Plant Biotechnology Journal (2012) 10, pp. 249–268 doi: 10.1111/j.1467-7652.2011.00664.

Review article

Recent advances towards development and

commercialization of plant cell culture processes for the
synthesis of biomolecules
Sarah A. Wilson and Susan C. Roberts*
Department of Chemical Engineering, University of Massachusetts Amherst, Amherst, MA, USA

Received 8 July 2011; Summary

revised 14 September 2011;
accepted 19 September 2011.
*Correspondence (fax 413 545 1647;
email sroberts@ecs.umass.edu)
Keywords: plant cell culture, second-
ary metabolites, recombinant pro-
teins, bioreactors, metabolic
engineering, commercialization.
Plant cell culture systems were initially explored for use in commercial synthesis of several
high-value secondary metabolites, allowing for sustainable production that was not limited by
the low yields associated with natural harvest or the high cost associated with complex chem-
ical synthesis. Although there have been some commercial successes, most notably paclitaxel
production from Taxus sp., process limitations exist with regards to low product yields and
inherent production variability. A variety of strategies are being developed to overcome these
limitations including elicitation, in situ product removal and metabolic engineering with single
genes and transcription factors. Recently, the plant cell culture production platform has been
extended to pharmaceutically active heterologous proteins. Plant systems are beneficial
because they are able to produce complex proteins that are properly glycosylated, folded and
assembled without the risk of contamination by toxins that are associated with mammalian or
microbial production systems. Additionally, plant cell culture isolates transgenic material from
the environment, allows for more controllable conditions over field-grown crops and pro-
motes secretion of proteins to the medium, reducing downstream purification costs. Despite
these benefits, the increase in cost of heterologous protein synthesis in plant cell culture as
opposed to field-grown crops is significant and therefore processes must be optimized with
regard to maximizing secretion and enhancing protein stability in the cell culture media. This
review discusses recent advancements in plant cell culture processing technology, focusing on
progress towards overcoming the problems associated with commercialization of these pro-
duction systems and highlighting recent commercial successes.

Introduction growth hormones and can be adapted to reactor systems (Giri

In 1902, Gottlieb Haberlandt first attempted to initiate plant cell
cultures (Haberlandt, 1902). Although he was unable to induce
cell division, he is recognized as the founder of plant cell culture
science (Georgiev et al., 2009). Plant cell cultures can now be
created from virtually any plant through the isolation of plant
tissue (Figure 1). Explants (i.e. isolated plant tissue) are sterilized
using an exogenous chemical treatment and plated on solid
growth media that is formulated to contain the growth hor-
mones and nutrients necessary for each species (Hall, 2000).
With correct media composition, explants proliferate into a cal-
lus of dedifferentiated cells, which can be screened for prod-
uct(s) of interest and then isolated and transferred to liquid
medium to create suspension cultures. Differentiated organ cul-
tures of roots, shoots and embryos can also be created and
maintained in vitro. Organ cultures often produce similar con-
centrations of secondary metabolites to the full plant but are
limited by the prohibitive expense of large-scale culture (Ver-
poorte et al., 2002). Hairy root cultures are generated through
infection of plant material with Agrobacterium rhizogenes.
Whereas regular root cultures grow slowly and require exoge-
nous phytohormone supply, hairy root cultures can stably pro-
duce high levels of secondary metabolites, do not require
and Narasu, 2000; Srivastava and Srivastava, 2007).
Plant cell culture was initially considered a potential option
for the production of high-value secondary metabolites, which
is limited commercially owing to environmentally unsustainable
natural harvest and complex chemical synthesis (Roberts, 2007).
Recently, commercial plant cell culture has been extended to
the production of heterologous proteins. Plant systems are ben-
eficial for protein production because they allow for proper pro-
tein assembly, folding and glycosylation, without the threat of
contaminants such as endotoxins, pathogens and oncogenic
DNA associated with microbial and mammalian cell culture (Ma
et al., 1995; Commandeur et al., 2003; Twyman et al., 2003).
Plant cell culture also allows for isolation of genetically modified
plants from the environment, eliminating risk of transgene
migration (Fox, 2006). Conditions are more easily controlled
compared to whole plant systems, allowing for more consistent
yields (James and Lee, 2001), and secretion of proteins to the
culture media can significantly decrease downstream processing
costs (Shih and Doran, 2009). The production of both second-
ary metabolites and proteins via plant cell culture is limited by
low product yields and unpredictable scale-up. Additionally, sec-
ondary metabolite accumulation over time within a single cell
line and among cultures of the same cell line is often variable,

ª 2011 The Authors

Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd 249
250 Sarah A. Wilson and Susan C. Roberts
(a)
(c)
which is likely related to cellular heterogeneity (Kolewe et al.,
2008). Despite these limitations, several secondary metabolites
and heterologous proteins have been successfully produced on
a commercial scale using plant cell culture (Table 1).
Plant secondary metabolites and production
routes
Plant secondary metabolites are low-molecular weight com-
pounds that aid in the adaptation of plants to their environ-
ment but do not directly affect growth and development.
Because of their significant biological activity, plant secondary
metabolites have been used in traditional medicine for centu-
ries. Currently, over 60% of anticancer drugs and 75% of
drugs for infectious disease are either natural products or ana-
logues of natural products (Newman et al., 2003; Cragg and
Newman, 2009). Secondary metabolites are also commonly
used as insecticides, dyes, flavours and fragrances (Srivastava
and Srivastava, 2007).
Natural harvest
One of the main problems associated with the commercial sup-
ply of secondary metabolites is limited compound availability.
Secondary metabolites typically represent <1% dry weight of
the plant, so natural harvest is often impractical (Georgiev
et al., 2009). For instance, 340 000 kg of Taxus bark or 38 000
trees were required to meet the 25 kg per year demand for the
anticancer drug paclitaxel (Taxol; Bristol-Myers Squibb, New
York, NY) (Cragg et al., 1993). Harvesting is also limited by sea-
sonal availability, species abundance and plant growth rate
(Roberts, 2007). Despite these challenges, several compounds
continue to be harvested from their native plant owing to lack
of better commercialization options (Table 2). As an alternative
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
(b)
(d)
Figure 1 Development of a plant cell suspension
culture. Sterile explants from the whole plant (a)
are plated on solid culture medium. With the
correct nutrients and hormones combination,
explants grow into a callus of dedifferentiated
cells (b). Callus cells are transplanted into liquid
media, creating a suspension culture (c), which
can be scaled up for growth and production in a
controlled bioreactor (d). To avoid contamination,
aseptic technique must be used to maintain the
cell cultures, resulting in increased manufacturing
costs.
to natural harvest, secondary metabolites can also be synthe-
sized and supplied through three general approaches: total or
partial chemical synthesis, heterologous expression of the bio-
synthetic pathway in other organisms and in situ production via
plant cell culture.
Chemical synthesis
Many plant secondary metabolites are produced economically
through total chemical synthesis. Vanillin is the most popular
flavour compound, but <1% of the annual demand is met
through extraction from Vanilla planifolia. Because of the lim-
ited availability and cost of the natural compound, it is most
commonly chemically synthesized from the substrates guaiacol,
eugenol or lignin (Schwab et al., 2008). One limiting factor for
chemical synthesis of many secondary metabolites is their large
size and presence of multiple chiral centres (Figure 2). For
instance, morphinan alkaloids (e.g. morphine and codeine) have
five centres of chirality, so the complex chemical synthesis route
is not cost efficient (Gerardy and Zenk, 1993). Semisynthesis of
the antimalarial compound artemisinin from both artemisinic
acid and arteannuin B has been achieved, but even though
artemisinic acid is present at concentrations 8–10 times higher
than artemisinin in Artemisia annua, these routes are still not
commercially feasible (Roth and Acton, 1989; Nowak and
Lansbury, 1998). Two different routes for the total synthesis of
paclitaxel were developed in 1994, but the processes involved
over 40 reactions, utilized harsh solvents, and had low product
yields, making them economically and environmentally unfa-
vourable (Holton et al., 1994a,b; Nicolaou et al., 1994). Com-
mercial paclitaxel was achieved through semisynthesis from two
paclitaxel precursors that could be extracted from the needles
of Taxus, which is discussed below in Success Stories: Phyton
Biotech, Inc. (Ahrensburg, Germany).
ª 2011 The Authors
 Development and commercialization of plant cell culture 251

Table 1 Products produced commercially via plant cell culture (Kolewe et al., 2008; Obembe et al., 2010)

