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Biotechnology Advances 29 (2011) 278299

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Towards high-yield production of pharmaceutical proteins with plant cell


suspension cultures
Jianfeng Xu a,b,, Xumeng Ge a, Maureen C. Dolan a
a
b

Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401, USA


College of Agriculture and Technology, Arkansas State University, Jonesboro, AR 72401, USA

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

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

4.

Other challenges to overcome . . .


4.1.
Non-mammalian glycosylation
4.2.
Genetic instability . . . . . .
5.
Perspective and conclusion . . . . .
Acknowledgements . . . . . . . . . . .
References . . . . . . . . . . . . . . .

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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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platform is providing a readily scalable system, this platform does


have several drawbacks including long development times for stable
transgenics, instability of product yield and quality, issues with
possible fertilizer contamination, impact of pests, diseases and
weather conditions on the production factory, and most importantly
challenges in applying good manufacturing practice (GMP) to the
whole-plant production pipeline (Hellwig et al., 2004; Karg and Kallio,
2009; Shih and Doran, 2009). Plant cell suspension culture integrates
many of the merits of the whole-plant system with those of microbial
and animal cell cultures (Hellwig et al., 2004), and holds tremendous
promise as a factory for valuable pharmaceutical proteins (Fig. 2).
Like the microbial cells, undifferentiated plant calli can be separated
and propagated under a controlled, liquid media environment to
generate stable cell suspension cultures. This culture technology
developed in the 1950s, has been primarily used for the production of
valuable secondary metabolites such as paclitaxel, shikonin, artemisinin, digoxin, ginsenosides and ajmalicine (Georgiev et al., 2009; Rao
and Ravishankar, 2002; Smetanska, 2008; Weathers et al., 2010; Xu et
al., 1998a). Commercial production of paclitaxel in Taxus cell
suspension cultures developed by Phyton Biotech (http://www.
phytonbiotech.com/) has provided Bristol-Myers Squibb with a
secure, sustainable and environmentally-friendly source of paclitaxel
for Taxol since 1995 (Roberts and Shuler, 1997). With considerable
advances in plant molecular biology over the subsequent 15 years, as
well as an ever increasing demand for protein therapeutics that meet
regulatory and safety standards, plant cell culture has emerged as a
viable alternative production platform for pharmaceutical proteins.
The benets of plant cell culture in achieving human-like protein
production have long been recognized. Like bacteria, plant cells have
relatively rapid doubling times and can be grown in simple synthetic

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.

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J. Xu et al. / Biotechnology Advances 29 (2011) 278299

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.

media using conventional bioreactors. However like mammalian cells,


plants are eukaryotes and can perform nearly all post-translational
modications that occur in human cells. As such, plant cell culture is
considered comparable to conventional mammalian cell culture including
Chinese hamster ovary (CHO) cells, which are routinely used for the
production of complex protein therapeutics. However, the most salient
advantage the plant cell system has over its mammalian counterpart is
plant cells do not harbor any known human pathogens. This issue has
received signicant consideration and review after Genzyme experienced
interruption (June, 2009) in their CHO cell production of Cerezyme, the
commercial Gaucher disease therapeutic, due to infection of their
production cell line with a calicivirus (Rybicki, 2010). This serious safety
concern for human patients and an immediate need to ensure a
sustainable supply chain for this orphan drug highlights the importance
and urgency for continued efforts to improve the performance of plant cell
culture bioproduction system for therapeutic proteins in the future.
Of signicant relevance to this review, the world's largest drug
company, Pzer, recently acquired licensing rights from Protalix
Biotherapeutics (Israel) for their carrot cell-based production system
of glucocerebrosidase, an orphan drug for Gaucher disease (Aviezer
et al., 2009a, 2009b; Shaaltiel et al., 2007). This followed with Protalix
receiving a Phase III Fast Track Designation for this drug in August,
2009 from the U.S. Food and Drug Administration (FDA). The Pzer
licensing transaction, which exceeded $100 million, highlights the
commitment of big pharmaceutical companies and their condence in
the commercial viability of plant cell-based production for pharmaceutical proteins. This product was approved for use in the treatment
of Gaucher disease in France by the French regulatory authority in
July, 2010, thus becoming the rst plant-made human pharmaceutical
to enter the commercial sector. Approval by FDA for use of this drug in
the USA is expected to come in early 2011. Further illustrating the
sustainability of this production platform, Protalix announced in June,

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

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

with cell doubling time as short as one day; 2) improved consistency


of protein product with the use of controlled bioreactors which are
less prone to biotic- and abiotic-induced variations that commonly
hamper eld/greenhouse grown plant-based protein production;
3) fewer regulatory and environmental compliance hurdles for ensuring product quality and safety and limited to no GMO (Genetically
Modied Organism) release issues and; 4) simple procedure for
secreted protein isolation/purication from inexpensive, well-dened
medium that lacks exogenous proteins which complicate the
purication process (Lee et al., 2000). Notably, plant suspension cell
cultures are composed of dedifferentiated cells that lack fully
functional plasmodesmata, and hence systemic post-transcriptional
gene silencing (PTGS) may be reduced since PTGS is generally
transmitted via plasmodesmata and the vascular system (Crawford
and Zambryski, 1999; Doran, 2000; Su and Lee, 2007). More detailed
discussions of the advantages of plant cell-based production platform
can be found in several recent reviews (Hellwig et al., 2004; Huang
and McDonald, 2009; Shih and Doran, 2009).
2.2. Pharmaceutical proteins produced by plant cell cultures
Following expression of the rst complex recombinant protein
(human serum albumin) in tobacco cell culture (Sijmons et al., 1990),
a diverse array of pharmaceutical proteins including antibodies,
vaccines, hormones, growth factors and cytokines have been
produced in plant cells with the majority having demonstrated
bioactivity (Kwon et al., 2003b; Lienard et al., 2007; Magnuson et al.,
1998; Matsumoto et al., 1993; Smith et al., 2002). Tobacco BY-2
(Bright yellow-2) and NT-1 (Nicotiana tabacum-1) cells are the most
frequently chosen host lines used for recombinant protein expression.
These closely related cell lines are fast growing (specic growth
rate up to 0.044 h 1), robust, and readily undergo Agrobacteriummediated transformation and cell cycle synchronization (Hellwig
et al., 2004; Ozawa and Takaiwa, 2010; Su and Lee, 2007; Tremblay
et al., 2010). Other plant hosts used for establishing cell cultures have
included rice (Huang et al., 2005; Torres et al., 1999), soybean (Smith
et al., 2002), alfalfa (Daniell and Edwards, 1995) and tomato (Kwon
et al., 2003a). Plant cell lines derived from common edible crop
species, may in fact be more favorable than tobacco cells in terms of
by-product levels and regulatory compliance (Hellwig et al., 2004). An

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extensive list of recombinant pharmaceutical proteins expressed


using plant cell-based platforms has been recently reviewed by Huang
and McDonald (2009). Table 1 summarizes the pharmaceutical
proteins produced at high yields (N10 mg/L or N1% TSP) in plant cell
suspension cultures to date.
2.2.1. Antibodies
Antibodies have emerged as the dominant class of recombinant
proteins in the pharmaceutical industry (De Muynck et al., 2010).
Since expression of the rst full-size monoclonal antibody (mAb) in
tobacco over 20 years ago (Hiatt et al., 1989), plant-produced
antibodies (Plantibodies) have received considerable interest (Ko
et al., 2009; Russell and Fuller, 2000; Stoger et al., 2002a; Tavladoraki
et al., 1993). Assembly of a number of functional immunoglobulin G
(IgG, primarily IgG1) and immunoglobulin A (IgA) antibodies has
been demonstrated in Nicotiana, Arabidopsis and other plants (De
Jaeger et al., 2000; De Wilde et al., 1998; Lai et al., 2010; Larrick et al.,
2001; Ma et al., 2003; Ramirez et al., 2003; Tremblay et al., 2010). For
several of the complex molecular forms of immunoglobulins including
secretory IgA (sIgA) and IgG4, plants in fact offer the only
commercially viable system for large-scale production to date (De
Muynck et al., 2010; Ma et al., 2003; Shaaltiel et al., 2009; Stoger et al.,
2002a).
There is an increasing interest in plant cell-based production
platforms for functional mAbs due to the rising concern about
potential safety issues. Examples include the Guy's 13 IgG1 (Fischer
et al., 1999b; Sharp and Doran, 2001a, 2001b), a human mAb against
hepatitis B virus surface antigen (HBsAg) (Yano et al., 2004), a human
anti-rabies virus mAb (Girard et al., 2006), and most recently a human
anti-HIV mAb (Holland et al., 2010) all of which have been reported to
be expressed in tobacco cell suspension cultures. Challenges do
remain where completely assembled IgG antibodies while actively
secreted into the culture media and fully functional, were shown to
be susceptible to rapid proteolytic degradation (Sharp and Doran,
2001a). With the exception of full-size mAbs, plants or plant cells
have also been quite successful at expressing several antibody
derivatives of therapeutic value, including Fab fragments, singlechain Fvs (scFv), bispecic Fvs, diabodies, minibodies, and single
variable domains (Almquist et al., 2006; De Wilde et al., 1998; Ma
et al., 2003; Ramirez et al., 2002, 2000, 2001; Shaaltiel et al., 2009;

Table 1
High-yield expression of pharmaceutical proteins in plant cell suspension cultures.
Therapeutic proteins

Host cells

Protein yields

Promoter

Localization

Reference

Human interferon 2b (hIFN 2)


Human growth hormone (hGH)

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

Shin et al. (2003)

O. sativa cv Taipei 309


O. sativa (rice)
O. sativa (rice)
N. tabacum cvNT-1
N. tabacum cvNT-1
N. tabacum cv. BY-2.
N. tabacum cv BY-2
Acanthopanax senticosus
N. tabacum cv BY-2

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

Huang et al. (2002b)


Huang et al. (2005)
Shin et al. (2010b)
Becerra-Arteaga et al. (2006)
Wongsamuth and Doran (1997)
Holland et al. (2010)
Fu et al. (2009)
Jo et al. (2006)
Choi et al. (2003)

Hepatitis B surface antigen (HBsAg)


mAb against HBsAg
Bryodin 1
Human 1-antitrypsin (rAAT)

Human cytotoxic T lymphocyte antigen


4-immunoglobulin (hCTLA4Ig)
Human granulocyte-macrophage colony
stimulating factor (hGM-CSF)
Human lysozyme
Human serum albumin (HSA)
Human interlukin-12 (IL-12)
Human alkaline phosphatase
Mouse IgG anti Streptococcus surface antigen
Human anti-HIV antibody 2G12
Human -iduronidase
Human lactoferrin
*TSP: total soluble proteins.

