1. The
lower cell concentration in mammalian cell culture causes
the volumetric production rate to be lower than that in the
microbial cell culture. To overcome this disadvantage, various types of perfusion culture system have been developed.
Hollow-fiber cell cultivation system could attain the highest
cell concentration (more than 5
107 cells mL-1) [14]; however, this cell concentration is still one-20th of the cell concentration in mammalian tissue.
2. CELL LINE DEVELOPMENT AND RECENT
OMICS TECHNOLOGY
The development of a manufacturing process for a recombinant protein in mammalian cells usually follows a
well-established scheme [2]. From transfection of an expression vector into a host CHO cell line to the establishment of
a manufacturing process, it takes more than one year [15].
The most time-consuming and critical steps are cell line
screening, subcloning and gene amplification. The transfected vector is normally randomly integrated into the CHO
genome by homologous recombination. The cells containing
transgene stably can survive under selective conditions. The
surviving CHO cells have a wide variety of productivity.
Among these heterogeneous cell pools, stable and highly
productive CHO cell lines should be established. In CHO
cells, the two most commonly used selection systems are
based on the amplifiable selection markers: dihydrofolate
reductase (DHFR) and glutamine synthesis (GS) [16]. Gene
amplification, which normally results in an increased productivity, is achieved by exposing surviving cells to increasing concentrations of an inhibitor of the selection protein;
methotrexate (MTX) and methionine sulphoximide (MSX)
are used for DHFR and GS, respectively [17]. Surviving
CHO cells mostly produce a high level of the selection protein by the increased copy number in the CHO genome. Interestingly, the copy numbers of the target genes are also
increased at the same integration site of the chromosome.
The surviving heterogeneous cell pools should be cloned
according to specific protein productivity and cell growth
rate. This step is routinely performed by cell cloning using
limiting dilution or cloning machines. Candidate cell clones
should be checked to ensure a constant level of protein production during the expansion culture because productivity
often tends to decrease during long-term cultivation.
Why is such instability or heterogeneity of constructed
CHO cell lines observed during the construction process?
Siminovitch commented that CHO cells are not strictly diploid in the functional sense, and that many genes are present
in the hemizygous state [18]. According to Siminovitch, this

Current Pharmaceutical Biotechnology, 2010, Vol. 11, No. 3

235

condition was termed functional hemizygosity. This means

that a mutant CHO cell line could be easily constructed by
comparison with other cell lines. Holliday concluded that
such functional hemizygosity is due to differences in de novo
methylation in host cells [19]. Recently, the detailed genetic
characteristic of the CHO DG44 cell line has been investigated using karyotype analysis [20]. The CHO DG44 cell
line is a DHFR-deficient strain constructed by Urlaub and
Chasin [21,22] and is a major CHO host cell line for commercial production. Despite its importance, the detailed
karyotype was firstly analyzed using G-banding in 2006
[20]. CHO DG44 cells have 20 chromosomes and only seven
are normal Chinese hamster chromosomes. G-banding is a
particularly useful procedure of initial screening for the
chromosomal aberrations, because the entire genome can be
evaluated in a single experiment. However, G-banding is not
directly linked to the genomic sequence. Recently, fluorescence in situ hybridization (FISH) imaging using bacterial
artificial chromosome (BAC) clones as hybridization probes
(BAC-FISH) has been developed for the construction of a
physical map of a genome. A physical map is a representation of the positions of genes on each chromosome expressed
in base pairs, with the genes placed in a known order and at
known distances from other genes. We constructed the CHO
genomic BAC library and analyzed CHO DG44 chromosomes by the BAC-FISH method [23]. As determined by
BAC-FISH analysis, only one pair of chromosomes is maintained in CHO DG44 cell lines and the other 18 chromosomes are markedly rearranged.
Genomic information is indispensable for the development of a productive cell line. However, no public genome
project of Chinese hamster has been proposed until now because of its gigantic genome size. A BAC library, which
represents the entire genome of an organism without cloning
artifacts or rearrangements, can reduce the complexity of the
genome and provide clones physically separated in an addressable format. BAC libraries also provide scaffolding
information for mapping sequence contigs to localized genomic regions using a direct genomic shotgun sequencing
approach. The total size of the inserts of our CHO genomic
BAC library was estimated to be approximately 15.1 Gb,
which was estimated to cover five times the CHO genome
size [23]. It is expected that the genome-wide analysis of
CHO will be performed by next-generation sequencing of
this CHO BAC library. Despite the slow progress of CHO
genome analysis, studies using omics approaches including cDNA analysis using an expressed sequencing tags
(ESTs) database and proteomic analysis by two-dimensional
electrophoresis (2-DE) are actively performed to understand
the development of a cell line and cell culture processes.
More than 68,000 ESTs were analyzed and 28,000 unique
CHO transcripts were identified by the University of Minnesota and A-star institute groups [24,25]. They investigated
the cDNA expression in antibody-producing CHO cell lines
under various cell culture conditions [26,27]. For instance,
the gene expression in mouse hybridoma and recombinant
CHO cell lines under butyrate treatment conditions were
analyzed and both cell lines showed a similar response to
butyrate treatment [28]. Doolan et al. investigated the transcriptional profiling of gene expression in a high-level secreting CHO cell line by their customized CHO oligo WyeHamster2a microarray [29]. Their microarray contains 3,714