Product Species Manufacturer Use ⁄ notes

Secondary metabolites
Anthocyanins Euphorbia milii Nippon Paint Co., Ltd., Osaka, Japan Textile dyes
Aralia cordata Colouring agents for fruit juices, wine and
other beverages
Arbtin Catharanthus roseus Mitsui Chemicals, Inc., Tokyo, Japan Pigment
Antiseptic
Berberines Coptis japonica Mitsui Chemicals, Inc. Anticancer
Thalictrum minus Antibiotic
Anti-inflammatory
Most commonly used for bacterial diarrhoea
and intestinal parasite and eye infections
Betacyanins Beta vulgaris Nippon Shinyaku Co., Ltd., Red to red-violet pigment
Kyoto, Japan Food colourant and dye
Carthamin Carthamus tinctorius Kibun Foods, Inc., Tokyo, Japan Red pigment
Food colourant and dye
Echinacea polysaccharides Echinacea purpurea Diversa, Ahrensburg, Germany Immunostimulant
Echinacea angustifolia Anti-inflammatory
Geraniol Geraminea spp. Mitsui Chemicals, Inc. Essential oil
Primary component of rose, palmarosa and
citronella oils
Ginseng Panax ginseng Nitto Denko Corporation, Osaka, Japan Dietary supplement
Wild ginseng stem cells Unhwa Biotech Corp., Jeonbuk, Korea Dietary supplement
Cosmetics
Paclitaxel Taxus spp. Phyton Biotech, Inc. Anticancer
Genexol – Samyang Genex, Seoul, FDA approved for the treatment of ovarian,
South Korea breast and lung cancers
Largest application of commercial plant cell culture
Podophyllotoxin Podophyllum spp. Nippon Oil, Tokyo, Japan Anticancer
Starting compound for the anticancer agents
etoposide and teniposide (Chattopadhyay
et al., 2002)
Rosmarinic acid Coleus blumei A. Nattermann & Cie. GmbH, Anti-inflammatory
Cologne, Germany
Scopolamine Duboisia spp. Sumitomo Chemical Co., Ltd., Anticholinergicum
Tokyo, Japan Antimuscarinic
Used in the treatment of motion sickness,
nausea and intestinal cramping
Shikonin Lithospermum erythrorhizon Mitsui Chemicals, Inc. Red pigment
Antibiotic
Heterologous proteins
Vaccines
HN protein of Newcastle Tobacco suspension cultures Dow AgroSciences Vaccine for Newcastle disease in poultry
disease virus LLC, Indianapolis, IN First FDA approved plant-derived vaccine
Currently not on market
Therapeutic proteins
Human glucocerebrosidase Carrot suspension cultures Protalix Biotherapeutics* Enzyme replacement therapy for Gaucher’s disease
(ULYPSO) Licensed by Pfizer, Inc.
Completed Phase III clinical trial in September 2009
Awaiting FDA approval
Recombinant a-galactosidase-A Carrot suspension cultures Protalix Biotherapeutics* Potential Fabry disease treatment
(PRX-102) In preclinical development
PEGylated recombinant human Carrot suspension cultures Protalix Biotherapeutics* Potential use in biodefence against
acetylcholinesterase (PRX-105) nerve agent attacks
Completed Phase I clinical trials
Anti-tumour necrosis factor Carrot suspension cultures Protalix Biotherapeutics* Potential therapeutic for autoimmune diseases,
(PRX-106) such as rheumatoid arthritis
In preclinical development

*http://www.protalix.com.

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
252 Sarah A. Wilson and Susan C. Roberts
Table 2 Examples of secondary metabolites commercialized through natural harvest
Use Compound ⁄ product
Anticancer agent Camptothecin
Vinblastine
Vincristine
Analgesic Morphine
Codeine
Malaria treatment Artemisinin
Insecticide Pyrethrins
Neem (Azadirachtin)
(a) (b) (c)
(d) (e)
Figure 2 Examples of the complex structures of commercially available
secondary metabolites. (a) Vanillin, a food flavouring derived from
Vanilla planifolia; (b) artemisinin, an antimalarial compound from Arte-
misia annua; (c) morphine, an analgesic from Papaver somniferum; (d)
camptothecin, an anticancer compound from Camptotheca acuminata;
and (e) paclitaxel, an anticancer compound from Taxus spp.
Heterologous production
Heterologous synthesis of plant secondary metabolites has been
investigated in bacteria, yeast and alternative plant species.
Engineered microbial species such as Escherichia coli and Sac-
charomyces cerevisiae are ideal because of their fast doubling
times compared to plant species (minutes vs. days), inexpensive
carbon sources, ease of genetic modification and well-estab-
lished scale-up technologies (Chang and Keasling, 2006; Chang
et al., 2007; Roberts, 2007). Microbial production routes could
overcome the inherent production variability associated with
plant suspension cultures, but synthesis of terpenes (e.g. arte-
misinin and paclitaxel) and other complex compounds through
transfer of complete pathways in E. coli is limited by the diffi-
culty of cytochrome P450 (CYP450) expression. CYP450s often
lose functionality in E. coli owing to improper folding, transla-
tion and insertion into the cell membrane, inefficient cofactor
pools and a lack of CYP450 reductases (Chang et al., 2007;
Ajikumar et al., 2010). For functional CYP450 expression, the
membrane anchor can be engineered to achieve proper mem-
brane translation or CYP450 chimeras can be created which
mimic proteins found in the native plant (Chemler and Koffas,
2008). This approach has been successfully employed for the
production of hydroxylated flavonoids (Leonard et al., 2006)
and isoflavones (Leonard and Koffas, 2007). The secondary
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Plant source Yield (% dry weight) References
Camptotheca acuminata 0.4–0.5 Lopezmeyer et al. (1994)
Catharanthus roseus 0.01 Ishikawa et al. (2009)
C. roseus 0.0003 Ishikawa et al. (2009)
Papaver somniferum 0.05–0.1 Odell et al. (2008)
P. somniferum 0.01–0.1 Odell et al. (2008)
Artemisia annua 1.5 Covello (2008)
Tanacetum cinerariifolium 1.8–2.5 Morris et al. (2006)
Azadirachta indica 0.1–0.9 Morgan (2009)
metabolic pathways for compounds such as paclitaxel have also
been introduced into other plant systems, such as Arabidopsis
(Besumbes et al., 2004), tomato (Kovacs et al., 2007) and
Physcomitrella (Anterola et al., 2009). The heterologous produc-
tion of plant secondary metabolites has recently been reviewed
in (Chemler and Koffas, 2008; Zhang et al., 2011). The main
limitation of heterologous production is the lack of fully charac-
terized secondary metabolic pathways. In addition to using
microbes for the production of natural products, analogues of
natural products have been created by introducing genes with
modified substrate specificity to plant-derived pathways that
can be expressed in microbes, such as the flavonoid (Katsuyama
et al., 2007; Werner et al., 2010) and carotenoid pathways
(Schmidt-Dannert et al., 2000). Similar studies have also been
conducted on the more complex alkaloid biosynthetic pathway
in Catharanthus roseus cell cultures (Runguphan and O’Connor,
2009; Runguphan et al., 2009).
Since 2000, biosynthetic vanillin has been produced and
commercialized through the microbial fermentation of ferulic
acid, with molar yields as high as 54.5% in Streptomyces sp.
(Hua et al., 2007) and 86.6% in E. coli (Lee et al., 2009). This
production route is more expensive than the chemical synthesis
of vanillin, but consumer value is increased because it can be
labelled as a natural flavouring (Schwab et al., 2008). To
decrease biosynthesis costs, vanillin production using glucose
($0.30 ⁄ kg) as a substrate, rather than ferulic acid ($5 ⁄ kg), is
being investigated. Vanillin was first produced from glucose
through the conversion of glucose to vanillic acid by heterolo-
gous E. coli, which was then converted to vanillin by aryl alde-
hyde dehydrogenase extracted from Neurospora crassa (Li and
Frost, 1998). Complete biosynthesis from glucose was achieved
in S. cerevisiae and Schizosaccharomyces pombe, reaching van-
illin concentrations of 45 and 65 mg ⁄ L, respectively (Hansen
et al., 2009). In silico design was used to engineer S. cerevisiae
for higher production levels, and experimental implementation
of the optimized strains resulted in a maximum production level
of 500 mg ⁄ L (Brochado et al., 2010).
Early genes in the paclitaxel biosynthetic pathway, which con-
tains 19 putative pathway steps from geranylgeranyl diphos-
phate (GGPP) (Croteau et al., 2006), have been introduced into
S. cerevisiae and E. coli. The introduction of GGPP synthase and
taxadiene synthase (TASY) into S. cerevisiae resulted in the
production of 1 mg ⁄ L of taxadiene (DeJong et al., 2006).
Additional pathway genes were introduced in this study, but
expression levels of the first CYP450 enzyme, taxadiene
5a-hydroxylase (T5aH), were low, resulting in only 25 lg ⁄ L of
the next intermediate, taxadiene-5a-ol. By eliminating the effect
of native metabolism on the heterologous biosynthetic pathway
ª 2011 The Authors
 Development and commercialization of plant cell culture 253