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J. Xu et al. / Biotechnology Advances 29 (2011) 278299

Tavladoraki et al., 1993). For example, an anti-phytochrome scFv


(Firek et al., 1994), a scFv against human spleen ferritin (Semenyuk
et al., 2003) and a mouse Fab against surface protein antigen of
Streptococcus mutants serotype c (Yano et al., 2006) have been
reported to express in tobacco cells with full function.
2.2.2. Vaccines
Plants have emerged as an attractive production system for
subunit vaccines which unlike traditional whole pathogen-based
vaccines, present only select immunogenic epitopes of the pathogen
for eliciting production of antibodies in the host organism (Chichester
et al., 2009; Daniell et al., 2009; Franconi et al., 2010; Rybicki, 2010;
Tiwari et al., 2009; Walter, 2008; Wu et al., 2009; Yu and Langridge,
2001). One of the unique features of plant-produced vaccines is
the concept that plants not only serve as the production system
but also as the delivery vehicle for oral vaccines (Tiwari et al., 2009;
Wang and Coppel, 2008). A complete list of vaccine antigens
expressed in plants can be seen in several recent reviews (Rybicki,
2010).
Plant cells offer most of the advantages of whole-plant system for
the expression of vaccines, along with controlled, sterile, reproducible
production that is aligned with GMP manufacturing requirements
(Tiwari et al., 2009). An example of a commercial plant cell platform is
the Concert Plant-Cell-Produced system developed by Dow AgroSciences for delivering vaccines to poultry and livestock (http://www.
dowagro.com/animalhealth/). Multiple antigens have been expressed
using this platform including heat-labile toxin from E. coli, hemagglutinin neuraminidase (HN), Newcastle disease virus (NDV), hemagglutinin (HA) from avian inuenza and VP2 from infectious bursal
disease (IBD). These plant-based vaccines have shown effective
immune responses in poultry and swine and provide a high-level
protection for animals challenged with the various disease pathogens
(Mihaliak and Webb, 2005). Plant-based platforms have garnered
particular interest for the production of the HBsAg antigen (Guan
et al., 2010). In addition to whole-plants, expression of the HBsAg has
been reported in both soybean and tobacco cell suspension cultures
(Ganapathi et al., 2007; Kumar et al., 2005, 2007; Sojikul et al., 2003).
Another example of a plant cell-produced vaccine targets the intimin
gene product of E. coli O157:H7 (Judge et al., 2004). Mice immunized
intraperitoneally with the recombinant intimin or fed with the
transgenic plant cells expressing this protein could generate an
intimin-specic mucosal immune response and exhibited a reduced
duration of E. coli O157:H7 fecal shedding following challenge (Judge
et al., 2004).
2.2.3. Cytokines, growth factors and growth hormones
Cytokines play a major role in orchestrating virtually every aspect
of the immune system, and thus have emerged as an interesting class
of promising immunotherapy agents (Cannon, 2000). As one of the
most important family of cytokines, bioactive interleukins of human
and animal origin have been produced in multiple plant species
including tobacco, tomato, rice and potato plant systems (GutierrezOrtega et al., 2004, 2005; Ma et al., 2005; Medrano et al., 2009, 2010;
Park and Cheong, 2002; Wang et al., 2008). In the context of plant cellbased expression, tobacco cells have been used to express bioactive
human interleukin-2 and -4 (hIL-2 and -4) (Magnuson et al., 1998),
human interleukin-18 (hIL-18) (Sharma et al., 2006), and human
interleukin-12 (hIL-12) (Kwon et al., 2003b). Of particular signicance to this review is the recent report of improved expression of
hIL-12 using a rice cell culture-based expression platform (Shin et al.,
2010b). Rice cells appear to exhibit lower associated proteolytic
activity on hIL-12 than all other plant-based platforms tested for
expression of this cytokine to date.
In addition to the interleukins, human granulocyte macrophage
colony stimulating factor (hGM-CSF) is another important cytokine
for which plants and plant cells have received much attention as a

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

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

protein yields remain problematic; typically ranging from 0.01 to


200 mg/L (Huang and McDonald, 2009; James and Lee, 2001; Su and
Lee, 2007). Recent advances in plant molecular biology have greatly
improved protein yields well beyond the threshold of 10 mg/L that is
generally regarded as the requisite expression range for entry into
commercial process development (Hellwig et al., 2004). However
the need for additional research and development is necessary to
further boost the production levels in the order of 5- to 10-fold to
attain the desired prot margin for plant-based recombinant protein
expression. To reach this goal, a systematic strategy is required to
maximize the efciency of all stages of the production pipeline from
gene expression to cell culture, process development, and nally to
downstream protein purication (Ponstein et al., 1996).
In the past two decades, numerous approaches have been
exploited to enhance the production of heterologous protein in
plant cell cultures. Categorized based upon production stages, these
approaches can be classied into four groups that include molecular,
cell culture, process engineering (bioreactor culture) and downstream processing approaches (Fig. 3). It is necessary that these
approaches are properly integrated into the production pipeline to
achieve the desired commercial success of plant cell-based bioproduction of recombinant proteins. A similar approach has been
systematically adopted by the mammalian cell culture industry over
the past 20 years, leading to its high productivity today.

283

3.1. Molecular approaches for increased expression


The most critical strategies in enhancing the transgene expression
levels in plant cell culture systems incorporate molecular methodologies. These approaches target mainly the two genetic information
transfer processes dened by the central dogma: transcription and
translation (Desai et al., 2010; Sharma and Sharma, 2009; Streateld,
2007). Great strides have been made over the past two decades to
improve heterologous protein expression in plant cells through
enhanced gene transcription, proper mRNA processing, improved
translation efciency, and incorporation of novel protein fusion
technology (Desai et al., 2010; Ponstein et al., 1996; Streateld,
2007). In addition, improved post-translational protein stability can
play a pivotal rule in achieving high protein yields (Doran, 2006a).
3.1.1. Enhancing gene transcription
Increasing the transcription rate of stably transformed gene
sequences is the most direct and efcient approach for boosting
protein expression. This is best achieved by using a strong constitutive
or inducible promoter and understanding the promoter elements and
factors that can be used to modulate transgene transcription. In
general the goal is to generate target mRNA yields approaching 10
20% of the total mRNA (Hellwig et al., 2004). In the context of plant
cell culture, the characteristics of various promoters used for

Fig. 3. Systematic strategies to achieve high-yield protein production with plant cell suspension cultures.

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J. Xu et al. / Biotechnology Advances 29 (2011) 278299

expressing foreign genes in this system has been summarized by


Huang and McDonald, 2009. The major plant expression promoters
are also discussed extensively in recent reviews (Desai et al., 2010;
Sharma and Sharma, 2009), though the focus is placed on the wholeplant expression system.
3.1.1.1. Constitutive promoters. Constitutive promoters directly and
continually drive the expression of the transgene in all plant tissues
and are generally not dependent of developmental stage of the
production host. The best-known and most widely used constitutive
promoter in plant cell culture is derived from the Cauliower Mosaic
Virus (CaMV35S) and is theoretically capable of continuously driving
recombinant protein expression until the plant cells reach a
stationary phase in growth (Huang and McDonald, 2009). The
CaMV35S promoter sequence has been duplicated and/or modied
to further improve the gene transcription efciency up to 10-fold
(Fischer et al., 1999b; He et al., 2008; Kay et al., 1987). Alternative
constitutive promoters used in plant cell culture include the ubiquitin
promoter and the (ocs)3mas promoter (Hellwig et al., 2004). The
ubiquitin promoter, isolated from a variety of plants including maize,
Arabidopsis, potato, sunower, tobacco and rice (Sharma and Sharma,
2009), has been used to express scFv antibody and insulin-like
growth factor-1 (IGF-1) in rice cells (Panahi et al., 2004; Torres et al.,
1999). The hybrid (ocs)3mas promoter, constructed from octopine
synthase (ocs) and mannopine synthase (mas) promoter sequences,
was used for the expression of HBsAg in soybean cell culture, which
resulted in high levels of secreted protein on the order of 22 mg/L
(Smith et al., 2002). However constitutive expression is regarded as
a growth-associated process and thus may present signicant
metabolic burden on the plant cell during critical stages of cell
growth and thus a reduced specic growth rate. In addition, the
constant accumulation of the foreign product may create a toxic
environment that negatively impacts host cell physiology and
metabolism (Huang and McDonald, 2009). Facilitating extracellular
secretion of the foreign protein is often an effective strategy in
alleviating this response.
3.1.1.2. Inducible promoters. Regulated or inducible promoters have
been increasingly used in recent years, particularly those which allow
external regulation by chemical stimuli such as alcohol, steroid, salts
and sucrose, or environmental factors such as temperature, light,
oxidative stress and wounding (Boetti et al., 1999; Corrado and
Karali, 2009; Cramer et al., 1996; Huang and McDonald, 2009; Lee
et al., 2006; Padidam, 2003; Tang et al., 2004). Inducible expression is
advantageous as this allows protein production to be separated
from cell growth, thus enabling independent optimization of both
processes. Such regulated expression is especially benecial when the
synthesized proteins are lethal to host cells. In a highly efcient
inducible expression system, the inducer/promoter pair should meet
the following criteria: high specicity, rapid response of promoter
upon induction, rapid switching off promoter upon inducer withdrawal, non-toxic to plant cells and easy scale-up (Sharma and
Sharma, 2009).
The most successful example of an inducible promoter used in
plant cell-based protein expression utilized the rice -amylase 3D
(RAmy3D) promoter which is induced by sucrose starvation. This
promoter has been used for achieving high-level expression in rice
cell suspension culture of many therapeutic proteins including rAAT,
hGM-CSF, hG-CMS, hGH, bryodin-1, lysozyme and human serum
albumin (see Table 1). Virtually every recombinant protein expressed
in this rice suspension cell system has produced signicantly higher
secreted protein levels than attained with any other plant cell
expression system tested. At 247 mg/L, the rAAT protein is the
highest secreted protein recovery reported from plant cell cultures to
date (McDonald et al., 2005); this yield in fact approaches the
production levels delivered by the mammalian cell culture. While