236 Current Pharmaceutical Biotechnology, 2010, Vol. 11, No. 3

transcripts including 2,835 derived from CHO sequences.

They investigated the paired amino acid cleaving enzyme
(PACE)-expressing CHO cell line using their microarray and
observed 1,076 significantly different genes in the highly
productive cell line. Recently, Kuroda et al. have constructed
a cDNA microarray on the basis of 12,281 CHO-K1-based
unique sequences from 45,312 EST sequences and analyzed
gene expression using suspension fed-batch culture [30].
Proteomics approaches are also effective for process development. 2-DE combined with mass spectrometry is a
powerful tool for the analysis of protein expression. The 2DE protein map of CHO DUKX cell line, which is a dhfrdeficient cell line, was firstly reported by Champion et al.
[31]. More than 1000 silver-stained protein spots were obtained and 25 major spots were identified. Subsequently,
Hayduk et al. reported a detailed 2-DE map of CHO K1 using fluorescence staining [32]. They reported 224 protein
identifications, as determined from 274 2-DE gel spots, and
phosphoproteome results using phosphorylation-specific
stains.
The combination of a transcriptome and a proteome gives
a better understanding of a cell culture process. Meleady et
al. also analyzed PACE-expressing CHO cells by 2-DE and
reported that more than 60 proteins showed differential expression [33]. A GFP-expressing CHO cell line was analyzed
by transcriptome and proteome profiling [26]. Microarray
analysis revealed 77 differentially expressed genes, with 53
genes up-regulated and 24 genes down-regulated. Proteomic
analysis yielded 75 and 80 proteins for the mid-exponential
and stationary phases, respectively. The integrated approach
could show the key factors related to the general mechanism
underlying high productivity in CHO cells.
The modification of metabolic networks of biochemical
reactions is defined as metabolic engineering [34]. Metabolic
engineering is concerned with bioreaction networks, pathway
synthesis issues, pathway flux and flux control. Some investigations have resulted in considerable cell culture process
improvement towards a specific goal [35]. Zupke and
Stephanopoulos first proposed the concept of metabolic flux
analysis in mammalian cell culture [36]. Our group also reported that simplified metabolic flux analysis is effective to
improve the energy metabolism and antibody production
[37]. To elucidate such complex data, the systems biology
approach is indispensable. The systems biology approach is
methodology for understanding biological systems including
genes, proteins, metabolism, cells, tissues and organs. The
key point of the systems biology approach is to separate each
component that composes complex biological systems and
investigate the relationships among these components similarly to unit operation analysis in chemical engineering. Zhao
and Kurata proposed a new concept, the maximum entropy
principle, for estimating elementary mode coefficient in
metabolic pathway analysis on the basis of systems engineering [38]. They applied this method to E. coli, S. cerevisiae
and CHO cells to increase prediction accuracy.
3. CELL ENGINEERING FOR IMPROVEMENT OF
SECRETION, FOLDING AND GLYCOSYLATION
Mammalian cell hosts can correctly fold, assemble and
glycosylate polypeptides [39]. These significant advantages