and expressing a codon-optimized TASY, taxadiene levels of
8.7 mg ⁄ L were ultimately achieved (Engels et al., 2008). In
E. coli, the two precursors to GGPP, isopentenyl pyrophosphate
(IPP) and dimethylallyl pyrophosphate (DMAPP) are synthesized
through the 1-deoxyxylulose-5-phosphate (DXP) pathway. To
increase the availability of GGPP, enzymes from the DXP path-
way were coexpressed with GGPP synthase and TASY in E. coli,
leading to production of 1.3 mg ⁄ L of taxadiene (Huang et al.,
2001). By balancing precursor supply with GGPP conversion to
maximize taxadiene production while minimizing inhibiting
effects of indole accumulation, a final level of 1.02 g ⁄ L of tax-
adiene was achieved (Ajikumar et al., 2010). Similar taxadiene
yields of 0.87 g ⁄ L were achieved using in silico analysis of the
DXP pathway in E. coli (Meng et al., 2011). Through codon
optimization, modification of the N-terminal transmembrane
region and the generation of chimera enzymes fused with a
Taxus CYP450 reductase, the expression of a Taxus CYP450 in
E. coli was recently achieved to allow for the 5a-oxidation of
taxadiene to taxadiene-5a-ol (Ajikumar et al., 2010). Expression
of the optimized CYP450 resulted in the production of nearly
60 mg ⁄ L taxadiene-5a-ol.
Plant cell culture
Although plant cell culture technology allows for sustainable
production of secondary metabolites, research is being directed
at overcoming problems associated with low product yields and
cell culture variability. Dedifferentiated cell cultures (i.e. callus or
suspension) often produce low levels of secondary metabolites
compared to differentiated cells (i.e. shoots or roots) (Lorence
and Nessler, 2004; Liu et al., 2006; Pasqua et al., 2006). Recent
work in our laboratory and others have shown that secondary
metabolite production can be unstable over long periods of
time in suspension cultures (Ketchum and Gibson, 1996; Kim
et al., 2004; Naill and Roberts, 2005b; Sanchez-Sampedro
et al., 2009), which may be correlated with variations in the cel-
lular ploidy level (Baebler et al., 2005). This variation in ploidy
has been linked to differences in gene expression, with higher
ploidy resulting in an increase in the number of silenced genes
(Mittelsten Scheid et al., 1996). Gene silencing within a second-
ary metabolic pathway could significantly affect metabolite
production.
Cell culture conditions can influence secondary metabolite
production in plant cell cultures. For example, large variations
in paclitaxel accumulation have been seen across cell cultures
that were derived from the same parent culture (Ketchum and
Gibson, 1996). High paclitaxel-producing cultures can exhibit
growth inhibition when subcultured, which was linked to accu-
mulation of paclitaxel and related taxanes in the media (Kim
et al., 2004; Expósito et al., 2009). The tendency of plant cells
to grow in aggregates can lead to the development of hetero-
geneous subpopulations in culture where cells differ metaboli-
cally and morphologically (Naill and Roberts, 2005a,b; Kolewe
et al., 2010). Individual cells within a culture accumulate vari-
able levels of a product. For instance, only 10% of cells in a
culture of C. roseus were found to produce the secondary
metabolite anthocyanin (Hall and Yeoman, 1987), and a broad
distribution of paclitaxel accumulation was observed in cul-
tured Taxus cells (Naill and Roberts, 2005b). The existence of
cell subpopulations necessitates the study of suspension cul-
tures at the single-cell level to fully understand and ultimately
engineer cultures for optimal performance (Kolewe et al.,
2008).
Whereas single cells are typically around 20–40 lm in diame-
ter, larger aggregates can be several millimetres in diameter.
Oxygen and nutrient diffusion limitations lead to the develop-
ment of microenvironments within a larger aggregate (Hulst
et al., 1989; Naill and Roberts, 2004). The average aggregate
size of a culture often varies over the culture period and can
affect secondary metabolite accumulation. The majority of
research aimed to link aggregate size to secondary metabolite
accumulation involves filtration and collection of different
aggregate size classes and subsequent product extraction from
biomass. No conclusive trends have been found which span all
plant species (Kolewe et al., 2010). A new strategy was used to
study the accumulation of secondary metabolites in cultures
with different sized aggregates that enabled consideration of
both cell- and media-associated accumulation (Kolewe et al.,
2011). Cultures composed of small aggregates of Taxus cuspi-
data cells were found to produce up to 20-fold more paclitaxel
after methyl jasmonate elicitation than cultures composed of
larger aggregates. The mechanism for the increase in paclitaxel
accumulation is currently under investigation.
Increasing yields of secondary metabolites
in plant cell culture
Elicitation
Elicitation is a traditional method used to increase secondary
metabolite accumulation in plant cell cultures (Bourgaud et al.,
2001). Many plant secondary metabolites are linked to plant
defence against pathogens and herbivores, so cellular metabo-
lism can be shifted to favour secondary metabolism through the
introduction of chemical or physical stresses (Pauwels et al.,
2009). Various environmental conditions (e.g. plant disease, UV
light, air quality) and chemical treatments (e.g. CuSO4, NaCl,
H2SO4) have been found to affect furanocoumarin content in a
variety of plant species (Bourgaud et al., 2001). Jasmonic acid
and its conjugates and precursors, collectively known as jasmo-
nates, are lipid-derived compounds involved in plant growth
and development (Wasternack, 2007). They are able to induce
a metabolic shift from growth to defence in many species,
allowing a plant to defend itself from a hostile environment
(Pauwels et al., 2009). Jasmonic acid and its methyl ester,
methyl jasmonate, were first shown to increase the accumula-
tion of secondary metabolites in suspension cultures of 36 dis-
tinct plant species (Gundlach et al., 1992), and exogenous
methyl jasmonate addition increased paclitaxel production up to
46-fold over unelicited cells (Mirjalili and Linden, 1996; Yuki-
mune et al., 1996; Ketchum et al., 1999). In Arabidopsis thali-
ana suspension cultures, methyl jasmonate was found to first
induce genes involved in transcriptional regulation and jasmonic
acid biosynthesis, followed by repression of cell cycle genes and
induction of genes leading to phenylpropanoid production
(Pauwels et al., 2008). Elicitation can also be used to under-
stand the regulation of secondary metabolic pathways through
differential gene expression studies of cultures that accumulate
low ⁄ no versus high levels of secondary metabolites.
In situ product removal
The accumulation of secondary metabolites can be limited by
both feedback inhibition of product synthesis and product
degradation (Wang et al., 2001). In situ product removal from
the culture media using two-phase solid–liquid or immiscible

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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
254 Sarah A. Wilson and Susan C. Roberts
liquid–liquid systems can overcome these limitations and
increase culture productivity, while simplifying product recovery
and allowing for semicontinuous operation (Yan et al., 2005).
Product adsorption using a nonaromatic resin led to a signifi-
cant increase in the production of trans-resveratrol and isotopi-
cally labelled trans-resveratrol in Vitis vinifera after elicitation
with either salicylic or jasmonic acid (Yue et al., 2011). Product
accumulation increased mainly as a result of introduction of the
adsorbent, rather than the elicitor, which suggested that the
rate-limiting step occurred after biosynthesis of trans-resveratrol.
The use of a hydrophobic macroporous resin in the culture of
Salvia miltiorrhiza hairy root cultures along with semicontinuous
operation resulted in tanshinone production 7.4 times higher
than batch culture productivity (Yan et al., 2005). Production
levels were only increased by adsorbent addition when the con-
centration of product in the media was high, suggesting that
production was limited by feedback inhibition at high concen-
trations. Two-phase systems can also help to simplify purifica-
tion of products from the media. For example, a sixfold
increase in paclitaxel production was achieved in Taxus chinen-
sis suspension cultures in aqueous-organic two-phase systems
with sucrose feeding, with 63% of the product released to the
media (Wang et al., 2001).
Immobilization
Immobilization of plant cells can be used to overcome some of
the problems associated with suspension cultures, such as low
and variable product yields, high susceptibility to shear and slow
growth rates, because of the potential for higher cell densities
and continuous product removal (Brodelius, 1985; Dornenburg
and Knorr, 1995). Cells have been successfully encapsulated in
calcium alginate, agar, agarose, gelatin, carrageenan, polyacryl-
amide, polyurethane foam and hollow fibre membranes, with
the most popular technique being encapsulation in an alginate
gel (Smetanska, 2008). Immobilization of Taxus baccata cells in
calcium alginate beads in a bioreactor resulted in total (i.e. cell-
associated and extracellular) paclitaxel yields of 43.43 mg ⁄ L
after 16 days, representing a production rate of 2.71 mg ⁄ L ⁄ day
(Bentebibel et al., 2005). These were the highest paclitaxel lev-
els reported to date achieved in a laboratory-scaled bioreactor.
Elicitation of these cells led to an increase in the production of
paclitaxel and its precursor, baccatin III, but the compounds
were maintained within the alginate beads (Bonfill et al., 2007),
which complicates purification. On the other hand, immobiliza-
tion of Solanum chrysotrichum cells in a similar matrix resulted
in an overall decrease in spirostanol saponin; however, there
was an increase in metabolite secretion to the extracellular
medium (Charlet et al., 2000), simplifying purification. In sys-
tems that secrete the secondary metabolite of interest, encapsu-
lation or immobilization can simplify downstream processing
and allow for continuous product removal.
Metabolic engineering
Both transient and stable transformation procedures can be
used to study the effect of the up-regulation or down-regula-
tion of biosynthetic pathway genes or transcription factors on
metabolite profiles or gene expression. Transient transformation
techniques such as particle bombardment, tissue electropora-
tion, DNA injection, agroinfiltration and viral expression systems
lead to higher gene expression levels and transformation effi-
ciencies in nearly all plant species over stable transformations
but only allow for short-term expression (Lessard et al., 2002;
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Komarova et al., 2010). Stable transformation techniques, such
as Agrobacterium-mediated transformation, are more time-con-
suming than transient techniques, but because genes are incor-
porated into the plant genome, expression can be maintained
over time (Newell, 2000). Although this transformation tech-
nique is ideal for metabolic engineering of plant cell cultures,
there are many industrially important species, such as soy
beans, cereal grains and tree species, that are recalcitrant to
Agrobacterium-mediated transformation (Gelvin, 2003b). As an
alternative, stable gene expression can be achieved through
direct gene transfer techniques such as particle bombardment,
silicon carbide fibres and electroporation. While transient gene
expression is achieved when DNA coated particles are trans-
ferred into the cell, stable gene expression requires integration
of the DNA into the genome, which is a much less frequent
event (Altpeter et al., 2005). As a result, a selection marker,
such as an antibiotic resistance, must be transformed into the
cells to allow for the isolation and scale-up of transformed cells
for multiple months post-transformation. Once the transgenic
line has been established, transgene expression, which can be
affected by the site of integration into the genome, gene silenc-
ing and the promoter utilized, must be studied to identify highly
expressing cell lines that maintain expression over time (Page
and Minocha, 2004). Efficient transformation protocols must
first be established for a plant species to enable metabolic engi-
neering studies. Both transient and stable transformation tech-
niques, including particle bombardment and Agrobacterium-
mediated transformation, have been developed for the Taxus
(Ketchum et al., 2007; Vongpaseuth et al., 2007) and Catha-
ranthus species (Goddijn et al., 1995; van der Fits and Meme-
link, 1997; Guirimand et al., 2009).
One major limitation to the metabolic engineering of plant
cells for increased secondary metabolite accumulation is the
lack of fully defined metabolic pathways. The rate-influencing
genes within a metabolic pathway must be identified to enable
targeted metabolic engineering approaches. Several techniques
are commonly used for the identification and characterization
of unknown metabolic pathway genes, including precursor
feeding, gene overexpression and inhibition, mutant selection
or differential gene expression studies using elicitation. Many of
the steps of the paclitaxel biosynthetic pathway have been elu-
cidated using these techniques. Precursor feeding led to identifi-
cation of the first committed step of the paclitaxel biosynthetic
pathway, the cyclization of GGPP to taxadiene by TASY (Koepp
et al., 1995). Using degenerate primers based on the sequences
of similar enzymes in other species, a hybridization probe was
identified and used to screen a cDNA library to obtain the full
sequence of TASY (Wildung and Croteau, 1996). The identified
gene sequence was expressed in E. coli and found to be catalyt-
ically active on GGPP. The use of differential gene expression
studies on elicited and unelicited cells allowed for discovery and
characterization of many of the genes involved in the 19 puta-
tive steps of paclitaxel biosynthesis, as reviewed in Croteau
et al. (2006); Vongpaseuth and Roberts (2007). In addition,
these studies identified putative genes that could be involved in
the undefined regions of the pathway. Transcript profiling of
Taxus suspension cultures using RNA gel blot analysis and
RT-PCR was used to identify potential rate-influencing steps in
paclitaxel biosynthesis (Nims et al., 2006). Recently, 454 pyrose-
quencing was used to create an expressed sequence tag library
from T. cuspidata needles (Wu et al., 2011). This represents the
largest EST collection for Taxus to date and could be used to
ª 2011 The Authors