these examples highlight the promise of cell culture systems for


pharmaceutical protein production, the growth rates and characteristics of the rice cell lines are considered inferior to tobacco BY-2 and
NT-1 cell lines (Hellwig et al., 2004), and their viability is negatively
impacted when cultivated in a sucrose-starvation medium (Huang
and McDonald, 2009). Furthermore, requisite medium exchange to
create a sucrose-starvation environment in the cultures presents
signicant technical difculties when considering large-scale production (Park et al., 2010; Su and Lee, 2007).
The oxidative stress-inducible peroxidase (SWPA2) promoter has
also been used for high-yield expression of human lactoferrin (up to
4.3% TSP) in both tobacco BY-2 and Siberian ginseng cell cultures (Choi
et al., 2003; Jo et al., 2006). The SWPA2 promoter has demonstrated
30-fold higher activity than the constitutive CaMV35S promoter in
driving foreign protein expression in plant cells (Lee et al., 2006).
Other chemically inducible promoters used in plant cell cultures
include an estradiol-inducible chimeric XVE transcription activator
(Zuo et al., 2000) and a dexamethasone-inducible pOp/LhG4 transcription activator (Samalova et al., 2005). In addition, plant viralbased expression system that is more efcient for the iterative
replication of viral vector in host cells (Matsuo et al., 2007) have been
developed for inducible expression of foreign genes. Examples include
rAAT expression using the estradiol-inducible cucumber mosaic virus
viral amplicon (CMViva) in tobacco (N. benthamiana) cells (Huang
et al., 2010), GFP expression with a estradiol-inducible tomato Mosaic
Virus (ToMV) amplicon in tobacco BY-2 cells (Dohi et al., 2006) and
Norwalk virus capsid protein expression with an ethanol-inducible
Bean Yellow Dwarf Virus (BeYDV) amplicon in tobacco NT-1 cells
(Zhang and Mason, 2006).

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.

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

3.1.2. Improving translation efciency


The translational efciency of a transgene is largely determined by
the proper mRNA processing (capping, splicing and polyadenylation)
and stability. The 5- and 3-untranslated region (UTR) of the plant
expression cassettes play critical roles in properly processing mRNA and
maintaining mRNA stability (Cowen et al., 2007). While the 5-UTR or
leader sequence is very important for 5-capping and facilitating
translational initiation, the 3-UTR generally is important for transcript
polyadenylation which strongly inuence the stability of mRNA (Abler
and Green, 1996; Chan and Yu, 1998). These untranslated sequences can
be manipulated for the optimization of foreign protein expression
(Sharma and Sharma, 2009). Classic examples include the translational
elements from tobacco etch virus (TEV), tobacco mosaic virus (TMV)
and alfalfa mosaic virus (AMV). These 5-leader sequences used in plant
expression systems enhance transgene expression by several folds by
enabling more efcient translation of the downstream transgene (Datla
et al., 1993; Gallie et al., 1995; Hood et al., 1997; Kang et al., 2004; Wang
et al., 2001). Recently, a cow pea mosaic virus (CPMV) translational
element was developed for extremely high-level and rapid transient
protein expression in plants (up to 25%30% TSP) (Sainsbury et al.,
2008; Sainsbury and Lomonossoff, 2008). Use of this translational
enhancer in N. benthamiana cell culture resulted in an increase in GUS
protein production similar to that obtained with the TEV 5'-UTR
(presented at 2010 Society In vitro Biology meeting; W. Curtis et al.). In
addition to leveraging 5-leader sequence, utilization of a heterologous
3-UTR derived from plants or plant viruses generally stabilizes the
transcript and in turn improves overall protein expression levels
(Cowen et al., 2007; Hood et al., 1997; Staub et al., 2000). In addition,
it has been demonstrated that some AU-rich sequences destabilize
mRNA and removal of this repetitive run from 3-UTR is requisite in
avoiding rapid degradation of the mRNA (De Rocher et al., 1998;
Schillberg et al., 2003).
Another potentially useful approach for improving translational
efciency involves optimization of recombinant gene codon usage to
better match that used by the host production (Gustafsson et al.,
2004; Hershberg and Petrov, 2008). The usage frequency of the
genetic code in plants in fact differs not only from that of animals and
microorganism (Desai et al., 2010), but also between monocots and
dicots (Kawabe and Miyashita, 2003; Liu and Xue, 2005; Murray et al.,
1989). Therefore, using preferred codons and/or removing rare
codons that favor the host plant cell has been shown to be an efcient
means of improving recombinant protein yields (Jabeen et al., 2010).
Among the more striking examples of this codon optimization
strategy was reported for human acetylcholinesterase expression in
tobacco plants which resulted in a 5- to 10-fold increase in
recombinant protein accumulation as compared to expression using
the native human sequence (Geyer et al., 2007).
3.1.3. Minimizing post-translational protein degradation
Evidence of post-translational protein degradation by proteases
within plant cells has been reported in many studies (Callis, 1995;
Doran, 2006a; Schiermeyer et al., 2005; Sharp and Doran, 2001a,
2001b; Stevens et al., 2000; Yang et al., 2005). Loss of synthesized
protein as a result of proteolytic degradation along the secretory
pathway is an important but often overlooked contributing factor to
low protein yields (Doran, 2006a; Shih and Doran, 2009). However,
proteolytic degradation may be effectively minimized by targeting the
foreign proteins to sub-cellular compartments such as the endoplasmic reticulum (ER). Since proteolysis is more likely to occur in the
apoplast and cytosol than the ER, retaining the expressed protein in
ER with the use of an ER retrieval signal (e.g. KDEL, HDEL) has been
commonly used to improve foreign protein stability (Desai et al.,
2010; Doran, 2006a; Sharma and Sharma, 2009). Furthermore, the ER
contains many molecular chaperones that facilitate nascent proteins
folding and assembly (Nuttall et al., 2002) and thus has often been
regarded as an ideal compartment for accumulating many classes of

285

foreign proteins. With ER retrieval, functional protein yields have


been reported to improve by 10- to 100-fold as compared to
recombinant proteins allowed to process through the secretory
pathway uninterrupted (Doran, 2006a; Faye et al., 2005; Hellwig
et al., 2004; Torres et al., 1999; Yasuda et al., 2006). One of the more
dramatic examples was shown for KDEL-targeted human EGF in
tobacco cells where a 104-fold increase in protein yields was
attributed directly to ER retention of the recombinant protein
(Wirth et al., 2004). However, this strategy is not always appropriate,
as is the case with some therapeutic proteins that require signicant
post-translational processing in the Golgi for functionality (Gomord
and Faye, 2004; Kaufman, 1998; Shaaltiel et al., 2007).
Many other strategies have been used for reducing the effects of
proteolytic degradation in plant cells including: co-expression of
recombinant protein with protease inhibitors, co-expression with
protein co-factor and subunit, knockout mutations in the genes
encoding specic proteolytic enzymes, and removal of proteasespecic sites from foreign proteins using genetic engineering
techniques. Reports utilizing co-expression of protease inhibitors
have shown no signicant inhibitory effects on plant growth and
development (Van der Vyver et al., 2003; Xu et al., 2004). Coexpression of recombinant serine proteinase inhibitor II (sPI-II) has
proven successful in increasing hGM-CSF yields in a rice cell
suspensions by 2-fold (Kim et al., 2007; Kim et al., 2008b). Likewise,
co-expression of protease inhibitor (Bowman-Birk Serprotease inhibitor) in transgenic tobacco root cultures resulted in increased
accumulation of a recombinant monoclonal antibody (Komarnytsky
et al., 2006). Protein degradation may be circumvented with some
foreign heteromeric proteins when cofactors and/or subunits are
allowed to be co-expressed (Desai et al., 2010; Doran, 2006a). In the
case of the ricin A-chain when expressed in tobacco cells without its
partner B-chain was easily degraded, while co-expression of both A
and B chains resulted in the formation of disulde-linked heterodimers that were secreted and recovered from the media fraction
(Frigerio et al., 1998). Similarly, co-expression of antibody with its
antigen was reported to increase the antibody yields possibly because
the antibody was stabilized by antigen binding (Stoger et al., 2002b).
While strategies leveraging knockout mutations in the genes encoding select proteinases and removal of protease-specic sites from
target proteins may be effective at reducing post-translational protein
degradation and improving protein stability in plant cell cultures
(Doran, 2006a), few studies in planta have been reported corroborating these approaches.
3.1.4. Stabilizing protein fusions and Hyp-Glyco technology
Expression of a heterologous protein as a peptide- or proteinbased fusion can be used to stabilize the structure of the target protein
in achieving higher protein yields. Utilizing a strategy that incorporates as the fusion partner that is a highly-expressed and stable plant
protein is one such approach (Streateld, 2007). A C-terminal fusion
of a single plant ubiquitin coding unit to -glucuronidase (GUS)
increased recombinant GUS protein levels by 10-fold (Garbarino et al.,
1995). Recently, a new and attractive fusion protein approach, known
as Hydroxyproline-Glycosylation (Hyp-Glyco) technology has been
shown to dramatically increase secreted protein yields from plant cell
culture systems (Kieliszewski and Xu, 2006; Kieliszewski et al., 2006).
This technology involves expressing heterologous proteins as a fusion
with a novel, Hyp-glycomodule tag comprised of Hyp-rich peptides
(HypRP) such as tandem repeats of Ser-Hyp or Ala-Hyp (Fig. 4). These
HypRP tags direct extensive Hyp-O-glycosylation in plant cells
resulting in arabinogalactan polysaccharides populating this repetitive peptide fusion (Kieliszewski, 2001; Shpak et al., 1999; Tan et al.,
2003) and signicantly facilitating the secretion of the expressed
protein from cultured plant cells. Heterologous proteins including
enhanced green uorescence protein (EGFP), human interferon 2
(hIFN2) and hGH have been expressed in tobacco and Arabidopsis