Omasa et al.

of mammalian cell expression systems are due to the development of post-translational steps in mammalian cells. Glycosylation is the most extensive of all the post translational
modifications and functionally important in biological activities in vivo and in vitro. Glycosylation occurs by sequential
intracellular enzyme reactions in a particular subcellular organelle and essentially introduces heterogeneity into a glycoprotein., as illustrated in Fig. (2). In general, the oligosaccharide heterogeneity of glycoproteins from recombinant
CHO cells has been shown to vary with culture conditions
[40,41]. Culture conditions such as nutrient content, pH,
temperature, and the concentrations of dissolved oxygen or
ammonia may have a significant impact on the distribution
of glycan structures. Cell specific growth rate also affects the
glycan structures. The control of environmental conditions is
an important issue from the viewpoint of quality control.
A more constructive approach is to manipulate CHO
cells genetically. The most successful example is alpha-1,6
fucosylation of glycoprotein. Alpha-1,6 fucosylation of glycoprotein is catalyzed by alpha-1,6 fucosyltransferase
(FUT8) (Fig. 2). Fucosylation is closely related to glycoprotein function, i.e., antibody-dependent cellular cytotoxicity
(ADCC) in antibody and antithrombin-heparin affinity in
antithrombin III [42,43]. To produce a non fucosylated
therapeutic protein, the construction of a FUT-8 knockout
CHO cell line or down-regulation of the FUT8 enzyme is
effective. Yamane-Ohnuki et al. established the FUT8deficient host CHO cell line and produced a non-fucosylated
antibody using this host CHO cell line [44]. An alternative
approach to defucosylation is to decrease the supply of nucleotide sugars for the fucosylation enzyme in the Golgi apparatus. GDP-fucose is the substrate of FUT8 in the Golgi
apparatus (Fig. 2). In mammalian cells, GDP-fucose is synthesized through two distinct pathways, i.e., the de novo and
salvage pathways. The fucosylation substrate GDP-fucose is
primarily synthesized from GDP-mannose via the de novo
pathway, because the media for mammalian cell culture generally contain no fucose. Among the key enzymes for oligosaccharide defucosylation are GDP-mannose 4,6-dehydratase
(GMD), GDP-4-keto-6-deoxymannose-3,5-epimerase-4reductase (GMER), GDP-fucose transporter (GFT), and
FUT8 (Fig. 2). Kanda et al. reported that the introduction of
GMD and FUT8 siRNA expression plasmids into CHO cells
was effective in inhibiting fucosylation of recombinant IgG
[45]. Another methodology is decreasing the fucose transport
from cytosol to Golgi. We focused on the GDP-fucose transport through the Golgi membrane for the defucosylation of
recombinant AT-III in CHO cells. GFT is down-regulated by
siRNA expression and the fucosylation could be inhibited by
decreasing Golgi-GDP-fucose transport [43].
Other important post-translational steps are folding, assembly and secretion. Fig. (3) shows the post-translational
steps in mammalian cells. An effective approach to improving productivity is to improve the transcription step. Approaches to increasing mRNA levels leads in CHO cells include 1) using a strong promoter and a stable element for
mRNA, 2) transcriptional activation employing enhancer
sequences and 3) increasing the gene copy number by gene
amplification. Recently, cell engineering studies focusing on
the secretory pathway of CHO cells have been carried out.
Mammalian cells have a strict quality control system to pro-