further elucidate the metabolic pathway and identify potential
transcription factors involved in the regulation of taxane
production.
Transcription factors are regulatory proteins that have the
ability to control multiple genes within a metabolic pathway
through protein–protein interactions or the binding of specific
DNA sequences (Broun, 2004). They have been considered a
viable alternative to single-gene metabolic engineering because
they can be used to redirect metabolism by regulating the
expression of multiple genes within a biosynthetic pathway
(Gantet and Memelink, 2002; Grotewold, 2008). In addition,
transcription factor engineering does not require complete
knowledge of the biosynthetic pathway of interest, rendering
this approach particularly useful for complex secondary metabo-
lites with undefined biosynthetic pathways. The use of tran-
scription factors for plant metabolic engineering has been
thoroughly reviewed (Gantet and Memelink, 2002; Broun,
2004; Grotewold, 2008; Iwase et al., 2009). Up-regulation of
both G10H, a gene responsible for the production of the terpe-
noid moiety, and Orca3, a transcription factor involved in ter-
pene indole alkaloid (TIA) biosynthesis (van der Fits and
Memelink, 2001), in C. roseus hairy root cultures resulted in a
6.5-fold increase in production of the TIA catharanthine (Wang
et al., 2010). The engineering of both transcription factors and
rate-influencing genes within the TIA biosynthetic pathway
could lead to a significant increase in TIA accumulation in C. ro-
seus (Liu et al., 2007). By affecting the expression of multiple
genes within a metabolic pathway, transcription factor expres-
sion can often overcome overall pathway flux limitations associ-
ated with single-gene metabolic engineering (Martin, 1996).
Plants as a production system for
heterologous proteins
Biopharmaceuticals are proteins, antibodies or nucleic acid-
based products used in the treatment of disease (Walsh, 2005).
There are over 200 biopharmaceuticals on the market, 58 of
which have been approved in the last 4 years. Of the 58 prod-
ucts recently approved, 55% are produced using mammalian
cell culture, 29% using E. coli and the remaining 16% using
yeast (S. cerevisiae and Pichia pastoris) and nontraditional sys-
tems, such as transgenic animals or baculovirus-insect cells
(Walsh, 2010). Bacterial systems have very short production
times, and yields are typically 0.5–0.8 g ⁄ L; higher yields on the
order of multiple grams per litre have been reported with addi-
tional engineering and process control (Joly et al., 1998; Choi
et al., 2000; Chen et al., 2004). This system is limited by
contamination by bacterial toxins and inability to support post-
translational modifications, such as protein folding and glycosyl-
ation, which can lead to protein aggregation or loss of function
(Gomord and Faye, 2004). Yeast systems are able to overcome
some problems associated with post-translational modifications
and offer relatively high yields between 0.1 and 1.0 g ⁄ L (James
and Lee, 2001), but proteins can be hyperglycosylated, which
can influence protein folding and function (Hamilton and Gern-
gross, 2007). Mammalian cells are capable of proper post-trans-
lational modifications and glycosylation and offer the highest
production levels, between 1 and 3 g ⁄ L (Boehm, 2007). On the
other hand, commercialization of mammalian cell culture is
costly and complicated, owing to expensive media components,
difficult handling and the potential for viral and oncogenic
contamination (Boehm, 2007; Yin et al., 2007).
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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Development and commercialization of plant cell culture 255
Heterologous proteins were first introduced into plants just
over two decades ago (Hiatt et al., 1989; During et al., 1990;
Sijmons et al., 1990). Since this time, the use of whole plants
and plant cell culture as production systems has been widely
explored. Plants are able to avoid some of the problems associ-
ated with mammalian and microbe-based production systems.
For instance, plant systems do not produce endotoxins, patho-
gens or oncogenic DNA and are able to fold and assemble com-
plex proteins (Commandeur et al., 2003; Twyman et al., 2003).
This was demonstrated in tobacco with the expression of the
four protein monomers of secretory immunoglobulin A which
were assembled into the functional, high-molecular weight
complex (Ma et al., 1995). While heterologous plant-produced
proteins and native human proteins have similar post-transla-
tional modifications, some differences exist. Engineering strate-
gies have been applied to further humanize the glycosylation
patterns, enabling the production of fully functional proteins in
plants (Gomord and Faye, 2004; Schähs et al., 2007; Gomord
et al., 2010). Although whole plant farming of pharmaceutical
proteins has been utilized, there are benefits to the use of plant
cell culture as a production platform. The US Department of
Agriculture’s concern about transgene migration and contami-
nation of the human food chain has been a major limitation to
the use of field-grown crops, especially when food crops are
the system of choice (Fox, 2006; Murphy, 2007). Plant cell cul-
ture allows for the isolation of genetically modified plant cells,
reducing the risk of contaminating food sources. They can also
have more consistent yields under controlled conditions, elimi-
nating the threat of crop destruction and inconsistency owing
to unpredictable environmental conditions or pathogen contam-
ination (James and Lee, 2001). For the recovery of foreign pro-
tein from field-grown corn, downstream processing costs
represent 94% of the operating costs annually, with only 83%
product purity (Evangelista et al., 1998). Although the material
and operating costs for aseptic plant cell culture that meets
cGMP demands are significantly higher than those for agricul-
tural production, the secretion of proteins in vitro has the
potential to significantly decrease downstream processing costs
(Shih and Doran, 2009). Additionally, the use of bioreactor sys-
tems allows for the use of expression systems that utilize chemi-
cally inducible promoters or viral vectors that are limited in
field-grown crop applications. The use of plant cell culture for
the production of heterologous proteins is on the rise, with
multiple biopharmaceuticals in preclinical and clinical develop-
ment (Table 1). To increase the commercial feasibility of plant
cell culture production, research is being conducted to increase
protein yields, which typically vary from 0.005 to 200 mg ⁄ L,
through the development of optimized expression systems and
enhancement of protein stability after production (Hellwig
et al., 2004).
Increasing yields of heterologous proteins
in plant cell culture
Gene transformation strategies
Heterologous genes can be introduced into plants both tran-
siently and stably. For transient transformations, gene expres-
sion is typically maintained for 6–14 days, allowing for
screening of expression systems, small-scale production for
protein characterization and large-scale manufacturing (Vaquero
et al., 1999; Huang and McDonald, 2009; Komarova et al.,
256 Sarah A. Wilson and Susan C. Roberts
2010). Transient gene expression can be accomplished in plant
cell culture through the use of Agrobacterium infiltration or
viral vectors (Fischer et al., 1999; Gleba et al., 2007). This pro-
cedure produces protein yields that are comparable to those
found in the Agrobacterium infiltration of intact plant tissue
and is a better platform for scale-up under aseptic conditions,
where the culturing conditions can be more easily controlled
(Andrews and Curtis, 2005). Additionally, the Agrobacterium
strain containing the viral expression system can be more easily
contained in vitro compared to whole plants grown in an open
field (Shih and Doran, 2009). It is hypothesized that transient
transformations allow for high levels of gene expression prior to
the onset of post-translational gene suppression (PTGS). As a
result, some transient transformations have resulted in higher
protein yields than stable transformations (Wroblewski et al.,
2005). Agrobacterium infiltration was used to demonstrate
transient expression of an antibody directed against the carcino-
embryonic antigen before stably introducing the production sys-
tem into Nicotiana tabacum. Expression levels in the transiently
transformed cells were approximately five times higher than
those found in the stably transformed plant (Vaquero et al.,
1999, 2002). Stable transformations can be accomplished
through the use of Agrobacterium-mediated transformation
(Gelvin, 2003a), particle bombardment (Christou, 1992) or elec-
troporation (Joersbo and Brunstedt, 1991). Because these stably
transformed cells take months to years to establish, protein
expression studies often begin with transient expression. Once
an expression system is established, the genes of interest can
be integrated into the cells using a stable transformation tech-
nique to allow for expression over longer culture periods
(Vaquero et al., 1999). Stably transformed cell lines are benefi-
cial for larger-scale production of proteins but can be prone to
genetic instability, resulting in the loss of transgene expression
(Gao et al., 1991). As a result, seed stocks can be established
using cryopreservation to limit production variability among
batches (Van Eck and Keen, 2009).
Expression systems
Both constitutive and inducible promoter systems can be uti-
lized to regulate gene expression for heterologous protein syn-
thesis in plant cell culture systems. Constitutive promoters allow
for gene expression throughout the entire growth phase of the
cell culture. The cauliflower mosaic virus 35S (CaMV 35S) pro-
moter is one of the most commonly used because it allows for
high expression levels; however, viral promoters can result in
transgene-induced gene silencing (Odell et al., 1985; Vaucheret
et al., 1998). To minimize this potential problem, plant-derived
promoters, such as ubiquitin and actin, have been developed
(Lessard et al., 2002). If the protein product affects cell growth
or viability, an inducible promoter can be used to create an
expression platform where genes are expressed in response to
specific and timely chemical, metabolic or physical stimuli
(Huang and McDonald, 2009). For plant cell culture, chemically
induced promoters are commonly used because inducers such
as tetracycline, alcohol or sugars can be added directly to the
bioreactor (Fleißner and Dersch, 2010).
Viral vectors offer a platform for the high expression of het-
erologous proteins using transient agroinfiltration. In addition to
expressing wild-type genes, viral vectors express the gene of
interest under the control of a strong viral promoter (Gleba
et al., 2007). A summary of the viral vector expression systems
utilized in plants can be found in Shih and Doran (2009).
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Through the cocultivation of Nicotiana glutinosa suspension cul-
tures with Agrobacterium, a transient viral vector expression
system was successfully scaled up to a 51-L stirred tank bioreac-
tor with no reduction in protein production levels (M. O’Neill
et al., 2008). A cucumber mosaic virus inducible viral amplicon
(CMViva) was developed that, upon induction by estradiol,
amplifies production of the CMV coat protein, allowing for high
levels of gene expression at specific cell growth conditions
(Sudarshana et al., 2006). Heterologous protein production
using this system in the plant tissue of Nicotiana benthamiana
was 30 times higher than production using the CaMV 35S pro-
moter, with a 170-fold increase in functional protein levels. This
system also led to a fourfold increase in protein production lev-
els in N. benthamiana suspension cultures, with a twofold
increase in functional protein (Huang et al., 2009). Scale-up of
the transgenic N. benthamiana cultures to a 2-L semicontinuous
bioreactor resulted in a 25-fold increase in protein production
over batch systems (Huang et al., 2010). The protease activity
and phenolic concentration in the extracellular medium were
lower than in batch cultures and the system allowed for sus-
tained production levels and steady-state bioreactor operation.
Co-expression of viral gene silencing suppressors in the bioreac-
tor system could decrease PTGS and allow for even higher
productivity levels (Mallory et al., 2002; Voinnet et al., 2003).
Subcellular targeting and secretion
Proteolytic degradation within plant cells is believed to be an
important factor leading to low heterologous protein yields
(Benchabane et al., 2008). To minimize degradation, signalling
tags are used to target protein production to specific regions
within the cell (Fischer et al., 2004). For more information on
localization and targeting of proteins in plants, see Mackenzie
(2005). Subcellular targeting can enhance protein stability
owing to differences in the redox potential, pH, and the pres-
ence or absence of chaperones, glycosyltransferases and proteo-
lytic enzymes in different organelles (Doran, 2006a;
Benchabane et al., 2008). The endoplasmic reticulum (ER) is
often targeted because it is an oxidizing environment that pro-
motes the formation of disulphide bridges. In addition, there is
a high concentration of chaperones in the ER, which aid in
complex protein folding, and a low concentration of proteases
(Fischer et al., 2004), which result in decreased protein degra-
dation. Storage vacuoles also have lower levels of proteases
(Neuhaus and Rogers, 1998). The presence of enzyme activity
in the storage vacuole that leads to exposure of terminal man-
nose residues on complex glycans (Lerouge et al., 1998) was
exploited for the production of a functional recombinant
human glucocerebrosidase in carrot suspension cultures, as dis-
cussed in Success Stories: Protalix Biotherapeutics (Shaaltiel
et al., 2007).
One benefit of plant cell culture over whole plant systems is
that heterologous proteins can be secreted into the cell culture
medium to simplify purification. For secretion, a signal peptide
is used to target unfolded peptides to the ER for folding and
assembly. After folding, the protein is transported from the ER
through the secretory pathway, enters the Golgi apparatus and
is secreted from the cell (Kermode, 1996). Some of the benefits
to protein secretion are: (i) no need to destroy the cell, which
results in complicated purification strategies owing to release of
intracellular compounds to the media; (ii) cells can be easily
filtered from the liquid media; (iii) plant cells do not secrete
many proteins, so purification is simplified because there are
ª 2011 The Authors

