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a r t i c l e
i n f o
Article history:
Received 25 July 2010
Received in revised form 24 December 2010
Accepted 2 January 2011
Available online 12 January 2011
Keywords:
Bioreactor
Bioprocess engineering
Plant cell culture
Protein production
Recombinant protein
Therapeutic protein
a b s t r a c t
Molecular farming in plants with signicant advantages in cost and safety is touted as a promising platform
for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals
account for a signicant portion of the preclinical and clinical pipeline, plant cell suspension culture, which
integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more
compliant alternative factory. However, low protein productivity remains a major obstacle that limits
extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and
recent progress in plant cell culture technology and outlines viable strategies at both the biological and
process engineering levels for advancing the economic feasibility of plant cell-based protein production.
Approaches to overcome and solve the associated challenges of this culture system that include nonmammalian glycosylation and genetic instability will also be discussed.
2011 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plant cell cultures as alternative bioproduction platform for pharmaceutical
2.1.
Advantages over whole-plant system . . . . . . . . . . . . . . .
2.2.
Pharmaceutical proteins produced by plant cell cultures
. . . . .
2.2.1.
Antibodies . . . . . . . . . . . . . . . . . . . . . . .
2.2.2.
Vaccines . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3.
Cytokines, growth factors and growth hormones . . . . .
2.2.4.
Therapeutic enzymes and others . . . . . . . . . . . . .
2.2.5.
Other pharmaceutical proteins . . . . . . . . . . . . .
Strategies for achieving high-yield production of pharmaceutical proteins .
3.1.
Molecular approaches for increased expression . . . . . . . . . .
3.1.1.
Enhancing gene transcription . . . . . . . . . . . . . .
3.1.2.
Improving translation efciency . . . . . . . . . . . . .
3.1.3.
Minimizing post-translational protein degradation . . . .
3.1.4.
Stabilizing protein fusions and Hyp-Glyco technology . . .
3.2.
Cell culture approaches . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Higher order testing of culture medium . . . . . . . . .
3.2.2.
Cell immobilization . . . . . . . . . . . . . . . . . . .
3.2.3.
In situ protein removal . . . . . . . . . . . . . . . . .
3.3.
Bioreactor engineering approaches . . . . . . . . . . . . . . . .
3.3.1.
Optimized bioreactor design and operation . . . . . . .
3.3.2.
Advanced bioreactor culture strategies . . . . . . . . . .
3.3.3.
Adoption of disposable bioreactors . . . . . . . . . . .
3.4.
Downstream processing . . . . . . . . . . . . . . . . . . . . .
. . . .
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Corresponding author. Arkansas Biosciences Institute, Arkansas State University, P.O. Box 639, State University, AR 72467, USA. Tel.: + 1 870 680 4812; fax: + 1 870 972 2026.
E-mail address: jxu@astate.edu (J. Xu).
0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.01.002
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1. Introduction
Protein therapeutics have revolutionized modern medicine and
are currently in use for the treatment of many human diseases
including diabetes, anemia, hepatitis and cancer (Leader et al., 2008;
Woodnutt et al., 2008). To date, nearly 100 proteins with human
therapeutic applications have entered the market (Hacker et al., 2009)
and in excess of 370 are currently under development. Included
among this group of proteins are mainly vaccines, antibodies and
antibody derivatives in addition to several serum-derived proteins
including cytokines, growth hormones, interleukins, and interferon.
Worldwide protein drug sales currently estimated to be more than
$90 billion per year, are expected to reach $125 billion by the year
2015 (Decision Resources 2009 at http://www. decisionresources.
com/). Commercial production of such pharmaceutical proteins has
traditionally relied on bacterial fermentation or mammalian cellbased production, however, limitations including cost, scalability,
safety and protein authenticity with these expression systems have
prompted research into alternative platforms (Boehm, 2007; Streateld,
2007). Plant-based molecular farming has emerged as a promising
approach with signicant advantages in both cost and safety over
other eukaryotic expression systems (Basaran and Rodriguez-Cerezo,
2008; Doran, 2000; Fischer et al., 2004; Lau and Sun, 2009; Ma et al.,
2003; Sharma and Sharma, 2009; Streateld, 2007; Tremblay et al.,
2010). Fig. 1 compares several production characteristics of plant-based
production platforms with those of other systems when used for
pharmaceutical protein expression.