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J. Xu et al. / Biotechnology Advances 29 (2011) 278299

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).

cells using the Hyp-Glyco technology. Upwards of 120 mg/L of EGFP,


35 mg/L of hGH and 28 mg/L of hIFN2 were secreted into cell culture
medium, representing a 500- to 1500-fold greater recovery than
their non-glycosylated controls (Shpak et al., 1999; Tan et al., 2003;
Xu et al., 2010, 2005, 2007, 2008; Zhao et al., 2002). Glycosylation of
HypRP tags also greatly extended the serum half-life of small therapeutic proteins including hGH and hIFN2 by as much as 13-fold
without signicantly affecting their respective bioactivity (Gomord
et al., 2010; Xu et al., 2010, 2007). While most therapeutic
applications will require cleavage of this HypRP tag from the fusion
protein and a concomitant increase in downstream processing costs,
this plant-centric Hyp-Glyco technology is extremely promising for
overcoming current limitations linked with low protein yields of
plant-based bioproduction.
3.2. Cell culture approaches
While on average molecular strategies typically increase transgene
expression levels by two to three orders of magnitude (Desai et al.,
2010; Huang and McDonald, 2009), the nal protein productivity of a
plant cell culture system can also be signicantly impacted by the
culture conditions. Factors including media composition, temperature, pH, light and presence of growth regulator or stress factors in
the cultures, are all important determinants of cell growth and
protein production (Rao and Ravishankar, 2002). A high volumetric
productivity is fundamental to pursuing cell culture process development and is crucial for the ultimate commercialization of this
technology. Unlike eld-grown plants, plant cells are cultured in
conned environments, and thus culture conditions can be altered
and manipulated much more readily than in the eld. Consideration
for not only culture conditions but aspects related to retrieval and
recovery of high quality recombinant protein product is an essential
rst step in considering scale-up and process development of plant
cell culture systems.
3.2.1. Higher order testing of culture medium
Although standard plant cell/tissue culture medium such as MS
(Murashige and Skoog, 1962), SH (Schenk and Hildebrandt, 1972),
Gamborg B5 (Gamborg et al., 1968) and LS (Linsmayer and Skoog,
1965), provide sufcient nutrients necessary for plant cell growth,
protein productivity can be further improved by manipulating the
medium composition (Zhong, 2001). The components that have been
routinely adapted include components of the basal medium (nitrogen
and phosphorus macronutrients as well as other micronutrients), the
carbon source (sucrose and glucose) and the phytohormones (auxin
and cytokinin). Since plant cell species often require distinct optimal

conditions for cell growth and foreign protein production, statistical


experimental design approaches are typically employed for customizing the culture medium (Kawana and Sasamoto, 2008; Omar et al.,
2004; Prakash and Srivastava, 2005; Weathers et al., 1997; Xu et al.,
1998b). Holland et al. (2010) reports a 1020-fold increase in the
production of the full-size human anti-HIV antibody in tobacco BY-2
cell cultures through an extensive media analysis and optimization,
which was signicantly impacted by the nitrogen source. In the case
of IgG1 production in tobacco cell culture, medium manipulation has
also been reported to result in a 5-fold increase in yield (Doran, 2000).
In addition, strategies that address potential limitations in the protein
production components such as amino acids can overcome production hurdles; when these protein synthesis precursors were supplemented into the plant cell culture system, a transient 3-fold increase
in antibody levels was observed (Fischer et al., 1999b).
In addition to various nutrients and phytohormones essential to
plant cell growth, other agents are sometimes required for improving
recombinant protein quality and recovery. In particular agents
including bovine serum albumin (BSA), gelatin, polyethylene glycol
(PEG) and polyvinylpyrrolidone (PVP), have been historically used to
stabilize heterologous proteins secreted into the media (Fischer et al.,
1999b; Hellwig et al., 2004; Tsoi and Doran, 2002). While secretion of
a heterologous protein into cultured plant cell medium is potentially
advantageous for downstream processing, these recombinant products can become more susceptible to degradation by proteolytic
enzymes or destabilization due to the simplistic make-up of plant cell
culture medium (Bateman et al., 1997; Doran, 2006a; Huang and
McDonald, 2009; Shih and Doran, 2009). The use of the proteinstabilizing agent PVP (0.75 g/L) in the culture medium resulted in a
35-fold increase of heavy chain (HC) antibody yields compared to that
obtained from control medium lacking PVP (Lee et al., 2000;
Magnuson et al., 1996). Non-proteinaceous agents such as PVP
(LaCount et al., 1997; Shin et al., 2010b; Wongsamuth and Doran,
1997), PEG (Lee et al., 2002), dextran (Lee et al., 2000), Pluronic F-68
(Lee and Kim, 2002), dimethylsulfoxide (DMSO) (Wahl et al., 1995),
as well as proteinaceous agents including gelatin (Kwon et al., 2003b;
Shin et al., 2010b; Wongsamuth and Doran, 1997), BSA (James et al.,
2000) and bacitracin (Bateman et al., 1997) have been utilized in plant
cell culture foreign protein expression. When selecting proteinstabilizing agents, these compounds should be non-phytotoxic at
the concentrations employed and preferably do not interfere with cell
viability, growth and division or the downstream purication process
(Lee et al., 2000).
The precise mechanism by which many such protein-stabilizing
agents function to enhance protein recovery is not clearly understood.
In general these compounds can protect the protein from hydrolytic

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

degradation, prevent protein precipitation, and block nonspecic


interactions between the secreted polypeptide and the glass or plastic
walls of the culture vessel (Doran, 2006b; Hellwig et al., 2004; Lee
et al., 2000). Although protease inhibitors can be added into culture
medium to mitigate protein degradation, these reagents are generally
short-lived in medium and can be cost-prohibitive for large-scale
culture production (Huang and McDonald, 2009). In addition,
exogenously added compounds to the plant cell cultures may also
increase the actual rate of protein synthesis as has been reported for
both gibberellic acid and haemin (Hellwig et al., 2004; Tsoi and Doran,
2002). Indeed signicant benet can be gained by identifying a
culture medium environment early in the production timeline that
addresses the integrity and stability of the specic foreign protein
being produced.
3.2.2. Cell immobilization
The use of cell immobilization has found widespread utility in
plant cell culture. This technique was originally developed to enhance
the production of secondary metabolites, but has proven useful for the
production of heterologous proteins as well (Koyama and Seki, 2004).
Immobilization approaches tested in plant cell cultures include barrier
retention of cells using at membranes and hollow ber membranes
(Jose et al., 1983; Kim et al., 1989; Shuler et al., 1986), encapsulation
or entrapment of cells within a porous matrix such as calcium
alginate, agar and polyurethane foam (Bodeutsch et al., 2001;
Brodelius and Mosbach, 1982; Cappelletti et al., 1999; Gillet et al.,
2000; Liu et al., 1999; Osuna et al., 2008; Ziyad-Mohamed and Scragg,
1990), and surface immobilization of plant cells on the brous
material such as glass bers (Archambault et al., 1989, 1996;
Campostrini et al., 1996). Immobilization has several associated
benets over free cell culture including protection of the cells from
hydrodynamic shear stress, prevention of unintended cell removal
with the efuent in continuous cultures, and convenient recovery of
the cells from culture broth for repeated use without the necessity of
inoculation. It has been shown that cell encapsulation in alginate,
carrageenan or agar could increase the production of hGM-CSF in
tobacco cells by 50% (Bodeutsch et al., 2001), with alginate exhibiting
the highest quality beads and most reproducible growth results. The
mechanism of action is speculated to be most likely due to the
extended exponential growth phase of the cells trapped within the
alginate beads. However, there are limitations with several of these
immobilization techniques currently in use. Issues of both cell leakage
and bead disruption as culture growth progresses have been
associated with porous matrix beads such as calcium alginate.
Likewise, polymeric matrices and barrier membrane systems impose
additional limits on both nutrient and metabolite mass transfer
(Hooker and Lee, 1990). The limitations of immobilization plant cell
culture systems remain a challenge and need to be addressed for this
technology to become commercially viable.
3.2.3. In situ protein removal
Although stabilizing agents have been shown to be effective in
preventing the loss of foreign proteins from attack by proteases,
potential for producing harmful effects on the cells and negatively
impacting downstream processing remains an issue (Joo et al., 2006).
In situ protein removal represents an alternative approach to improve
the recovery of the secreted foreign proteins. This can be achieved
through aqueous two-phase culture (Dutta et al., 1994; Hooker and
Lee, 1990; Ilieva et al., 2001), supplementation of protein-binding
resins (Sharp and Doran, 2001b), and perfusion culture (Lee and Kim,
2006; Su et al., 1996). An aqueous two-phase system (ATPS) is
formulated by mixing two different water-soluble polymers such as
PEG and dextran, or a polymer and a salt such as PEG and phosphate
or sulfate (Cabral, 2007; Ilieva et al., 2001). ATPS which has been used
in a variety of applications including protein extraction (Cabral, 2007;
Hooker and Lee, 1990; Lee et al., 2004a), and may be advantageous for