few contaminating proteins; and (iv) plant cells can be reused
or maintained within the bioreactor, allowing for additional pro-
duction cycles (Fischer et al., 2004; Huang and McDonald,
2009). A major drawback to secretion is that proteins can be
degraded by proteolytic enzymes in the culture media (Doran,
2006a). In N. tabacum suspension cultures, functional heterolo-
gous protein was identified in samples containing both cells
and media, but not in media-only samples (Schiermeyer et al.,
2005). Endogenous proteases secreted to the medium degraded
the protein, and addition of a protease inhibitor or EDTA to the
media was found to reduce degradation. The conversion of cal-
lus cultures to suspension cultures resulted in the rapid loss of
hepatitis B surface antigen expression in Glycine max (Gana-
pathi et al., 2007). By adding protease inhibitors to the culture
media, product recovery was restored by 50% to 12.1 ng ⁄ g of
fresh weight.
Increasing secreted protein stability
Secreted proteins are also not always stable in the media envi-
ronment (Tsoi and Doran, 2002). To prevent degradation or
protein unfolding, stabilizing agents such as dimethyl sulfoxide,
polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, bovine
serum albumin or protease inhibitors can be added to the cul-
ture media (Franconi et al., 2010). In transgenic N. tabacum
suspension cultures, functional heterologous protein production
was found to correlate with cell growth until stationary phase,
where functional protein levels decreased significantly (Becerra-
Arteaga et al., 2006). Total extracellular protein in these sam-
ples was found to either increase or remain constant over time,
suggesting that loss of function was owing to denaturation
rather than proteolytic degradation. The addition of two known
protein stabilizers, PVP and bacitracin, led to a reduction in the
loss of activity in stationary phase (Becerra-Arteaga et al.,
2006). Surface adsorption of secreted proteins can decrease
protein levels in the media and be mitigated by coating glass
vessels with a protein-resistant polymer (Doran, 2006b). Effec-
tive monitoring and adjusting of culture conditions can lead to
increased yields of secreted proteins. An increase in the media
pH of N. benthamiana suspension cultures resulted in a fourfold
increase in recombinant protein production, with a twofold
increase in the ratio of functional to total protein (Huang et al.,
2009). At higher pH, protein stability was increased through a
decrease in proteolytic activity, resulting in maximal protein
recovery.
In situ protein removal has also been effective at increasing
functional protein recovery. Aqueous two-phase systems (ATPS)
are produced by mixing two water-soluble polymers in water.
Nutrients, metabolites, proteins, cell particles and whole cells
can be effectively partitioned between the two phases based
on size, protein confirmation (folded vs. denatured) and the
charge and ionic composition of the molecule (Albertsson,
1970; Hooker and Lee, 1990). Extractive fermentation using
ATPS has been used successfully for a diverse range of com-
pounds, from low-molecular weight products to high-molecular
weight enzymes (Banik et al., 2003). The use of ATPS with
plant cell culture was first demonstrated using suspension cul-
tures of N. tabacum in a 3% PEG and 5% crude dextran sys-
tem (Hooker and Lee, 1990). Culture growth rates and cell
densities were comparable to those measured in standard sus-
pension cultures. Enzyme production of N. tabacum in ATPS
cultures was explored using a 4% polyethylene glycol and
7.5% dextran system (Ilieva et al., 1996). Despite the reduced
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Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Development and commercialization of plant cell culture 257
growth rate observed, the extracellular acid and alkaline phos-
phomonoesterase yields were increased by 18- and 10-fold,
respectively, although the overall enzyme yield was comparable
to the control system.
Protein-binding resins can also be used to remove proteins
in situ. A hydroxyapatite resin leads to a 20%–21% increase in
the recovery of functional monoclonal antibody from hairy root
and suspension cultures of N. tabacum (Sharp and Doran,
2001). Protein production was increased through more frequent
harvestation of the resin from the media. An affinity chroma-
tography bioreactor circulates media from a bioreactor through
a column to remove secreted proteins during cell growth. This
system was used with protein G and iminodiacetic acid metal
affinity resin for suspension cultures of N. tabacum (James
et al., 2002). Production of the heavy chain of a mouse mono-
clonal antibody was increased by eightfold, while a twofold
increase was observed in the production of a HIS-tagged
human granulocyte macrophage colony-stimulating factor.
These increases were attributed to the removal of proteins from
the degrading media and prevention of product inhibition
pathways.
Industrial production using plant cell culture
For plant cell culture, the transition from shake flask to bioreac-
tor is complicated. As a result, a wide variety of bioprocessing
strategies have been specifically designed for large-scale cultiva-
tion of plant cells. Bioreactors for plant suspension cultures (Eibl
and Eibl, 2008; Georgiev et al., 2009; Huang and McDonald,
2009) and hairy root cultures (Kim et al., 2002b; Georgiev
et al., 2007; Srivastava and Srivastava, 2007) have been recently
reviewed. This section will focus on technologies used for com-
mercial bioprocessing of plant cell culture.
Bioreactor design and operation
Plant cells (20–50 lm in diameter and 100–500 lm in length)
are significantly larger than bacterial (<1 lm in diameter), fun-
gal (5–10 lm in diameter and <100 lm in length) and mamma-
lian cells (10–100 lm in diameter), with intracellular vacuoles
that account for up to 90% of cell volume (Huang and McDon-
ald, 2009). As a result, plant cell suspensions are subject to
shear sensitivity, which limits the mechanical agitation tech-
niques available to meet oxygen demands for cell growth.
Oxygen transport in plant cell bioreactors has been modelled,
accounting for the transfer of oxygen through the gas, liquid
and solid phases (Curtis and Tuerk, 2006). Although cell culture
medium typically behaves as a Newtonian fluid, excretion of
extracellular polysaccharides can significantly increase medium
viscosity (Georgiev et al., 2009). Culture broth rheology can also
be affected by aggregate size distribution, cell morphology, bio-
mass concentration and culture conditions (Huang and McDon-
ald, 2009). Changes in these culture properties can lead to
diffusion and mixing problems, so reactor conditions (e.g. agita-
tion, aeration, temperature, pH, etc.) must be optimized to
maximize cell growth. Azadirachta indica was grown in a 3-L
stirred tank bioreactor with both a setric and centrifugal impel-
ler (Prakash and Srivastava, 2007). Owing to a decrease in shear
and increase in oxygen transfer, biomass accumulation (15.5 vs.
18.7 g dry weight per L) and azadirachtin production (0.05 vs.
0.071 g ⁄ L) were increased when using the centrifugal impeller.
Response surface methodology was used to optimize elicitor
treatment of Azadirachta indica cells, and azadirachtin yields
258 Sarah A. Wilson and Susan C. Roberts