Cell Engineering and Cultivation of Chinese Hamster Ovary (CHO) Cells

Current Pharmaceutical Biotechnology, 2010, Vol. 11, No. 3

237

Fig. (2). Biosynthesis of GDP-fucose and protein fucosylation in mammalian cells. Cytosol GDP-mannose is converted to GDP-fucose by
GDP-mannose dehydratase (GMD) and GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (GMER). Synthesized GDP-fucose is
transported through the Golgi membrane by GDP-fucose transporter (GFT). In the Golgi apparatus, fucose is finally transferred to N-linkedtype complex glycoprotein by
-1,6-fycosyltransferase (FUT8). Approaches to defucosylation are classified into two methods; the control of
GDP-fucose supply and the inhibition of fucosyltransferase reaction.

Fig. (3). Cell engineering for the improvement of product quality and cellular productivity. Improvement of transcription and translation steps
is aimed at increasing mRNA lecel and regulating translation, respectively. Translation and post-transcriptional modification steps are also
important to improve the quality and cellular productivity by improvement of folding, assembly, secretion and glycosylation.

238 Current Pharmaceutical Biotechnology, 2010, Vol. 11, No. 3

Omasa et al.

Fig. (4). UPR signaling pathway in mammalian cells. UPR originates from PERK, ATF6 and IRE1 proteins, which detect the accumulation of
an unfolded protein in the endoplasmic reticulum (ER).

tect them against the stress of misfolded proteins in the endoplasmic reticulum (ER). Fig. (4) illustrates the signaling of
unfolded protein response (UPR) via protein kinase R-like
ER kinase (PERK) to aplha-subunit of eukaryotic translation
initiation factor 2 (eIF2alpha) and activating transcription
factor 4 (ATF4) in mammalian cells [39-46]. Some researchers have attempted to maximize the translational or secretory
capacity of a host cell by engineering the secretory pathway.
The ectopic expression of the spliced form of XBP-1 (X-box
binding protein 1) (XBP-1s), which is one of the key regulators in the mammalian UPR system, has been shown to enhance the production of recombinant proteins in CHO cells
[47,48]. The concentrations of SEAP, antibodies, interferongamma, and EPO increased by 2-5 times with XBP-1s expression during the construction of transient and/or stable
expression in CHO cells.
Overexpression of ATF4 also improves the production of
recombinant AT-III in CHO cells [49]. ATF4 encodes the
activating transcription factor 4 and its transcription is induced by stress signals including anoxia/hypoxia, endoplasmic reticulum stress, amino acid deprivation, and oxidative
stress [46-50]. ATF4 has been reported to enhance the expression of subsets of UPR target genes including GADD34
and CHOP (C/EBP-homologous protein) (Fig. 4). GADD34
was originally isolated as a UV-inducible transcript in CHO
cells [51]. ATF4 can induce the expression of GADD34 in

mouse embryonic fibroblast cells [52]. Mouse GADD34

forms a complex with the catalytic subunit of protein phophatase 1 (PP1c) that specifically promotes the dephosphorylation of eIF2alpha [53]. Dephosphorylation of eIF2alpha
induces translation attenuation in response to ER stress.
ATF4 overexpression could enhance the dephosphorylation
of eIF2alpha via GADD34 resulting in the induction of translation attenuation. GADD34 overexpression also enhances
the dephosphorylation of eIF2alpha, which leads to the increase in the specific production rate of AT-III without reducing the protein quality [54]. These ATF4-related expression factors are likely effective to increase productivity in an
established CHO cell line.
CONCLUSIONS
With the development of biological science including
omics and information technologies, the understanding and
application of mammalian cells themselves are markedly
improved. The intra- and extracellular reaction profiles can
be analyzed in detail now. The construction of a cell factory
by cell engineering could not be attained without these informations. However, it seems that the lack of systems engineering approaches integrating various omics technologies
may inhibit the development of effective cell engineering
technologies. Integrated approaches are likely to be an important issue in next-generation cell engineering.