Molecular farming encompasses the use of either whole-plants or
in vitro cultured plant cell/tissues for the production of recombinant
proteins. While one of the seminal features of a whole-plant based
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292
292
293
294
294
294
Fig. 1. Comparison of key production parameters of the different expression systems used for recombinant therapeutic proteins. Note: the purication cost of the plant cell culture
system will become low if recombinant proteins are secreted into medium.
280
Fig. 2. Schematic depicting the plant cell suspension culture process for producing recombinant proteins. Production plant cell lines can be established from either the transgenic
whole-plant (upper-pathway) or by direct transformation of cultured plant cells with the target genes (lower-pathway). Scale-up production and downstream purication
methodologies adapted from other commercialized cell-based bioproduction systems can be used in delivering plant-made pharmaceuticals and proteins to the marketplace.
2010 that a second protein candidate produced through plant cell culture
system, PRX-105 that is a human acetylcholinesterase for treatment of
nerve-gas and pesticide poisoning, has been completed Phase I clinical
trials, and another two plant cell-produced enzymes, PRX-12 (galactosidase enzyme replacement therapy for Fabry disease) and pr-antiTNF
(biosimilar version of Enbrel) are in its preclinical pipeline (http://www.
protalix.com). Protalix's landmark success in commercializing plant cellmade human proteins was in fact preceded by Dow AgroSciences who
received the world's rst regulatory approval by US Department of
Agriculture (USDA) for tobacco cell-based vaccine against Newcastle
disease virus in 2006. The commercialization success of plant cell
bioproduction by these two companies undeniably sets the stage for
entry of other plant-made pharmaceuticals into the marketplace.
The molecular farming industry is still in its early development
(Sharma and Sharma, 2009). While plant biotechnology continues to
drive increasing numbers of whole-plant produced biopharmaceuticals into the preclinical and clinical development pipeline (Karg and
Kallio, 2009), the plant cell culture system is clearly emerging as a
more immediate alternative plant-based factory for producing
complex pharmaceutical proteins (Hellwig et al., 2004; Shih and
Doran, 2009). This review highlights the recent progress of this
promising bioproduction platform for pharmaceutical proteins and
outlines a range of strategies taken or underway to increase the
protein yields from plant cell cultures.
2. Plant cell cultures as alternative bioproduction platform for
pharmaceutical proteins
2.1. Advantages over whole-plant system
In comparison to whole-plant cultivation, plant cell culture
platforms have several notable advantages including: 1) rapid growth
281
Table 1
High-yield expression of pharmaceutical proteins in plant cell suspension cultures.
Therapeutic proteins
Host cells
Protein yields
Promoter
Localization
Reference
N. tabacum cv BY-2
N. tabacum cv BY-2
O. sativa L. cv. Donjin
Glycine max cv Williams82 (Soybean)
N. tabacum cv BY-2
N. tabacum cv NT-1
O. sativa (rice)
O. sativa cv Taipei 309
O. sativa (rice)
O. sativa (rice)
O. sativa (rice)
O. sativa (rice)
28 mg/L
35 mg/L
57 mg/l
22 mg/L
15 mg/L
30 mg/L
200 mg/L
85 mg/L
247 mg/L
110 mg/L
50 mg/L
76.5 mg/L
CaMV35S
CaMV35S
RAmy3D
(ocs)3mas
CaMV35S
CaMV35S
RAmy3D
RAmy3D
RAmy3D
RAmy3D
RAmy3D
RAmy3D
Secreted
Secreted
Secreted
Intracellular
~ 50% secreted
Secreted
Secreted
Secreted
Secreted
Secreted
Secreted
Secreted
Xu et al. (2007)
Xu et al. (2010)
Kim et al. (2008a)
Smith et al. (2002)
Yano et al. (2004)
Francisco et al. (1997)
Huang et al. (2001)
Terashima et al. (1999)
McDonald et al. (2005)
Trexler et al. (2005)
Trexler et al. (2002)
Park et al. (2010)
O. sativa (rice)
129 mg/L
RAmy3D
Secreted
3%4% TSP