were increased to 0.16 g ⁄ L after treatment with a combination
of salicylic acid, jasmonic acid and chitosan (Prakash and Srivast-
ava, 2008). A variety of bioreactors have been designed specifi-
cally for the culture of plant cell suspension cultures and are
summarized in Table 3.
The use of hairy root cultures in bioreactors adds additional
complexities for bioprocess design. The branching of hairy roots
creates a tight matrix within a culture, leading to nutrient trans-
port limitations that result in areas of senescent cells (Srivastava
and Srivastava, 2007). These nutrient gradients also attribute to
variability in root growth and productivity (Eibl and Eibl, 2008).
Hairy roots are sensitive to shear stress, and mechanical agita-
tion can result in root wounding (Srivastava and Srivastava,
2007). Three types of bioreactors are typically used for the culti-
vation of hairy root cultures: (i) liquid-phase reactors, where
roots are submerged in liquid media; (ii) gas-phase reactors,
where media are either sprayed onto the roots or delivered as a
mist; and (iii) hybrid reactors (Kim et al., 2002a). Hybrid reactors
combine the properties of both liquid- and gas-phase reactors,
allowing an initial growth phase to occur in a liquid reactor
before transitioning to a gas-phase system (Srivastava and Sri-
vastava, 2007). The reactor type can significantly affect culture
growth and product accumulation. Growth and productivity of
a mammalian immunomodulator, interleukin 12, in N. tabacum
hairy roots were compared in shake flask, air-lift and mist biore-
actors (Liu et al., 2009). The highest root quality was found in
the shake flask and mist bioreactors. Sections of dark roots
were found in the air-lift reactor, which suggests that roots
were nutrient starved. The highest protein levels were found in
the mist bioreactor at 5.3 lg ⁄ g DW, which was 49.5% higher
than that produced in the air-lift bioreactor. Arachis hypogaea
hairy root biomass production was examined in different scales
of mist bioreactors utilizing disposable culture bags (Sivakumar
et al., 2010). Production levels of 12.75 g dry weight per litre

Table 3 Bioreactors designed for plant cell suspension cultures (Huang and McDonald, 2009)

Bioreactor types Features ⁄ advantages Disadvantages

Stirred tank bioreactor
Pneumatic bioreactor:
bubble column
Pneumatic bioreactor:
air-lift and modified air-lift
Membrane bioreactor
Wave bioreactor
Commonly used for all cell types High shear stress around the impeller
Easy to scale up High capital and operational costs
Useful for high-viscosity cell culture Heat generated from mechanical mixing
Able to achieve high oxygen transfer High energy cost owing to mechanical agitation
Good fluid mixing Contamination risk with mechanical seal
Alternative impellers available
Ease of compliance with cGMP requirements
Suitable for plant and animal cells Poor oxygen transfer capabilities
Easy to construct and scale up Poor fluid mixing in highly viscous cultures
Low operational cost High levels of foaming under high-aeration conditions
Low contamination risk
Low shear stress
No heat generation owing to lack of mechanical agitation
Suitable for plant and animal cells Relatively poor oxygen transfer capabilities
Easy to construct and scale up Poor fluid mixing for highly viscous cultures
Low operational cost High levels of foaming under high-aeration conditions
Low contamination risk
Low shear stress
No heat generation due to lack of mechanical agitation
Multiple choices of internal draft tubes
Better oxygen transfer than bubble column
Circulating flow patterns to aid in gas and nutrient transfer
Disposable equipment Difficult to scale up
Ability to concentrate biomass and protein product in Oxygenation required
membrane compartment Low heat transfer rate
Easy to withdraw extracellular product Difficult for online monitoring of culture conditions
Low shear stress Increased cost owing to disposability
Low operational cost
Simplified media exchange
Disposable equipment Difficult to scale up
Low shear stress Difficult to apply advanced cell culture
High oxygen mass transfer operational strategies
Useful for high cell density culture Low heat transfer rate
Low operational cost
Reduce cleaning, in-house sterilization requirements
Increased operational flexibility—batch, semi-batch,
perfusion configurations
Light weight

ª 2011 The Authors

Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268

were comparable to those in shake flask cultures (11.10 g dry
weight per litre), but scale-up to a 20-L reactor led to a
decrease in biomass accumulation to 7.77 g dry weight per
litre. The main factors affecting biomass accumulation upon
scale-up were flow rate to the nozzle, timing of flow rate
changes as biomass increased and an increase in the frequency
of misting.
The use of disposable bioreactors for plant cell culture is on
the rise and has been successfully implemented by Protalix Bio-
therapeutics with their ProCellEx production platform (see
Success Stories: Protalix Biotherapeutics). Benefits include high
flexibility, ease of handling, reduction in cross-contamination
and savings in both time and cost (Lim and Sinclair, 2007;
Mauter, 2009), which can be attributed to the presterility of
the container in which the cells are cultured (Eibl et al., 2010).
Many different classes of disposable bioreactors have been
designed for plant cell culture, including wave-mixed, stirred
and bubble columns, as reviewed in Eibl et al. (2009). Suspen-
sion cultures of transgenic N. tabacum produced three times
more human anti-rabies monoclonal antibody than the whole
plant system (Girard et al., 2006). Biomass and protein produc-
tion were comparable to shake flask production levels when
scaled up to 10 L in a plastic bag disposable bioreactor, demon-
strating that these inexpensive, disposable reactors are suitable
for promoting both biomass and heterologous protein synthesis.
The presterility of disposable bioreactors enables compliance
with strict good manufacturing practice standards associated
with bioreactor systems, and therefore these systems have the
potential to reduce manufacturing costs.
Bioreactor design depends on whether product formation is
growth- or non-growth-associated and where the product is
stored, either within the cell or secreted extracellularly. If a
metabolite is produced during exponential growth phase, one
reactor is usually suitable for growth and product recovery.
When the product is synthesized after cell growth, one reactor
is often used during exponential growth to increase cell num-
ber, while another reactor is used for metabolite production.
For products maintained within the cell, the reactor is usually
run in batch mode so that the cells can be permeabilized to
release the product after the run is completed. If the product is
secreted to the media, a continuous reactor can be used for
longer processing times and product can be removed as it is
synthesized (Bourgaud et al., 2001). A perfusion reactor was
used to culture transgenic Anchusa officinales suspension
cultures producing secreted acid phosphatase over a 4-week
period, with yields reaching 490 units ⁄ L ⁄ day, as opposed to
100–150 units ⁄ L ⁄ day for batch cultures (Su and Arias, 2003).
Batch or semi-batch production systems are used for the com-
mercialized products highlighted in this review, but coupling
protein or secondary metabolite secretion with semicontinuous
or continuous reactors could decrease the cost of both reactor
operations and downstream processing.
Product recovery
Secondary metabolite recovery and purification
Many secondary metabolites are hydrophilic, so they are primar-
ily stored in the cell vacuole; however, hydrophobic molecules
are often stored in the cell membrane, vesicles, dead cells and
extracellular sites, such as the cell wall (Guern et al., 1987). Pac-
litaxel accumulates within the cell wall (Choi et al., 2001), with
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Development and commercialization of plant cell culture 259
as little as 7%–10% of the product released to the extracellular
medium (Wickremesinhe and Arteca, 1994; Pestchanker et al.,
1996). The use of cell wall-digesting enzymes resulted in the
recovery of up to 90% of total paclitaxel in the extracellular
medium (Roberts et al., 2003). Cell walls can be ruptured using
homogenization, sonication, cell wall-digesting enzymes or
steam explosion, but all biomass is destroyed. To maintain cell
viability and allow for further use of biomass, cells can be per-
meabilized using pH shock (Thimmaraju et al., 2003) or chemi-
cal treatments (Brodelius, 1988; Park and Martinez, 1992). If
the product is secreted to the medium, cell rupture can be
avoided and biomass can be separated from the medium prior
to downstream processing. Promoting secretion by utilizing
ATPS, media exchange and resin adsorbents is beneficial for
product purification because unwanted by-products are
minimized.
Adsorption, precipitation, distillation, membrane separation
and extraction are examples of different techniques used for ini-
tial recovery of secondary metabolites from plant cell culture.
There are complications associated with each separation tech-
nique, such as protein fouling on membranes and low selectiv-
ity of extraction procedures owing to complex media
compositions (Schügerl, 2005). Extraction is the most common
technique used for this first stage of product purification. The
initial extraction of the product from biomass is typically
achieved through liquid–liquid extraction or ATPS (Romanik
et al., 2007). The extraction solvent must be chosen carefully
based on the physicochemical properties of the product of
interest (Georgiev et al., 2009). Solvent properties such as pH
and polarity affect product stability and separation efficiency. In
addition, interactions between the solvent and molecule of
interest sometimes lead to undesired structural changes in the
product (Maltese et al., 2009).
Following initial recovery, chromatography is typically per-
formed, and although this technique can be expensive and
complicated, it is rarely avoided (Georgiev et al., 2009). Coun-
tercurrent chromatography is commonly used for preparative
chromatography for all scales of product purification and has
been recently reviewed in Pauli et al. (2008). Because the liquid
and stationary phases compose a biphasic solvent mixture, there
is no irreversible adsorption onto either of the phases. The com-
position of the phases is nearly unlimited, allowing for high
product selectivities. The stationary phase has a loading capacity
that is only limited by the solubility of the product, and the
overall solvent consumption is lower than solid-phase chroma-
tography (Georgiev et al., 2009). High-speed countercurrent
chromatography is used for the preparative separation of both
natural and synthetic products and can purify multiple grams of
product in several hours (Ito, 2005). For the purification of
some compounds, such as anthocyanins, isoflavones and flava-
nols, countercurrent chromatography can be followed by high-
performance liquid chromatography to increase product purity
(Valls et al., 2009).
Protein recovery and purification
Downstream processing can account for 80% of total produc-
tion costs for heterologous proteins expressed in plant cell sys-
tems (Abranches et al., 2005). As a result, the protein
extraction and purification techniques must be optimized to
maximize protein recovery while minimizing processing
expenses, as recently reviewed (Wilken and Nikolov, 2011).
260 Sarah A. Wilson and Susan C. Roberts