Cell Engineering and Cultivation of Chinese Hamster Ovary (CHO) Cells

ACKNOWLEDGEMENTS
This work is partially supported by grants from NEDO of
Japan, the Program for the Promotion of Fundamental Studies in Health Sciences of the NIBIO, and a Grant-in-Aid for
Scientific Research from the JSPS.

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[22]

[23]

[24]

REFERENCES
[1]
[2]

[3]
[4]
[5]

[6]

[7]

[8]
[9]

[10]

[11]
[12]

[13]
[14]

[15]

[16]
[17]

[18]
[19]
[20]

[21]

Harrison, R. G. Observations on the living developing nerve fiber.

Proc. Soc. Exp. Biol. Med., 1907, 4, 140-143.
Wurm, F. M. Production of recombinant protein therapeutics in
cultivated mammalian cells. Nat. Biotechnol., 2004, 22(11), 13931398.
Walsh, G. Biopharmaceutical benchmarks 2006. Nat. Biotechnol.,
2006, 24(7), 769-776.
Shibuya, K.; Haga, R.; Namba, M. A serum substitute for fed-batch
culturing of hybridoma cells. Cytotechnology, 2008, 57(2), 187197.
Terada, S.; Nishimura, T.; Sasaki, M.; Yamada, H.; Miki, M.
Sericin, a protein derived from silkworms, accelerates the
proliferation of several mammalian cell lines including a
hybridoma. Cytotechnology, 2002, 40, 3-12.
Sakai, K.; Matsunaga, T.; Hayashi, C.; Yamaji, H.; Fukuda, H.
Effects of phosphatidic acid on recombinant protein production by
Chinese hamster ovary cells in serum-free culture. Biochem. Eng.
J., 2002, 10(2), 85-92.
Matsunaga, N.; Kano, K.; Maki, Y.; Dobashi, T. Culture scale-up
studies as seen from the viewpoint of oxygen supply and dissolved
carbon dioxide stripping. J. Biosci. Bioeng., 2009, 107(4), 412-418.
Matsunaga, N.; Kano, K.; Maki, Y.; Dobashi, T. Estimation of
dissolved carbon dioxide stripping in a large bioreactor using
model medium. J. Biosci. Bioeng., 2009, 107(4), 419-424.
Takuma, S.; Hirashima, C.; Piret, J. M. Dependence on glucose
limitation of the pCO2 influences on CHO cell growth, metabolism
and IgG production. Biotechnol. Bioeng., 2007, 97(6), 1479-1488.
Oguchi, S.; Saito, H.; Tsukahara, M.; Tsumura, H. pH Condition in
temperature shift cultivation enhances cell longevity and specific
hMab productivity in CHO culture. Cytotechnology, 2006, 52(3),
199-207.
Fujiwara, M.; Koizumi, S.; Takagi, M. Effect of static pressure on
intracellular pH of adhesive Chinese hamster ovary cells. J. Biosci.
Bioeng., 2007, 104(6), 510-512.
Hood, E. E.; Woodard, S. L.; Horn, M. E. Monoclonal antibody
manufacturing in transgenic plants--myths and realities. Curr.
Opin. Biotechnol., 2002, 13(6), 630-635.
Kelley, B. Very large scale monoclonal antibody purification: The
case for conventional unit operations. Biotechnol. Prog., 2007,
23(5), 995-1008.
Omasa, T.; Kobayashi, M.; Nishikawa, T.; Shioya, S.; Suga, K.;
Uemura, S.; Kitani, Y.; Imamura, Y. Enhancement of antibody
production by growth factor addition in perfusion and hollow-fiber
culture systems. Biotechnol. Bioeng., 1995, 48(6), 673-680.
Chartrain, M.; Chu, L. Development and production of commercial
therapeutic monoclonal antibodies in mammalian cell expression
systems: an overview of the current upstream technologies. Curr.
Pharm. Biotechnol., 2008, 9(6), 447-467.
Omasa, T. Gene amplification and its application in cell and tissue
engineering. J. Biosci. Bioeng., 2002, 94(6), 600-605.
Matasci, M.; Hacker, D. L.; Baldi, L.; Wurm, F. M. Recombinant
therapeutic protein production in cultivated mammalian cells:
current status and future prospects. Drug Discov. Today Technol.,
2009, 5(2-3), e37-e42.
Siminovitch, L. On the nature of hereditable variation in cultured
somatic cells. Cell, 1976, 7(1), 1-11.
Holliday, R. Mutations and epimutations in mammalian cells.
Mutat. Res., 1991, 250(1-2), 351-363.
Derouazi, M.; Martinet, D.; Besuchet Schmutz, N.; Flaction, R.;
Wicht, M.; Bertschinger, M.; Hacker, D. L.; Beckmann, J. S.;
Wurm, F. M. Genetic characterization of CHO production host
DG44 and derivative recombinant cell lines. Biochem. Biophys.
Res. Commun., 2006, 340(4), 1069-1077.
Urlaub, G.; Chasin, L. A. Isolation of Chinese hamster cell mutants
deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci.
USA, 1980, 77(7), 4216-4220.