287

plant cell cultivation since cultured cells are typically immobilized in


one phase along with nutrient substrates while the secreted products
are drawn into and collected from the other phase. This concept of
separate phases may in turn protect the secreted proteins from
degrading proteases associated with the alternate phase and thus
enhance foreign protein productivity. Compared with cell immobilization in/on solid phases, the growth of cells in ATPS is unhindered
since cells remain in a free suspension rather than bound within a
matrix or between barrier membranes (Hooker and Lee, 1990). In
addition, ATPS can achieve high degrees of phase contact which
diminishes limitations associated with mass transfer. Tobacco
suspension cells have been successfully grown in various ATPS
cultures with the highest observed growth rates occurring with a 3%
PEG (20,000)/5% dextran system, wherein the growth rate and cell
density approached that of cultures grown in standard medium
(Hooker and Lee, 1990). Ilieva et al. (2001) reported phosphodiesterases production in tobacco cell culture using an ATPS with 4% PEG
(20,000)/7.5% dextran where the phosphodiesterases partitioned
primarily to the bottom dextran phase and exhibited high specic
enzyme activity.
Addition of resins into culture medium for the binding and
recovery of target metabolites has long been used for enhancing
secondary metabolite production (Bisaria and Panda, 1991; Choi et al.,
2001; Roja et al., 2005). A similar strategy could also be harnessed
for improving recombinant protein recovery which in effect would
isolate the protein product from degrading inuences and/or remove
the protein from product inhibition pathways (James et al., 2002). In
the case of tobacco cells producing and secreting mouse HC mAb and
6xHis tagged hGM-CSF, in situ recovery using Protein G and iminodiacetic acid metal afnity resins, respectively proved quite effective. This approach resulted in as much as an 8-fold increase in HC
mAb production and a 2-fold enhancement of hGM-CSF yield when
compared to these proteins recovered under normal culture procedures (James et al., 2002). In another example, addition of
hydroxyapatite resin to a tobacco NT-1 cell culture with periodic
harvesting of the resin led to reduced susceptibility of the secreted
IgG1 antibody to hydrolytic degradation and an overall increase in
product recovered (Sharp and Doran, 2001b). Perfusion culture for
in situ removal of protein is typically carried out in a bioreactor, as will
be discussed in further detail in following section. However demonstrating proof-of-principle of this technology, a semi-continuous
perfusion culture of tobacco NT-1 cells conducted in shake ask
performed by replacing 33% of the medium every 12 h, allowed constant
levels of secreted HC mAb to be harvested (LaCount et al., 1997).
3.3. Bioreactor engineering approaches
Process development is a critical step in scaling-up plant cell
cultures to achieve commercial productivity. While similar to
bacterial fermentation or mammalian cell culture in that plant cells
can be grown in bioreactors as homogeneous suspensions, there are
some notable differences in terms of growth kinetics, obtainable cell
densities and nutritional requirements. Although plant cells are
readily cultured in most conventional bioreactors with minor
adjustments (Hellwig et al., 2004; Su and Lee, 2007), scaling-up the
plant cell culture from shake asks to bioreactors is not always
straightforward. Due to the dramatic change of cell growth environments between the two platforms in terms of hydrodynamic shear
forces and rheological properties (Curtis and Emery, 1993; Kieran
et al., 1997; Sajc et al., 2000), poor growth rates of cells and relatively
low product yields are common challenges (Sajc et al., 2000; Su and
Lee, 2007). Fortunately, most of these limitations can be addressed
with improved bioreactor design and optimizing key parameters of
the system including agitation speed, aeration rate and nutrient
supply. Other aspects include identifying plant species most amenable
to in vitro culturing and effective screening procedures for selecting

288

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

Table 2
Enhanced recombinant protein production in plant cell cultures with process engineering approaches.
Culture mode

Bioreactor type and strategies

Expressed protein

Host
plant cell

Yield or productivity enhancement

Reference

Batch
culture

2 L Stirred tank with a single pitched blade impeller.


Control higher medium pH

1-antitrypsin
(rAAT)

N. benthamiana

Huang et al.
(2009)

Fed-batch
culture

7 L and 15 L stirred tank with four-bladed hollow-paddle


impeller. 10 concentrated amino acids fed from day 7.

4.5 mg/L total and 136 ug/L functional


protein, up to 4-fold increase in
productivity
76.5 mg/L or 5.8 mg/L/day, 1.2-fold
increase compared to that of
two-stage cultures with medium
exchange
2-fold increase in volumetric productivity
compared with batch culture
Maximum 40 ug/L/day, 9.4-fold increase
in productivity compared with 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.

elite cell lines exhibiting satisfactory growth characteristics and


genetic stability (Parekh et al., 2008). In addressing and optimizing
operations, plant cell cultures in bioreactors producing hGM-CSF or
hCTLA4Ig have in fact shown signicantly better performance and
productivity than their shake ask controls (Lee et al., 2004b; Park
et al., 2010). Foreign protein production can be further improved by
applying advanced culture strategies such as fed-batch culture,
perfusion culture, continuous culture and various modications of
these platforms as outlined in Table 2 (Georgiev et al., 2009; Roberts
and Shuler, 1997; Su and Lee, 2007). Bioreactor engineering aspects of
plant cell cultures including bioreactor design criteria and optimization strategies have been recently reviewed (Georgiev et al., 2009;
Huang and McDonald, 2009; Parekh et al., 2008). Thus this section will
summarize some advanced culture strategies in the context of
enhancing foreign protein production with a particular focus on the
use of disposal bioreactors for plant cell cultures.
3.3.1. Optimized bioreactor design and operation
The goal in bioreactor design and operation is to provide a
prolonged and sterile culture environment with efcient mixing and
oxygen exchange in an effort to support optimized cell growth and
achieve high productivity (Su and Lee, 2007). Large-scale culturing of
plant cells has been conducted in variety of bioreactors including

Rice (O. sativa L.)

N. tabacum L. cv
Xanthi
N. tabacum L. cv
Havana SR

Anchusa
ofcinalis

~ 49 units/L/day, more than 3-fold


increase in productivity compared
with batch culture

Park et al.
(2010)

Liu et al.
(2001)
Lee et al.
(2004b)

Su and
Arias
(2003)

Up to 0.6 mg/L/day for individual


cycle, 25-fold increase in productivity
compared with batch culture
Rice (O. sativa L.) 312 mg/L/day for individual cycle;
overall productivities up to
7.7 mg/L/day.

Huang et al.
(2010)

N. tabacum
BY-2

Des Molles
et al. (1999)

N. benthamiana

500 unit/L/day,4-fold increase in


productivity compared with batch
culture

Trexler et al.
(2005)

standard stirred-tank bioreactor (STR) (Doran, 1999; Hooker et al.,


1990), bubble column bioreactor (BCB) (Kim et al., 2001; Min et al.,
2007), air-life bioreactor (ALB) (Hsiao et al., 1999; Su et al., 1996) and
other novel bioreactor types including the wave bioreactor (Eibl and
Eibl, 2006; Terrier et al., 2007), the membrane bioreactor (McDonald
et al., 2005), the hollow ber bioreactor (Kim et al., 1989) and the
rotating drum reactor (Shibasaki et al., 1992; Tanaka et al., 1983).
While lessons can be learned from scaled production of microbial
and mammalian cells, plant cells have many unique and distinguishing features that impose limitations on their large-scale growth
and process development (Table 3). Distinct properties of plant cell
cultures that necessitate special consideration in bioreactor process
development include: large cell size and complex morphology, tendency for aggregation, time-dependent rheological behavior, foaming and wall growth, shear sensitivity, and relatively low growth
and oxygen uptake rate (Parekh et al., 2008). The implications of
these factors on bioreactor design and operation have been comprehensively reviewed (Huang and McDonald, 2009; Sajc et al., 2000).
Some plant-centric factors of mention include the inherent
sensitivity of suspended plant cells to shear forces due to the majority
of their cell volume (~ 90%) being associated with intracellular
vacuoles and the rigid nature of their cellulose-based cell wall
(Dunlop et al., 1994). The shear-sensitivity of plant cells has been