77 mg/L
31 mg/L
27 mg/L
10.8 mg/L
~ 10 mg/L
10 mg/L
3.6% TSP
4.3% TSP
RAmy3D
RAmy3D
RAmy3D
CaMV35S
CaMV35S
CaMV35S
CaMV35S
SWPA2
SWPA2
Intracellular
Secreted
Secreted
Secreted
Secreted
Secreted
Secreted
Intracellular
Intracellular
282
production platform (James et al., 2000; Joo et al., 2006; Kwon et al.,
2003a, 2003c; Lee et al., 2004a; Shin et al., 2003). While very low
secreted protein yields of hGM-CSF (0.0070.07 mg/L) (Lee et al.,
2004b) were recovered when expressed in tobacco cells, high levels of
the protein (129 mg/L) were achieved using transgenic rice cell
expressed under an inducible rice amylase 3D (Ramy3D) promoter
(Shin et al., 2003). Conversely, functional human growth hormone
(hGH) has been expressed at high levels in both rice cells (57 mg/L)
(Kim et al., 2008a) and tobacco BY-2 cells (35 mg/L) (Xu et al., 2010).
Success of plant-based production may be protein specic however as
growth factors including human epidermal growth factor (hEGF)
(Parsons et al., 2010; Wirth et al., 2004) and insulin-like growth factor
(IGF-1) (Panahi et al., 2004, 2003) were produced at low protein
yields in plant cells.
2.2.4. Therapeutic enzymes and others
Several enzymes with therapeutic/diagnostic applications have
been produced in plant cell bioreactors. For example tobacco BY-2
cells have been shown to produce human tissue transglutaminase
(htTG), an enzyme used in the diagnosis of coeliac disease (Sorrentino
et al., 2005). Human lysozyme has been expressed in both rice cells
and rice grains for its potential utility as an antimicrobial food
supplement (Huang et al., 2002a, 2002b). In addition, several human
lysosomal enzymes have been produced in tobacco BY-2 cells
including human -iduronidase which has application as an enzyme
replacement therapy for treating mucopolysaccharidosis I (MPS I). Of
particular interest is the recombinant human glucocerebrosidase
enzyme (prGCD) that is being produced in carrot cells by Protalix
for commercial distribution in the treatment of Gaucher disease. The
carrot cell-produced prGCD naturally bears terminal mannose glycans
which eliminates the current requirement for post-production
chemical trimming of the sugars of the mammalian cell-expressed
recombinant GCD (sold under the trade name, Cerezyme). This new
prGCD is being developed for the human enzyme replacement
therapy market under the brand name of UPLYSO (Aviezer et al.,
2009a, 2009b; Shaaltiel et al., 2007). Preclinical and human clinical
trials of UPLYSO have shown the serum half-life of this plantexpressed GCD is signicantly longer than that of mammalian cellexpressed Cerezyme (http://www.protalix.com/). Other enzymes in
preclinical and clinical development using this plant cell platform
include -galactosidase for the treatment of Fabry disease; pr-antiTNF,
a biosimilar version of Enbrel and; acetylcholinesterase to treat nervegas and pesticide poisoning (Karg and Kallio, 2009).
2.2.5. Other pharmaceutical proteins
Other pharmaceutical proteins expressed in plant cell cultures
include dust mite allergens produced in tobacco BY-2 cell for use in
allergy diagnosis or immunotherapy (Lienard et al., 2007); a human
1-antitrypsin (AAT) produced in rice cell for treatment of hereditary
disorder in which a deciency of AAT leads to a chronic uninhibited
tissue breakdown (Terashima et al., 1999; Trexler et al., 2002, 2005); a
human lactoferrin produced in Siberian ginseng cell for use in advanced
nutritional products and in therapy for systemic infections; and a
thrombomodulin derivative produced in tobacco BY-2 cells for the
treatment of thrombotic disorders (Schinkel et al., 2005). As the
number of recombinant proteins shown to be successfully expressed
in plants increases, the production of pharmaceutical and diagnostic
proteins that leverage plant cell cultures as a commercially valid
platform is anticipated to gain broader acceptance and wider adoption.