[25]

[26]

[27]
[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]
[36]

[37]

[38]
[39]

[40]
[41]

[42]

239

Urlaub, G.; Kas, E.; Carothers, A. M.; Chasin, L. A. Deletion of the

diploid dihydrofolate reductase locus from cultured mammalian
cells. Cell, 1983, 33(2), 405-412.
Omasa, T.; Cao, Y.; Park, J. Y.; Takagi, Y.; Kimura, S.; Yano, H.;
Honda, K.; Asakawa, S.; Shimizu, N.; Ohtake, H. Bacterial
artificial chromosome library for genome-wide analysis of Chinese
hamster ovary cells. Biotechnol. Bioeng., 2009, 104(5), 986-994.
Wlaschin, K. F.; Nissom, P. M.; Gatti Mde, L.; Ong, P. F.; Arleen,
S.; Tan, K. S.; Rink, A.; Cham, B.; Wong, K.; Yap, M.; Hu, W. S.
EST sequencing for gene discovery in Chinese hamster ovary cells.
Biotechnol. Bioeng., 2005, 91(5), 592-606.
Kantardjieff, A.; Nissom, P. M.; Chuah, S. H.; Yusufi, F.; Jacob, N.
M.; Mulukutla, B. C.; Yap, M.; Hu, W. S. Developing genomic
platforms for Chinese hamster ovary cells. Biotechnol. Adv., 2009,
27, 1028-1035.
Nissom, P. M.; Sanny, A.; Kok, Y. J.; Hiang, Y. T.; Chuah, S. H.;
Shing, T. K.; Lee, Y. Y.; Wong, K. T.; Hu, W. S.; Sim, M. Y.;
Philp, R. Transcriptome and proteome profiling to understanding
the biology of high productivity CHO cells. Mol. Biotechnol., 2006,
34(2), 125-140.
Wlaschin, K. F.; Hu, W. S. A scaffold for the Chinese hamster
genome. Biotechnol. Bioeng., 2007, 98(2), 429-439.
De Leon Gatti, M.; Wlaschin, K. F.; Nissom, P. M.; Yap, M.; Hu,
W. S. Comparative transcriptional analysis of mouse hybridoma
and recombinant Chinese hamster ovary cells undergoing butyrate
treatment. J. Biosci. Bioeng., 2007, 103(1), 82-91.
Doolan, P.; Melville, M.; Gammell, P.; Sinacore, M.; Meleady, P.;
McCarthy, K.; Francullo, L.; Leonard, M.; Charlebois, T.; Clynes,
M. Transcriptional profiling of gene expression changes in a
PACE-transfected CHO DUKX cell line secreting high levels of
rhBMP-2. Mol. Biotechnol., 2008, 39(3), 187-199.
Kuroda, K.; Maseki, Y.; Nishida, R.; Yoneya, T.; Tsukahara, M. A
cDNA microarry for Chinese hamster ovary cell. In: The 21st
Anual and International meeting of the Japanese Association for
Animal Cell Technology, 2008.
Champion, K. M.; Arnott, D.; Henzel, W. J.; Hermes, S.; Weikert,