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

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

the subject of extensive investigation (Camacho et al., 2000; Namdev


and Dunlop, 1995; Sowana et al., 2001; Sun and Linden, 1999; Zhong
et al., 2009, 1994) however, the precise mechanism by which
shear stress induces cell damage is not well-understood and has
been very difcult to study due to the incredible diversity of plant cell
lines, the varying aggregate size distribution and their assorted cell
morphologies (Huang and McDonald, 2009). As such, susceptibility of
plant cells to shearing forces varies widely among different cell
lines. Even within the same culture line, the age of the culture and the
cultivation schedule can signicantly inuence the responses of the
cells to shear elds (Georgiev et al., 2009). The concept of critical
shear stress, above which cell viability is lost, has been an important
factor in establishing guidance for plant cell bioreactor design (Huang
and McDonald, 2009). The general solution to the shear-sensitivity
issue involves cultivating plant cells under low-shear stress environments such as those established in pneumatic bioreactors (air-lift and
bubble column bioreactor), centrifugal impeller bioreactors (CIB)
(Wang and Zhong, 1996a, 1996b), or stirred-tank bioreactors (STR)
with decreased impeller agitation speed or with a specially designed
low-shear impeller. In the case of STRs others have suggested that
shear sensitivity may not be as critical, and that signicant losses in
viability are not, in general, to be expected under normal operating
conditions in a conventional STR (Kieran et al., 1997).
3.3.2. Advanced bioreactor culture strategies
Batch culture is the simplest culture strategy and is most
frequently employed for large-scale plant cell cultures. Optimizing
culture conditions such as agitation speed (in STR), aeration rate, and
pH, has led to higher protein yields than those obtained from shake
asks (Lee et al., 2004b). However, due to the inherent limitations
of the batch culture mode, the protein productivity of plant cells in
this basic system cannot be further improved. The long lag phase,
the depletion of the key nutrients and the accumulation of inhibitory
substances/metabolites prevent the batch culture from achieving
desired productivity. In addition, signicant amounts of time are
required for sterilizing the culture system hardware and media as well
as lling, emptying and cleaning the system (Georgiev et al., 2009).
This time and labor-intensive issue has been the major driver in the
development of advanced culture strategies, including fed-batch
(Park et al., 2010), two-stage (Shin et al., 2003), perfusion (Wang
et al., 2010), semi-continuous (Su and Arias, 2003) and continuous
cultures (van Gulik et al., 2001) for improving the overall productivity
of plant cell culture technology (Georgiev et al., 2009; Roberts and
Shuler, 1997; Su and Lee, 2007). The integration of these advanced
culture systems for plant cells has been much more limited with
the majority of these linked to secondary metabolite production as
opposed to recombinant proteins. In addition, the choice of culture
strategy can be signicantly impacted by species-specic characteristics of the host cell (specic growth rate, aggregation, etc.), the
promoter used for driving the expression of the recombinant protein
(constitutive or inducible), and the localization and structural/
functional properties of the expressed proteins (intracellular or
secreted; stable or easily degradable; toxic or harmless to hosts)
(Huang and McDonald, 2009).
3.3.2.1. Fed-batch culture. Fed-batch cultures are characterized by the
addition of one or more nutrients continuously or intermittently
into the medium after the start of cultivation, or at some point during
the batch process (Georgiev et al., 2009). Simple implementation
makes this the most popular culture strategy for achieving high
cell density and prolonging the exponential growth phase (Sim
and Chang, 1999). Fed-batch is especially appropriate for growthassociated production systems such as those driven by a constitutive
promoter. Such a medium-feeding strategy demonstrated that
improved cell biomass and protein (GFP) production could be
achieved in a fed-batch culture of tobacco BY-2 cells (Liu et al.,

289

2001). Although fewer studies have been reported, fed-batch culture


has also been shown to improve productivity in an inducible
expression system. In the production of hCTLA4Ig expressed under
the inducible RAmy3D promoter, a fed-batch addition of concentrated
amino acids into the rice cell culture grown in the 15-L stirred-tank
bioreactor increased productivity of the foreign protein by 1.2-fold in
comparison to the more complex two-stage culture that implemented
a medium exchange (Park et al., 2010). However, like batch culture,
protein productivity in fed-batch cultures is also limited by accumulation of toxic or inhibitory metabolites and cell growth has been
shown to be retarded at higher cell densities (above 60% packed cell
volume (PCV)) (Su, 1995).
3.3.2.2. Perfusion culture. Perfusion cultures involve maintaining cells
within a bioreactor where fresh medium is continuously fed and the
spent, cell-free medium is constantly being removed. In comparison
to fed-batch cultures, perfusion culture offers additional process
exibility and advantages. In addition to growth inhibition caused by
the accumulation of inhibitory metabolites in the medium being
alleviated in perfusion culture, the cultured cells in a bioreactor can be
reused thereby minimizing turnaround time between batches and
thus a concomitant increase in the overall productivity of the system.
Perfusion culture has been exploited for the culture of plant cells
with notable success (De Dobbeleer et al., 2006; Su and Arias, 2003; Su
et al., 1996; Wang and Qi, 2009; Wang et al., 2010). Cell densities
of 31 g/L were obtained for perfusion cultures of Thalictrum rugosum
cell in a STR (Kim et al., 1991). A major challenge in establishing a
plant cell perfusion culture is maintaining healthy, robust and fully
viable cultured cells in the bioreactor while the cell-free spent
medium is continuously being removed and the fresh medium
supplemented (Su et al., 1996).
The most common approaches used for cell retention in perfusion
cultures include continuous centrifugation (Johnson et al., 1996),
in situ ltration (Kawahara et al., 1994) and gravitational sedimentation (Su and Arias, 2003). Because plant cells tend to grow as
aggregates, cell/medium separation can be accomplished with
relative ease using gravitational sedimentation. Development of an
efcient plant cell perfusion process by inserting a cylindrical bafe
into the STR vessel to create an annular settling zone wherein continuous cell/medium separation via gravitational sedimentation has
been achieved (Su and Arias, 2003). In this study, perfusion cultures
could be maintained at perfusion rates of 0.4 day 1 and a PCV of
60% while successfully achieving complete plant cell retention. In
order to establish a higher rate perfusion culture, (De Dobbeleer et al.,
2006) modied a STR tank for plant cell production by installing four
sedimentation columns vertically on the lid of the tank, which
enabled a maximum perfusion rate of 20.4 day 1 to be run from day 4
to day 6, and a reduced rate of 5 day 1 to be run on day 9 at a 55% PCV.
However, operation of the perfusion culture under extremely high
PCV (e.g. PCV N60%) for a prolonged period will lead to declined
oxygen uptake and reduced cell viability due to the agglomeration
and sedimentation of the plant cells (Huang et al., 2010). In this case,
continuously removing a certain percentage of cultured cells with
the medium in a process referred to as cell bleeding helps maintain
the best performance of the perfusion culture. Cell bleeding at
0.11 vvd (reactor volume per day) had been shown to stabilize the
perfusion culture of Anchusa ofcinalis cells maintained at a PCV below
60% and successfully used for the production of an acid phosphatase
(Su and Arias, 2003). In this study, both the specic oxygen uptake
rate and cell viability were shown to increase, leading to higher cell
biomass yields approaching 25 g/L and improved acid phosphatase
productivity of ~490 units/L/day. Although further optimization of
the culture operation is required, perfusion bioreactor systems are
emerging as the preferred advanced culture strategy relative to other
systems for achieving high productivity of foreign protein expressed
in plant cell culture.

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J. Xu et al. / Biotechnology Advances 29 (2011) 278299

3.3.2.3. Continuous culture/semi-continuous culture. In a continuous or


chemostat operation, fresh medium is continuously fed into the
bioreactor and the excess culture (spent medium and cells) is
simultaneously washed out, thereby permitting the maintenance of
cultures very close to their maximum growth rate. Due to long runtimes and the need for plant cells to be maintained in exponential
growth phase, continuous culture is attractive and holds promise as
an emerging advanced culture strategy (Georgiev et al., 2009).
However the limited research that exists on this continuous culture
system has primarily focused on secondary metabolite production
(Miller et al., 1968; Sahai and Shuler, 1984; van Gulik et al., 2001).
Some typical problems associated with this culture strategy include
low specic growth rate, cell aggregation, genetic instability and
excretion of sticky polysaccharides (Hellwig et al., 2004; van Gulik
et al., 2001). These problems make it difcult to maintain an ideallymixed suspension of cells which is requisite for establishing a
continuous culture system.
A modication of this system known as semi-continuous culture
is a hybrid of batch and continuous operations and may be a more
appropriate application for plant cell-based recombinant protein production. In a semi-continuous operation, a portion of the suspension
culture (spent medium and cells) is periodically harvested in exchange
for an equivalent volume of fresh medium. The advantages of such
a semi-continuous culture include: active recombinant proteins can
be harvested periodically and factors limiting cell growth and protein
stability in the culture broth are simultaneously removed (Huang
et al., 2010). Long-term chloramphenicol acetyltransferase (CAT)
production has been demonstrated with a 41-day, semi-continuous
culture of tobacco cells in a STR bioreactor (Hogue et al., 1990). Semicontinuous cultures may also be suitable for inducible production
system, where a two-stage culture is often practiced; rst stage
dedicated to cell growth and the second to protein production. An
example of this conguration was shown with a rice cell culture
system expressing a transgene under the inducible Ramy3D promoter
(Huang and McDonald, 2009). In this case an exchange to sugar-free
medium was required at a specic phase of cell growth to induce
the recombinant protein expression. The cyclical semi-continuous
operation enabled culture conditions to be alternated to support
either cell growth or production as well as the reuse of the cultured
rice cells in promoting a long-term operation. With a semi-continuous
culture of rice cells consisting of three cycles over a 2528 day period,
rAAT protein levels ranging from 4.5 to 7.7 mg/L/day were achieved
(Trexler et al., 2005). More recently, Huang et al. (2010) developed a
semi-continuous bioreactor operation for enhancing the production
of rAAT in N. benthamiana cell culture. In this optimized semicontinuous operation, plant cells transformed with the inducible
CMV viral amplicon and expressing rAAT maintained higher viability
and produced lower extracellular protease activity and phenolic
concentrations than when grown in a batch mode. Integration of
this semi-continuous culture method resulted in a 25-fold increase in
the production of rAAT that corresponded to 603 g/L of protein
harvested daily.
3.3.3. Adoption of disposable bioreactors
In recent years there has been a trend towards the use of
disposable bioreactors for large-scale plant cell culture with a goal
of reducing production costs and minimizing validation efforts under
GMP regulations (Ducos et al., 2010; Eibl et al., 2010; Hacker et al.,
2009). In contrast to traditional glass or stainless steel bioreactors,
disposable bioreactors are usually made of FDA-approved biocompatible plastics such as polyethylene, polystyrene, polytetrauorethylene, polypropylene and ethylene vinyl acetate (Eibl and Eibl, 2006;
Eibl et al., 2010). Since the bioreactor vessel is pre-sterilized and
discarded after harvest, clean-up and re-sterilization steps are
eliminated and associated risks with culture contamination are
reduced (Singh, 1999). To date the integration of disposable