3. Strategies for achieving high-yield production of
pharmaceutical proteins
While plant cell culture holds tremendous promise as an
alternative production platform for pharmaceutical proteins, several
bottlenecks limit the commercialization of this technology. Low
283
Fig. 3. Systematic strategies to achieve high-yield protein production with plant cell suspension cultures.
284
3.1.1.3. Other transcription modulating strategies. In addition to optimization of promoters, transcription activity can be further improved
by engineering enhancers, activators or repressors in locations up- or
downstream of the core promoter. Enhancers, which are DNA sequences
shown to increase gene expression when placed proximal to the
promoter, typically bind activator proteins and thereby facilitate the
binding of RNA polymerase II to the TATA box. For example, while the
CaMV35S promoter containing only 46 bp of 5-sequence (including
TATA box and the CCAAT consensus sequence) is sufcient for accurate
transcription initiation, sequences between 46 and 105 act as an
enhancer that signicantly increase the transcription level of the base
promoter (Lam et al., 1991). Enhancer sequences can also be stacked
to further augment gene transcription. In the case of the CaMV35S
promoter this enhancer sequence was duplicated (or double enhanced;
CaMV35SDE) and shown to increase the expression level of many
heterologous proteins in plants (Kay et al., 1987; Mishra et al., 2006;
Ruggiero et al., 2000; Sharm et al., 2008). Transcription has also been
shown to be enhanced by anking the transgene with sequence
encoding nuclear scaffold/matrix attachment regions (S/MARs) that are
important in mediating structural organization of eukaryotic chromatin (Halweg et al., 2005). In the context of plant transgene expression,
S/MARs have been shown to minimize silencing (Tang and Wang,
2006), increase plant cell transformation frequencies, and reduce
variance of transgene expression (Allen et al., 2000; He et al., 2008;
Petersen et al., 2002). In addition, synthetic super-promoters that
combine the most active sequences of several promoters is another
strategy for boosting gene expression (Sharma and Sharma, 2009). An
example of this approach resulted in a hybrid promoter containing
elements from both the CaMV35S promoter and the Agrobacterium Ti
plasmid mannopine synthetase promoter that increased GUS expression
by 3- to 5-fold over levels achieved with the double enhanced
CaMV35S promoter (Comai et al., 1990; Desai et al., 2010). Although
these strategies have been exploited most extensively in the wholeplant system in boosting heterologous protein expression, they are
generally applicable to plant cell-based systems.
285
286
Fig. 4. Schematics of Hyp-Glyco technology. Expressing therapeutic proteins as fusion with a HypRP tag comprised of tandem Ser-Pro repeats. A typical Hyp-arabinogalactan
polysaccharide (Hyp-glycan) consists of galactose, arabinose, rhamnose and glucuronic acid. The Hyp-glycan structure is from Xu et al. (2007).
287
288
Table 2
Enhanced recombinant protein production in plant cell cultures with process engineering approaches.
Culture mode
Expressed protein
Host
plant cell
Reference
Batch
culture
1-antitrypsin
(rAAT)
N. benthamiana
Huang et al.
(2009)
Fed-batch
culture
Human cytotoxic
Tlymphocyte antigen
4-immunoglobulin
(hCTLA4Ig)
Fed-batch
3 L stirred-tank bioreactor. 10 B5 medium with 300 g/L
Green uorescence
culture
of sucrose fed based on culture GFP uorescence
protein (GFP)
Perfusion
5 L (2 L working volume) stirred tank with 4-bladed
Human granulocyteculture
hollowed-paddle impeller. A 50 m stainless steel
macrophage colony
meshes as internal cell separator. Perfusion rates at
stimulating factor
-1
0.5, 1.0 and 2.0 day .