S.; Stults, J.; Vanderlaan, M.; Krummen, L. A two-dimensional
protein map of Chinese hamster ovary cells. Electrophoresis, 1999,
20, 994-1000.
Hayduk, E. J.; Choe, L. H.; Lee, K. H. A two-dimensional
electrophoresis map of Chinese hamster ovary cell proteins based
on fluorescence staining. Electrophoresis, 2004, 25(15), 25452556.
Meleady, P.; Henry, M.; Gammell, P.; Doolan, P.; Sinacore, M.;
Melville, M.; Francullo, L.; Leonard, M.; Charlebois, T.; Clynes,
M. Proteomic profiling of CHO cells with enhanced rhBMP-2
productivity following co-expression of PACEsol. Proteomics,
2008, 8(13), 2611-2624.
Tyo, K. E.; Alper, H. S.; Stephanopoulos, G. N. Expanding the
metabolic engineering toolbox: more options to engineer cells.
Trends Biotechnol., 2007, 25(3), 132-137.
Schugerl, K. Progress in monitoring, modeling and control of
bioprocesses during the last 20 years. J. Biotechnol., 2001, 85(2),
149-173.
Zupke, C.; Stephanopoulos, G. Intracellular flux analysis in
hybridomas using mass balances and in vitro(13)C NMR.
Biotechnol. Bioeng., 1995, 45(4), 292-303.
Omasa, T.; Furuichi, K.; Iemura, T.; Katakura, Y.; Kishimoto, M.;
Suga, K. I. Enhanced antibody production following intermediate
addition based on flux analysis in mammalian cell continuous
culture. Bioprocess Biosyst. Eng., 2010, 33(1),117-125.
Zhao, Q.; Kurata, H. Maximum entropy decomposition of flux
distribution at steady state to elementary modes. J. Biosci. Bioeng.,
2009, 107(1), 84-89.
Dinnis, D. M.; James, D. C. Engineering mammalian cell factories
for improved recombinant monoclonal antibody production:
lessons from nature? Biotechnol. Bioeng., 2005, 91(2), 180-189.
Jenkins, N.; Parekh, R. B.; James, D. C. Getting the glycosylation
right: implications for the biotechnology industry. Nat. Biotechnol.,
1996, 14(8), 975-981.
Butler, M. Optimisation of the cellular metabolism of glycosylation
for recombinant proteins produced by Mammalian cell systems.
Cytotechnology, 2006, 50(1-3), 57-76.
Mori, K.; Iida, S.; Yamane-Ohnuki, N.; Kanda, Y.; Kuni-Kamochi,
R.; Nakano, R.; Imai-Nishiya, H.; Okazaki, A.; Shinkawa, T.;
Natsume, A.; Niwa, R.; Shitara, K.; Satoh, M. Non-fucosylated

240 Current Pharmaceutical Biotechnology, 2010, Vol. 11, No. 3

[43]

[44]

[45]

[46]