During purification, there are benefits and disadvantages to et al., 2004). Chloroplast production of a HIS-tagged protein in
secreted verses intracellular proteins. Secreted proteins require tobacco allowed for one-step chromatography with >85%
no cell disruption, which minimizes the presence of contaminat- recovery (Leelavathi et al., 2003). By co-expressing an antibody
ing proteins, secondary metabolites and cell debris. On the with a fusion protein containing both an antibody-binding site
other hand, the product is dilute and prone to degradation and and a cellulose-binding domain, the product antibody was able
instability in the bulk cell culture medium. The first step in to be captured on cellulose beads (Hussack et al., 2010). Pro-
downstream processing is removal of bulk cell mass from the tein A, Protein G or Protein L chromatography can also be used
medium, typically using a decanter, disc-stack separator or for recovery of plant-derived antibodies, but these resins can be
semicontinuous or continuous centrifuge (Hellwig et al., 2004). expensive, and there is a risk of leaking of the proteinaceous
Intracellular proteins reduce the volume of material being pro- ligands, which can elicit an immune response in humans (Ter-
cessed downstream, resulting in higher product concentrations; man and Bertram, 1985; Platis and Labrou, 2006, 2008). Finally,
however, the feedstock is more complex, and there is a higher membrane systems such as ultrafiltration, microfiltration, perva-
concentration of proteases and oxidizing substances. For these poration and pertraction have also been considered for purifica-
proteins, the first processing step is to isolate the cell biomass tion of plant-derived protein products (Xu et al., 2011).
and disrupt cells using wet milling, sonication, pressure homog-
enization or enzymatic treatment (Xu et al., 2011). This process
Success stories
is nearly identical to the extraction required for the whole plant
(Hellwig et al., 2004). The optimal purification process for intra- Phyton Biotech, Inc.
cellular proteins is dependent upon the localization of the pro-
To explore the medicinal properties of natural products, the
tein, as demonstrated for protein targeted to the apoplast,
U.S. Department of Agriculture led the collection and screening
plasma membrane and ER in tobacco leaf tissue (Hassan et al.,
of plants for the discovery and development of plant-derived
2008).
anticancer agents between 1960 and 1982 (Cragg et al.,
After the initial step, downstream processing is the same for
1996). Approximately 35 000 plants were tested throughout
both intracellular and extracellular proteins, beginning with clar-
the United States and Mexico, and a number of anticancer
ification, which is typically accomplished using dead-end or
compounds, such as paclitaxel and camptothecin, were discov-
cross-flow filtration (Hellwig et al., 2004). As an alternative,
ered (Cragg et al., 1993). The extract of the bark of the Pacific
expanded bed adsorption (EBA) can be used to concentrate the
yew tree, Taxus brevifolia, was first isolated in 1963 and
protein of interest without having to perform upstream clarifica-
showed anticancer activity (Mountford, 2010). In 1964, the
tion (Bai and Glatz, 2003). EBA was used to achieve 60%
active ingredient was isolated, and the structure of Taxol (Bris-
recovery, with 90% purity of a recombinant protein from N. ta-
tol-Myers Squibb; generic name paclitaxel) was first published in
bacum, but clarification could not be avoided owing to an
1971 (Wani et al., 1971). Further investigation into the efficacy
interaction between the particulates and adsorbent (Valdes
of paclitaxel was not resumed until 1978 owing to limited solu-
et al., 2003). In contrast, EBA was successfully able to recover
bility in water and low yield (0.014%) from the bark of the yew
72% of a 34-fold purified recombinant protein from canola
tree (Kingston, 2000). In 1979, Horwitz determined that paclit-
with no need for clarification. The expanded bed diethylamino-
axel binds to cell microtubules, promoting microtubule assembly
ethyl (DEAE) resin was found to have similar binding and elution
into bundles and preventing mitosis (Manfredi et al., 1982). This
properties to the packed bed DEAE resin (Bai and Glatz, 2003).
discovery increased interest in the compound, and Phase I clini-
ATPS can be used during this primary protein recovery step to
cal trials began in 1983 (Cragg et al., 1993). Paclitaxel was
enrich the protein content while removing cell debris and other
approved for marketing as an anticancer agent in 1992 (Bristol-
contaminates (Aguilar and Rito-Palomares, 2010). An ATPS was
Myers Squibb), and with $1 billion dollars in commercial sales in
used to purify a recombinant protein from alfalfa, with 88% of
1998, it became the best-selling anticancer drug in history
the protein recovered in the top phase and 94% of the
(Kingston, 2000; McChesney et al., 2007). Used in the treat-
contaminant proteins at the interface or in the bottom phase
ment of ovarian, breast and lung cancers as well as AIDS-
(Ibarra-Herrera et al., 2011).
related Kaposi’s sarcoma, paclitaxel usage continues to increase
ATPS, along with chromatography and membrane filtration,
dramatically from the initial 25 kg per year required to treat
are often used for protein purification. When compared to ion-
ovarian cancer in the United States (Cragg et al., 1993;
exchange chromatography, ATPS led to a 37% decrease in puri-
Vongpaseuth et al., 2007). Demand could potentially exceed
fication costs, with 97% of a penicillin acylase recovered in a
200–300 kg per year as applications are being developed for
PEG-rich phase (Aguilar et al., 2006). Other chromatographic
paclitaxel in the treatment of Alzheimer’s and post-heart
methods include affinity chromatography, hydrophobic interac-
surgery patients (Cragg et al., 1993; Nims et al., 2006).
tion chromatography and size-exclusion chromatography (Xu
Initially, paclitaxel demand was met by harvesting bark of
et al., 2011). Utilizing four different immobilized affinity chro-
T. brevifolia. Because of the low paclitaxel content in the bark,
matography resins (histamine, tryptamine, tyramine and phenyl-
340 000 kg of Taxus bark or 38 000 trees were required to
amine), two therapeutic antibodies and a protease were
extract the desired 25 kg of drug per year (Cragg et al., 1993).
successfully purified from both tobacco and maize extracts,
Taxus trees are found in North America, Europe and Asia, but
showing that one binding resin is capable of isolating multiple
never in very high abundance, so availability is limited (Patel,
proteins (Platis and Labrou, 2008). For all resins, the maize
1998). Harvesting is also dependent upon seasonal variability
extract was purified more than the tobacco, possibly owing to
and the slow growth rate of the tree, making it nearly impossi-
the presence of phenolic substances binding to the column
ble to keep up with demands (Roberts, 2007). To prevent the
matrix. Affinity tags, such as glutathione S-transferases, malt-
extinction of Taxus species and decrease processing costs,
ose-binding proteins and polyhistidine, can be used to simplify
alternative paclitaxel production methods were investigated.
chromatographic methods and increase product purity (Fischer