(hGM-CSF)
Perfusion
3.3 L (working volume) stirred tank with a 6-bladed
Acid phosphatase
culture
Rushton turbine on top and a 3-bladed upward pumping (APase)
marine axial impeller on the bottom. A cylindrical bafe
as internal cell separator. Perfusion rate up to 0.4 day 1.
Semi-continuous 2 L (working volume) stirred tank with a single pitched
1-antitrypsin
culture
blade impeller. Medium exchange daily at dilution rates of (rAAT)
1
0.05, 0.1, 0.15, 0.175, and 0.2 day .
1-antitrypsin
Semi-continuous 5 L (4.5 L working volume) stirred tank with a single
culture
pitched blade impeller. Six complete medium exchanges (rAAT)
(three inductions and three additions of fresh growth
medium) for each run lasting ~ 30 days.
Continuous
10 L (9.6 L working volume) stirred tank with two
Carrot acidic
culture
six bladed stirrer turbines. Dilution rate at 0.25 day 1
invertase
1
for 22 day, then 0.2 day
afterwards.
N. tabacum L. cv
Xanthi
N. tabacum L. cv
Havana SR
Anchusa
ofcinalis
Park et al.
(2010)
Liu et al.
(2001)
Lee et al.
(2004b)
Su and
Arias
(2003)
Huang et al.
(2010)
N. tabacum
BY-2
Des Molles
et al. (1999)
N. benthamiana
Trexler et al.
(2005)
Table 3
Comparison of the characteristics among plants cells, microbes and mammalian cells in bioprocess development.
Characteristics
Plant cells
Mammalian cells
Bacteria
Shape
Size
Cell wall
Doubling time
Cell aggregation or clumping
Shear sensitivity
Oxygen uptake rate (OUR)
KL required in bioreactor operation
Protein yields
Protein localization
Scale-up capacity
Spherical/cylindrical
20200 m
Yes
20100 h
Aggregated to form cell clusters over 2 mm
High
510 mmol/L h
1050/h
0.01200 mg/L
Intracellular/secreted
Medium
Spherical
1050 m
No
2048 h
Not aggregated
Extremely high
0.055 mmol/L h
0.2510/h
110 g/L
Secreted
Medium
Spherical
b1 m
Yes
Less than 2 h
Not aggregated
Low
1090 mmol/L h
1001000/h
b1 g/L
Usually intracellular
High
289
290
bioreactors for plant cell culture and particularly for the production
of foreign proteins is surprisingly limited. However one example that
highlights the integration of this type of advanced culture technology
is the commercial production of the human recombinant glucocerebrosidase in carrot cells grown in exible plastic bags (www.protalix.
com). Inspired by the historical success of Protalix, adoption of
disposable bioreactors for plant cell culture at large-scale is
anticipated to increase due to the clear advantages of process
simplicity, safety and production exibility.
The various types of disposable bioreactors currently used for plant
cell culture include wave-mixed, orbitally shaken or stirred reactors
and have been recently reviewed (Eibl et al., 2010). Schematics and
applications of the major types of disposable reactors that have been
used for plant cell culture are listed in Table 4. The wave bioreactor
represents the most successfully developed type of disposable
bioreactor to date. Introduced about 10 years ago (Singh, 1999),
units with working volumes up to 300 L (Eibl and Eibl, 2008) have
been commercialized by Wave Biotech LLC, now part of GE Healthcare.
In the wave bioreactor operation, cells are grown in an inated bag
placed on a rocking platform that induces wave action of the liquid
medium (Singh, 1999). Although originally developed for mammalian
cell culture, the wave bioreactor has successfully been used for
culturing a range of plant suspension cell types sourced from tobacco,
grape, apple, yew and barley at culture volumes ranging from 0.4
to 10 L and with biomass productivity approaching 40 g(FW)/L/day
(Eibl and Eibl, 2008; Ritala et al., 2008). However, the volumetric
oxygen transfer efciency (kLa) of the wave bioreactor is typically low
(b4 h 1) (Singh, 1999), resulting in possible oxygen limitation with
high cell-density cultures.