[47]
[48]

therapeutic antibodies: the next generation of therapeutic

antibodies. Cytotechnology, 2007, 55(2-3), 109-114.
Omasa, T.; Tanaka, R.; Doi, T.; Ando, M.; Kitamoto, Y.; Honda,
K.; Kishimoto, M.; Ohtake, H. Decrease in antithrombin III
fucosylation by expressing GDP-fucose transporter siRNA in
Chinese hamster ovary cells. J. Biosci. Bioeng., 2008, 106(2), 168173.
Yamane-Ohnuki, N.; Kinoshita, S.; Inoue-Urakubo, M.; Kusunoki,
M.; Iida, S.; Nakano, R.; Wakitani, M.; Niwa, R.; Sakurada, M.;
Uchida, K.; Shitara, K.; Satoh, M. Establishment of FUT8
knockout Chinese hamster ovary cells: an ideal host cell line for
producing completely defucosylated antibodies with enhanced
antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng.,
2004, 87(5), 614-622.
Kanda, Y.; Imai-Nishiya, H.; Kuni-Kamochi, R.; Mori, K.; Inoue,
M.; Kitajima-Miyama, K.; Okazaki, A.; Iida, S.; Shitara, K.; Satoh,
M. Establishment of a GDP-mannose 4,6-dehydratase(GMD)
knockout host cell line: a new strategy for generating completely
non-fucosylated recombinant therapeutics. J. Biotechnol., 2007,
130(3), 300-310.
Ron, D.; Walter, P. Signal integration in the endoplasmic reticulum
unfolded protein response. Nat. Rev. Mol. Cell Biol., 2007, 8(7),
519-529.
Tigges, M.; Fussenegger, M. Xbp1-based engineering of secretory
capacity enhances the productivity of Chinese hamster ovary cells.
Metab. Eng., 2006, 8(3), 264-272.
Ku, S. C.; Ng, D. T.; Yap, M. G.; Chao, S. H. Effects of
overexpression of X-box binding protein 1 on recombinant protein

Received: October 10, 2009

Accepted: October 28, 2009

Omasa et al.

[49]

[50]
[51]

[52]

[53]

[54]

production in Chinese hamster ovary and NS0 myeloma cells.

Biotechnol. Bioeng., 2008, 99(1), 155-164.
Ohya, T.; Hayashi, T.; Kiyama, E.; Nishii, H.; Miki, H.;
Kobayashi, K.; Honda, K.; Omasa, T.; Ohtake, H. Improved
production of recombinant human antithrombin III in Chinese
hamster ovary cells by ATF4 overexpression. Biotechnol. Bioeng.,
2008, 100(2), 317-324.
Ameri, K.; Harris, A. L. Activating transcription factor 4. Int. J.
Biochem. Cell Biol., 2008, 40(1), 14-21.
Zhan, Q.; Lord, K. A.; Alamo, I., Jr.; Hollander, M. C.; Carrier, F.;
Ron, D.; Kohn, K. W.; Hoffman, B.; Liebermann, D. A.; Fornace,
A. J., Jr. The gadd and MyD genes define a novel set of
mammalian genes encoding acidic proteins that synergistically
suppress cell growth. Mol. Cell. Biol., 1994, 14(4), 2361-2371.
Ma, Y.; Hendershot, L. M. Delineation of a negative feedback
regulatory loop that controls protein translation during endoplasmic
reticulum stress. J. Biol. Chem., 2003, 278(37), 34864-34873.
Novoa, I.; Zeng, H.; Harding, H. P.; Ron, D. Feedback inhibition of
the unfolded protein response by GADD34-mediated
dephosphorylation of eIF2alpha. J. Cell Biol., 2001, 153(5), 10111022.
Omasa, T.; Takami, T.; Ohya, T.; Kiyama, E.; Hayashi, T.; Nishii,
H.; Miki, H.; Kobayashi, K.; Honda, K.; Ohtake, H.
Overexpression of GADD34 enhances production of recombinant
human antithrombin III in Chinese hamster ovary cells. J. Biosci.
Bioeng., 2008, 106(6), 568-573.