Beruflich Dokumente
Kultur Dokumente
233
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
Omasa et al.
Fig. (1). Typical upstream process of mammalian cell culture. Mammalian cell culture is scaled up from a working cell bank (WCB) to
production scale.
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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-
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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.
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
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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