In contrast to the wave bioreactor that rocks the whole bag, the
wave and undertow (WU) bioreactor moves only one section of the
bag up and down to form waves in the bag. This has resulted in
improved kLa values approaching 10 h 1 in 30 L WU bioreactor
cultures of tobacco BY-2 cells (Terrier et al., 2007). The slug bubble
(SB) bioreactor is made up of a vertical plastic cylinder lled up to
80% of its height with the cultured cell suspension (Ducos et al., 2010).
Single slug bubbles are generated at the bottom and rise up to the top
of the cylinder thereby providing agitation and aeration. Much higher
kLa values approaching 17 h 1 for a 50 L SB bioreactor culture of BY-2
cells have been obtained in this system (Terrier et al., 2007). The
plastic-lined bioreactor was rst reported by Hsiao et al. (1999) and
later patented for growing Hyoscyamus muticus cells (Curtis, 2004).
This type of bioreactor is constructed by inserting a sterilized plastic
liner into a vessel attached with a head plate. Aeration and mixing are
provided by an aeration tube passing through the head plate down to
the bottom of the cell culture. Similar to the plastic-lined bioreactor,
the stirred bag bioreactor consists of a plastic bag in a steel support
container equipped with aeration devices and rotating or bumbling
impellers. The suitability of a 50 L stirred bag bioreactor for growing
tobacco cells was demonstrated by Eibl et al. (2009). Finally, the
Osmotek bag bioreactor is made up of a simple culture bag without
any exterior supporting vessel. This system was developed originally
by Osmotek LTD (Rehovot, Israel) to grow tissues and more recently
has been modied by Protalix to accommodate large-scale plant cell
cultures used in producing therapeutic proteins (e.g. glucocerebrosidase) by employing an external open wire cage around the bag
(Shaaltiel et al., 2007; Weathers et al., 2010). This system in fact will
be the bioreactor used in producing the rst plant-made, human
therapeutic to be FDA-approved and successfully commercialized
(Desai et al., 2010).
3.4. Downstream processing
Although high productivity is critical in moving plant cell culture
technology towards commercialization, the efcient recovery and
purication of target recombinant proteins from either cultured cells
291
Table 4
Disposable bioreactors used for plant cell cultures.
Reactor type
Cultured cells
Products
Working volume
Yields/productivity
Reference
Wave bioreactor
Vitis vinifera
N. tabacum BY-2
Hordeum vulgare L.
Biomass
Biomass
Humancollagen I
alpha1
1L
10 L
4L
40 g(FW)/(L day)
32 g(FW)/(L day)
5.1 g/L (day 25)
N. tabacum BY-2
Biomass
10 L
20 L
30 L
100 L
13.6 g(DW)/L
12.8 g(DW)/L
12.6 g(DW)/L
13.0 g(DW)/L
N. tabacum BY-2
Biomass
10 L
20 L
50 L
70 L
17.2 g(DW)/L
13.7 g(DW)/L
14.2 g(DW)/L
12.9 g(DW)/L
Plastic-lined bioreactor
G
A
S
Hyoscyamus muticus
Biomass
28.5 L
100 L
N. tabacum BY-2
Biomass
25 L
Carrot cells
Glucocerebrosidase
N/A
N/A
Slug Bubble
AG
Osmotekbag bioreactor
G
Aair inlet; Gexhaust gas; Ssampling; DWdry weight; FWfresh weight; N/Anot available.
Fig. 5. A standardized procedure of protein separation and purication from plant cells
or cell culture medium.
292
Table 5
Examples of downstream processing studies with plant cell culture system.
Cell culture system
Expressed protein
Localization of protein
Separation/purication procedure
Purity achieved
Reference
N. tabacum BY-2
Secreted
Xu et al. (2007)
Secreted
N95%
N95%
Carrot cell
Glucocerebrosidase
Intracellular retention
N99%
Intracellular retention
N80%
Intracellular retention
N99%
O. sativa L. cv Tainung 67
Human interferon 2
(hIFN 2)
Human 1-antitrypsin
(rAAT)
Human serum albumin
N95%
Secreted
IEXion exchange chromatography; ACafnity chromatography; HIC hydrophobic interaction chromatography; SECsize-exclusion chromatography.
293
294
295
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