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

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

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)

Eibl and Eibl (2006)


Eibl and Eibl (2006)
Ritala et al. (2008)

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

Terrier et al. (2007)

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

Terrier et al. (2007)

Plastic-lined bioreactor
G
A
S

Hyoscyamus muticus

Biomass

28.5 L
100 L

7.02 g(DW)/L (day 13)


15.0 g(DW)/L (day 33)

Hsiao et al. (1999)


Curtis (2004)

Stirred bag bioreactor

N. tabacum BY-2

Biomass

25 L

13.5 g(DW)/L (day 5)

Eibl et al. (2009)

Carrot cells

Glucocerebrosidase

N/A

N/A

(Shaaltiel et al., 2007;


Weathers et al., 2010)

Wave and undertow bioreactor

Slug bubble bioreactor


G

Slug Bubble

AG

Osmotekbag bioreactor
G

Aair inlet; Gexhaust gas; Ssampling; DWdry weight; FWfresh weight; N/Anot available.

or extracellular medium is equally important. As in other cell-based


bioproduction systems, the downstream processing in plant cell
culture may account for as much as 80% of the total production costs
(Hellwig et al., 2004). The purication of proteins for therapeutic
application represents the most challenging and expensive process
in which protein of the highest purity (N99%) is required and purication procedures must meet the stringent regulatory standards
established that govern production of biopharmaceuticals (Hellwig
et al., 2004). Therefore, efforts to develop new and improved downstream processing methods can signicantly reduce the overall costs
associated with plant cell-based production systems (Nikolov and
Woodard, 2004).
In plant cell expression systems, the target proteins are either
retained inside the cells or secreted into the medium. Secretion
systems are advantageous to the downstream protein recovery
process in that typically two unit operation steps cell disruption
and clarication can be circumvented (Fig. 5). Generally lower in
phenolic compounds and other oxidizing substances, the medium
fraction enters the purication process containing a much lower
percentage of contaminating proteins and other metabolites relative
to cell extracts (Hellwig et al., 2004). On the other hand secreted
target proteins may be diluted and thus more unstable in the culture

Fig. 5. A standardized procedure of protein separation and purication from plant cells
or cell culture medium.

292

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

medium highlighting the need for efcient and rapid recovery


techniques for the foreign protein from large medium volumes. In
contrast, the advantages of intracellular retention for downstream
processing lie in the smaller volume of the harvested plant cells and
in some cases a higher concentration of the target protein in the
starting material. Major disadvantages associated with cell retention
of the foreign protein include a more complex composition of the
feed-stream and the liberation of proteolytic and oxidizing compounds during the cell disruption process (Hellwig et al., 2004).
Various considerations of downstream processing for transgenic
plant-derived proteins have been previously reviewed (Hellwig
et al., 2004; Menkhaus et al., 2004).
The downstream processing for plant cells where the protein is
intracellularly retained begins with cell disruption/clarication, and is
followed by concentration/capturing and chromatographic purications (Fig. 5). Some examples of downstream processing studies with
plant cell culture are shown in Table 5. While secreted proteins can
advance immediately to the concentration/capturing step of the
process, those retained inside cells must rst be released by breaking
the cell wall and membrane. Techniques available for plant cell
disruption include sonication, pressure homogenization, wet milling
and enzymatic lysis. While all methods work efciently in breaking
the plant cell wall, wet milling and sonication are in fact difcult to
scale-up and enzymatic lysis is cost-prohibitive at scale (Hellwig et al.,
2004). Thus, pressure homogenization is generally regarded as the
method of choice in large-scale processes. After cell disruption,
clarication of the cell extract is typically carried out by either deadend or cross-ow ltration for large-scale production or centrifugation at smaller scale. Alternatively, advanced chromatographic
technology Expanded Bed Adsorption (EBA) could be an efcient
approach for simultaneous clarication, concentration, and prepurication from either cell extract or cell culture medium (Bai and
Glatz, 2003; Valdes et al., 2003).
The purication steps that follow are generally common to the
protein recovery methods used in other production platforms. Several
chromatography options including ion exchange chromatography
(IEX), afnity chromatography (AC), hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) are used
for the enrichment of the recombinant protein (Fig. 5). Method
development from selection of the most effective chromatographic
matrices, to their arrangement in a suitable order and evaluation of
their efciency in product recovery is dependent upon the unique

properties of the target protein including molecular size, charge,


solubility, and stability as well as the protein background of the
production host. The use of afnity tags including GST (glutathione
S-transferases), MBP (maltose binding protein) and 6xHis can facilitate
the capture of the foreign protein but these tags may require removal
following purication to restore the native structure/function of the
protein or is required for market distribution of the product (Fischer
et al., 2004). In the case of recombinant antibody production in plants
(Plantibodies), Protein A or Protein G afnity chromatography provide
efcient and convenient choices for protein capture and purication
(Girard et al., 2006; Hellwig et al., 2004; Hussack et al., 2010; Park et al.,
2010). In addition to the column-based separation approaches
mentioned above, selection based on membranes such as ultraltration, microltration, pervaporation, and pertraction should also be
considered for use in protein purication. The advantages of membranes
include speed, easier scale-up and large interfacial area available for
mass transfer (Charcosset, 2006; Sajc et al., 2000). Ultraltration has
recently been used to purify the recombinant type I human collagen and
GFP from corn extracts with success (Aspelund and Glatz, 2010).
4. Other challenges to overcome
Outside of low productivity that could be addressed and improved
by implementing the strategies discussed above, other outstanding
challenges for recombinant protein production in plant cell culture
that remain to be addressed include non-mammalian glycosylation
and genetic instability (Doran, 2000; Gomord et al., 2005; James and
Lee, 2006; Shih and Doran, 2009).
4.1. Non-mammalian glycosylation
There are some distinct and important differences in the glycosylation processes of animal and plant cells that warrant consideration when producing recombinant proteins. Although the resulting
alteration of the plant glycosylation pattern may not affect the activity
of the recombinant protein, possible immunogenicity issues in using
plant-produced glycoproteins for animal/mammalian applications
need to be considered (Doran, 2000; Faye et al., 2005; Garcia-Casado
et al., 1996; Li and d'Anjou, 2009). As the issue of protein N- and Oglycosylation in plants and the limitations and advantages of plantspecic glycosylation on recombinant therapeutic proteins has been
reviewed in depth (Gomord et al., 2010), the following discussion

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%

Terashima et al. (1999)

N95%

Huang et al. (2005)

Carrot cell

Glucocerebrosidase

Intracellular retention

N99%

Shaaltiel et al. (2007)

N. tabacum cv. Xanthi

GFP and hGM-CSF-GFP

Intracellular retention

N80%

Peckham et al. (2006)

N. tabacum cv. Xanthi

anti-rabies virus mAb

Intracellular retention

Capturing with HIC;


Purication and polishing with SEC and RP-HPLC
One-step capturing and purication with AC
(with anti-AAT antibody)
Clarication with ammonium sulfate precipitation
and centrifugation;
Capturing and purication with anion IEX
Concentration with membrane ltration
Cell disruption with homogenization;
Clarication with centrifugation;
Capturing with cation IEX;
Purication and polishing with HIC and cation IEX
Cell disruption with ultra-sonication;
Clarication with ammonium sulfate precipitation
and centrifugation;
Capturing with HIC;
Purication with anion IEX
Cell disruption with French press;
Clarication with centrifugation;
Concentration with membrane cross-ltration;
One-step capturing and purication with protein
G-based AC

N99%

O. sativa L. cv Tainung 67

Human interferon 2
(hIFN 2)
Human 1-antitrypsin
(rAAT)
Human serum albumin

N95%

Girard et al. (2006)

O. sativa L. cv Taipei 309

Secreted

IEXion exchange chromatography; ACafnity chromatography; HIC hydrophobic interaction chromatography; SECsize-exclusion chromatography.

J. Xu et al. / Biotechnology Advances 29 (2011) 278299

summarizes this complex topic and specically highlights the issue of


glycosylation as it relates to foreign protein expression in the plant
cell culture platform.
The N-linked glycans produced in plants or plant cells differ from
their mammalian counterparts in the processing and modications of
the core glycan that occur in the Golgi apparatus (Faye et al., 2005;
Frey et al., 2009). There are two key modications on core glycan in
the plant Golgi generally considered the major obstacles limiting the
use of plant-made biopharmaceuticals (Frey et al., 2009): 1) the
absence of (1,4)-galactose, core (1,6)-fucose and terminal sialic
acids that characterize animal glycosylation and which may reduce
the clinical efciency of the plant-derived glycoproteins due to their
decreased serum half-life (Gomord et al., 2010; Lerouge et al., 1998)
and; 2) incorporation of (1,3)-fucose and (1,2)-xylose residues,
two epitopes not found in mammalian glycans that have been linked
with inducing immunogenicity (Bardor et al., 2003; Doran, 2000;
Wilson et al., 1998). Thus far, considerable progress has been made
towards the humanization of protein N-glycosylation in plant cells.
Strategies showing much promise include targeted expression of
therapeutic proteins retrieved from the early Golgi back into the ER
(Lai et al., 2010; Petruccelli et al., 2006), knockout of certain Golgi
enzyme glycosyltransferases (e.g., for xylose and fucose) (Cox et al.,
2006; Koprivova et al., 2004; Sourrouille et al., 2008; Strasser et al.,
2008), utilization of the N-glycosylation plant mutants (Downing
et al., 2006; Strasser et al., 2004) and the engineering of the mammalian glycosyltransferases (e.g. (1,4)-galactosyltransferase or
(1,4)-N-acetylglucosaminyl transferase III) (Bakker et al., 2001;
Frey et al., 2009; Gomez and Chrispeels, 1994). In addition, BecerraArteaga and Shuler (2007) report that culture medium supplementation with nucleotide-sugar precursors such as glucosamine can
alter signicantly the N-linked glycosylation of a recombinant protein
in tobacco cell culture. Most recently, Shin et al. (2010a) report that
sugar starvation conditions, e.g. growth medium with sucrose (+S) or
under sucrose starvation (S), also alter the N-glycan patterns of
the glycoproteins secreted from rice cell suspension cultures. These
studies provide another alternative strategy for the humanization of
N-glycosylated, plant-expressed recombinant proteins.
While N-glycosylation has been extensively addressed, very little
attention has been given to the O-glycosylation of plant cell-made
foreign proteins (Gomord et al., 2010). In general, O-glycosylation is
one of the most complexly-regulated, post-translational modications of proteins, and yet is one of the least understood processes.
Unlike N-glycosylation which occurs at a consensus sequence (Asp-XSer/Thr), there is no well-dened sequence for predicting protein Oglycosylation. In plant cells, O-glycosylation has been described
mainly for the hydroxyl groups of hydroxyproline (Hyp) Ser and
Thr residues, of which the O-glycosylation on Hyp residues is unique
to higher plants and green algae (Kieliszewski and Shpak, 2001;
Showalter, 1993). The Hyp-O-linked glycans are extremely abundant
in plant cells and in fact make a signicant contribution to the
structural properties of the plant extracellular matrix as distinct posttranslational modications of both the extensins and arabinogalactan
proteins (AGPs) (Cassab, 1998; Kieliszewski and Lamport, 1994).
Therefore, in the case of therapeutic proteins produced in plant cells,
the issue of these non-human Hyp-O-glycans being a source of
immunogenicity has been addressed in several studies (Leonard et al.,
2005; Willats et al., 1998; Yates et al., 1996). However to date, all
studies (Xu et al., 2010, 2007) have indicated that the Hyp-O-glycans
produced in tobacco BY-2 cells does not elevate the mouse serum
antibody titers beyond those marginal titers determined in preimmune sera, suggesting immunogenicity may be linked to the
specic glycan structure and size. Recently, Hyp-O-glycosylation of
the maize-expressed human IgA1 has been reported in which up to six
Hyp-O-arabinose residues were detected in the hinge region of this
recombinant IgG (Karnoup et al., 2005). So far, it is not yet known if
such modication has any impact on the stability, biological activity,

293

or efcacy of the IgA1 molecule, or whether these sugars could be a


source of immunogenicity or allergenicity in patients. Reports of
the presence of Hyp-O-glycosylation on plant-produced therapeutics
are likely to increase as plant-based protein production technologies
advance. Since O-glycosylation occurs mainly in the Golgi apparatus,
targeting the expression of foreign proteins to the ER would avoid this
modication. However, O-glycosylation does not necessarily convey a
negative impact on plant cell-expressed proteins. Further research is
needed to fully understand the impact of O-glycosylation on yields
and protein stability in planta as well as the half-life and pharmacodynamics of therapeutic proteins in the host (Gomord et al., 2010).
4.2. Genetic instability
The tendency towards genetic instability of suspended plant cells
poses another challenge to plant cell culture technology. Although
dedifferentiated plant cells lack of fully functional plasmodesmata
has reduced systemic post-transcriptional gene silencing (PTGS) and
are morphologically stable (Offringa et al., 1990; Shih and Doran,
2009), they have been frequently shown to suffer from genetic
instability. The underlying causes of this instability may result from
somaclonal variation (gene drift) (Cote et al., 2001), transgene loss
(Kononowicz et al., 1990; Ulker et al., 1999), transcriptional gene
silencing (Bruening, 1998; Chandler and Vaucheret, 2001; Weld et al.,
2001), or other undesirable genetic changes. Epigenetic transcriptional silencing is considered the dominant contributing factor to the
unstable expression of transgenes in plant cells that are maintained in
culture for extended periods (Matzke and Matzke, 1998; Phillips et al.,
1994). Transcriptional silencing is associated with meiotically
heritable epigenetic modications often, but not always, through
cytosine methylation (Matzke and Matzke, 1998). Due to genetic
instability, plant cell cultures will lose the capacity for high production
levels as cells with suppressed protein expression capacity begin to
dominate the culture (Gao et al., 1991; James and Lee, 2006; Shih and
Doran, 2009). For example, the production of a mAb (IgG1) has been
found to decline severely in tobacco cell cultures maintained for a
period of 3 years (Sharp and Doran, 2001b) in comparison with
relatively constant levels of this antibody expressed in tobacco hairy
root cultures over this same time period. In another example, hGMCSF protein production in tobacco NT-1 cell culture was shown to
decrease by more than 80% following 250 subculture events (James
and Lee, 2006) despite the NT-1 cell line itself being relatively stable.
Development of efcient techniques to preserve the elite cell lines,
namely cell banking, is considered a surmountable means of
overcoming this issue of genetic instability in plant cells. In general,
cell banking is considered a prerequisite for the reliable supply of
well-dened starting material for commercial recombinant protein
production on other production platforms. A three-tiered setup
consisting of a research cell bank, a master cell bank and a working
cell bank is generally accepted as the most practical approach for the
establishment and validation of the producer cell line (Parekh et al.,
2008). Cell banking by cryopreservation of elite plant cell lines has
been studied to allow the maintenance of a stable production system (Cho et al., 2007; Schmale et al., 2006). An alternative approach
is to rescreen the high-producing plant cell lines when the reduction
of protein yield is observed, provided that a reasonable fraction of
high-producing cells is still present in the cell culture. Recently, a
mathematical model for predicting the loss of productivity over
successive generations in plant cell culture has been developed
(James and Lee, 2006). This model presents a useful tool for
characterizing the stability of production cell lines and in planning
storage and rescreening efforts to ensure propagation of the high
expressing cells. In addition, co-expression of gene silencing suppressors (Baulcombe et al., 1997; Huang and McDonald, 2009) may
also be a useful strategy in maintaining high productivity of elite plant
cell culture lines.

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J. Xu et al. / Biotechnology Advances 29 (2011) 278299

5. Perspective and conclusion


The utility of plants as a commercially viable production platform
for pharmaceutical proteins has been a long-standing controversy in the
biotechnology industry. In the interest of addressing increased concerns
about regulatory compliance and product safety, there has been
renewed interest in plant cell cultures as a promising production
platform (Huang and McDonald, 2009; Shih and Doran, 2009). Since
plant cell cultures are inherently simpler, cheaper and safer than mammalian cell approaches and support eukaryotic processing (e.g. complex
glycosylation) absent in microbial systems, this technology has
continued to attract attention as an alternative host system. Although
the cost for plant cell culture in bioreactors is higher than an agriculture approach, the concept of being able to secret the protein of
interest into simple culture medium offers an important opportunity
for substantial cost savings (Doran, 2006a). In fact with completion of
Phase III clinical trials of plant cell-produced human glucocerebrosidase (UPLYSO) by Protalix, many in the industry are eagerly
anticipating the commercial debut of this technology early in 2011. As
plant-produced human glucocerebrosidase becomes a successful
reality, a new era in the biopharmaceutical industry will be dened
that promises to provide growth opportunity for the plant cell culture
platform.
The plant cell culture technology has made great strides in the last
two decades due to the advances in plant molecular biology and
bioreactor process engineering. As high as 247 mg/L secreted protein
yield has been achieved with rice cell culture (McDonald et al., 2005);
large-scale plant cell culture up to a volume of 100,000 L has been
successfully performed (Fischer et al., 1999a). In addition, new
bioreactor designs and advanced culture strategies (e.g. fed-batch,
perfusion culture, and continuous/semi-continuous culture) continue
to enhance the productivity of this system and facilitate the in situ
product recovery (Huang and McDonald, 2009). In recent years
signicant progress has also been made towards utilizing low-cost,
highly efcient and safe bioreactor congurations including largescale disposable bioreactors that are capable of meeting GMP
requirements (Ducos et al., 2010). Substantial advances in downstream processing including the application of advanced chromatographic technologies could signicantly enhance overall costeffectiveness (Georgiev et al., 2009). The key areas to ensure the
advancement of this technology will be in leveraging the molecular
and process engineering approaches to further increase the foreign
protein productivity, to humanize the glycans of plant glycoproteins
and to improve post-translational protein stability. With the ultimate
goal of achieving economically-feasible yields of pharmaceutical
proteins that are structurally and functionally equivalent to their
native counterparts, systematic and concerted research efforts that
are both biologically- and engineering-based will be critical to the
success of the plant cell culture protein production platform.
Acknowledgements
This work was supported by the Plant Powered Production (P3)
Center funds provided through an NSF RII Arkansas ASSET Initiative (AR
EPSCoR) grant and the Arkansas Biosciences Institute, the major
research component of the Arkansas Tobacco Settlement Proceeds Act
of 2000.
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