ª 2011 The Authors

Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268

Paclitaxel total synthesis has been accomplished, but reaction
schemes are complex with low yields (Holton et al., 1994a,b;
Nicolaou et al., 1994; Wender et al., 1997a,b). As a result, total
synthesis is economically and environmentally unfavourable. The
European yew tree, T. baccata, contains about 0.1% of two
precursors (baccatin III and 10-deacetylbaccatin III), which can
be synthetically converted into paclitaxel (Denis et al., 1988;
Patel, 1998; Wuts, 1998). Initially, semisynthesis yields were
approximately 50%, but a commercially viable semisynthesis
route was patented in 1992 (Holton, 1992). This process contin-
ued to depend upon seasonal availability but did not require
destruction of trees for harvesting (Heinig and Jennewein,
2009). With this process, paclitaxel synthesis became commer-
cially viable and Taxol went on the market in 1993 (Mount-
ford, 2010).
Semisynthesis required 11 chemical transformations using 13
solvents and 13 organic reagents, making it costly and harmful
to the environment (Vongpaseuth and Roberts, 2007). In 2002,
Bristol-Myers Squibb discontinued the use of the semisynthetic
production route, switching entirely to a plant cell culture fer-
mentation process for both environmental health and safety
reasons and a financial incentive for a green pharmaceutical
process (Mountford, 2010). This production system, developed
by Phyton Biotech, Inc., is the largest commercial application of
plant cell culture, utilizing the Chinese yew (T. chinensis) culti-
vated in 75 000-L bioreactors (Huang and McDonald, 2009)
and has recently been described in detail by Mountford (2010).
Taxus cells that have been cryogenically frozen in a production
cell bank are used to start each fermentation batch. These cells
are placed on solid culture media to form a callus, before being
transplanted into liquid medium for scale-up. Once the biomass
has been accumulated, cells are fed a production medium to
induce paclitaxel synthesis. Phyton Biotech, Inc. has quantified
paclitaxel production after elicitation by many compounds,
including jasmonic acid, methyl jasmonate, silver thiosulfate and
3,4-methylenedioxy-6-nitrocinnamic acid, an inhibitor of the IPP
pathway (Bringi et al., 1995). Paclitaxel is purified from the
medium using liquid–liquid extraction and chromatography, fol-
lowed by crystallization of the pure product (Mountford, 2010).
Whereas the semisynthetic production route decreased costs to
25% of that for natural harvest, the plant cell fermentation
process reduced costs to just 20% of that for natural harvest
(Mountford, 2010). Bristol-Myers Squibb was awarded a Presi-
dential Green Chemistry Challenge Award by the U.S. Environ-
mental Protection Agency, acknowledging the development
and use of an environmentally friendly, sustainable manufactur-
ing technique for paclitaxel (Tabata, 2006). Production of paclit-
axel through this plant cell culture platform is currently
sustainable, but demand for paclitaxel usage in combination
with chemotherapy is continuing to rise, while additional appli-
cations are being developed in the treatment of Alzheimer’s
and post-heart surgery patients. For these reasons, research
efforts need to be directed towards the development of supe-
rior plant cell culture processes (e.g. using metabolic engineer-
ing or establishment of optimal cell lines) to increase culture
yields and decrease production costs.
Protalix Biotherapeutics
Gaucher’s disease is the most common lysosomal disease,
caused by decreased activity of the lysosomal enzyme acid
b-glucosidase (glucocerebrosidase; GCD), resulting in lysosomal
accumulation of glucosylceramide, the enzyme’s main substrate
ª 2011 The Authors
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
Development and commercialization of plant cell culture 261
(Grabowski, 2008). The disease can lead to enlargement of the
liver and spleen, anaemia, a decrease in blood platelets and
skeletal deterioration (Grabowski and Hopkin, 2003; Jmoudiak
and Futerman, 2005). Since 2005, the main treatment option
for patients with severe Gaucher’s disease is enzyme therapy,
but treatment can cost from $100 000 to >$200 000 per year
(Barton et al., 1991; Weinreb et al., 2002, 2004; Grabowski,
2008). In May of 1994, Cerezyme (Genzyme Corporation,
Cambridge, MA), a recombinant GCD expressed in mammalian
Chinese hamster ovary (CHO) cells, was approved for use in
patients with Gaucher’s disease (Jmoudiak and Futerman,
2005). Proper glycosylation of GCD is required for optimal
enzyme activity and targeting to macrophages. As a result,
functional GCD cannot be produced by E. coli, and deglycosy-
lated GCD from the human placenta is not active (Furbish et al.,
1981; Grace and Grabowski, 1990). The GCD produced from
CHO cells must be enzymatically remodelled to expose the
mannose residues required for macrophage uptake, which sig-
nificantly increases production costs (Friedman et al., 1999).
Protalix Biotherapeutics (Carmiel, Israel) developed a recom-
binant human GCD, taliglucerase alfa, produced by suspension
cultures of transgenic carrot cells (Shaaltiel et al., 2007). GCD
expression is controlled by the CaMV 35S promoter followed
by a tomato mosaic virus omega translation enhancer element.
Protein expression is targeted to the storage vacuole through
the ER using a signal peptide from A. thaliana and a storage
vacuole target from tobacco. Expression in carrot cells results
in a functional protein that does not require enzymatic modifi-
cation after production, and the glycosylation patterns are
highly reproducible from batch to batch (Shaaltiel et al., 2007;
Aviezer et al., 2009). The recombinant plant-derived GCD
(prGCD) was found to be structurally homologous to
Cerezyme with comparable enzymatic activity and uptake in
macrophages (Shaaltiel et al., 2007). In December 2009, Pfizer,
Inc. (New York, NY) purchased the worldwide rights to the
drug, excluding Israel, for $115 million. With this deal, Protalix
Biotherapeutics maintained control of manufacturing and 40%
of expenses, receiving 40% of revenues in return (Ratner,
2010). The drug completed phase III clinical trials in September
2009 and Protalix Biotherapeutics submitted a new drug appli-
cation. In the meantime, both the FDA and European Medi-
cines Agency requested supply of prGCD for select patients
under an expanded access programme (Ratner, 2010). At the
time of writing this review, the protein is still awaiting
approval after the FDA issued a Complete Response Letter
requesting additional information regarding testing specifica-
tions and assay development in February 2011. The FDA did
not request data from additional clinical studies and found the
Protalix Biotherapeutics manufacturing facilities acceptable
(http://www.protalix.com). Two therapies are currently on the
market for the treatment of Gaucher’s disease: Genzyme’s
Cerezyme and Shire’s velaglucerase alfa (Vpriv), which was
approved in 2010 (Opar, 2011).
According to the Protalix Biotherapeutics’ website, the prGCD
is produced using a patented ProCellEx production platform
(http://www.protalix.com). This platform utilizes presterilized,
flexible polyethylene containers that are >400 L in volume for
culturing and harvesting in consecutive cycles. Cultures are
provided with oxygen, nutrients, inoculants and culture media,
and excess air and waste gases are easily removed. The temper-
ature, lighting, air and nutrients used for operation have been
optimized to maximize cell productivity. This reactor system was
262 Sarah A. Wilson and Susan C. Roberts
designed specifically for plant cell culture, is rapidly scalable at a
low cost, requires minimal initial capital investment and is easy
to use. As a result, Protalix Biotherapeutics is currently working
on the development of several other plant-produced protein
therapeutics using the ProCellEx production platform, as seen in
Table 1. The company is also developing lyophilized plant cells
as a drug delivery vehicle for the prGCD protein. The cellulose
of the cell wall protects the enzyme from degradation in the
digestive tract, aiding in the delivery of the active enzyme into
the blood stream.
Future outlook for plant cell culture
commercialization
Several plant cell culture systems have been used for the indus-
trial production of secondary metabolites and heterologous pro-
teins, but a further understanding of plant cellular metabolism
and the development of new optimization strategies could dra-
matically affect the field and enable more widespread use of
the technology. A better knowledge of the inherent variability
and heterogeneity associated with plant cell cultures could
allow product yields to be maximized by adjusting process con-
ditions. A new technique has been developed to determine the
aggregate size distribution of plant cell suspension cultures
using a simple Coulter counter (Kolewe et al., 2010). This tech-
nique can be used to rapidly analyse the aggregate size distribu-
tion of a culture over time, which has been widely shown to
affect metabolite production. To fully understand cell culture
variability and heterogeneity, cultures can be studied on the
level of the individual cell. Typical cell culture studies focus on
culture-averaged parameters (e.g. fresh weight, dry weight, cul-
ture productivity, etc.), where samples containing millions of
cells are analysed together, and no information is collected
regarding cell–cell differences. Flow cytometric techniques using
isolated single particles (e.g. cells, protoplasts, nuclei, mitochon-
dria and chromosomes) can be used to analyse cell cycle partici-
pation, genome size, ploidy level and metabolite accumulation
of individual cells (Galbraith, 2004; Gaurav et al., 2010). For
example, to study paclitaxel accumulation in single Taxus cells,
an immunostaining protocol was developed using an anti-paclit-
axel monoclonal antibody (Naill and Roberts, 2005b). Paclitaxel
accumulation was highly variable among cells in culture, and
approximately 20% of cells did not accumulate any cell-associ-
ated paclitaxel (Naill and Roberts, 2005b), even with elicitation
with methyl jasmonate.
One reason for plant cell culture heterogeneity is that tradi-
tional suspension cultures are created from dedifferentiated cal-
lus cultures that originated as a mixture of specialized cell types
isolated from plant tissue. These cells could remain heteroge-
neous over time and contribute to the variability associated with
suspension cultures (Roberts and Kolewe, 2010). To avoid this
dedifferentiation process, cambial meristematic cells (CMC)
have been isolated from T. cuspidata, Panax ginseng, Ginkgo
baloba and Solanum lycopersicum (Lee et al., 2010; Roberts
and Kolewe, 2010). Taxus cuspidata CMC were found to have
lower growth variability, smaller aggregate size and less sensitiv-
ity to shear in stirred tank and air-lift bioreactors than tradi-
tional Taxus suspension cultures. Additionally, paclitaxel
production levels were higher than those found in traditional
cultures, and process optimization could potentially create a
more homogeneous culture and further increase product yields
(Lee et al., 2010). Development and optimization of CMC
Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 249–268
cultures from a variety of species could expand the potential for
commercial plant cell culture.
Functional genomics approaches have been used to identify
key genes and transcription factors involved in the secondary
metabolism of genome-sequenced and unsequenced plant spe-
cies, as reviewed in Yonekura-Sakakibara and Saito (2009).
Metabolite and transcript profiling of methyl jasmonate-elicited
tobacco cells yielded both known and novel genes potentially
involved in Nicotiana secondary metabolism, resulting in the dis-
covery of a new nicotine transporter (Goossens et al., 2003;
Morita et al., 2009). A similar study conducted on elicited
C. roseus cultures identified several genes putatively involved in
TIA biosynthesis (Rischer et al., 2006). These functional genom-
ics techniques can be used to identify genes involved in the
biosynthesis of secondary metabolites, allowing for single-gene
metabolic engineering of plant cell cultures. They can also iden-
tify putative transcription factors involved in the regulation of
secondary metabolic pathways, allowing for the metabolic
engineering of cultures without fully elucidated biosynthetic
pathways. Metabolic engineering for increased productivity
could decrease production costs associated with existing com-
mercial plant cell culture systems as well as make other plant
cell culture systems commercially feasible.
The sales of biologics are projected to continue rising dramat-
ically, and it is predicted that four of the five top-selling drugs
will be protein therapeutics by the year 2013 (Goodman, 2009).
As patents begin to expire on existing biopharmaceuticals, the
production of biosimilars, or replicate versions of the original
therapeutic proteins or antibodies, is on the rise. Owing to their
complex structures, the development of generic biologics is far
more difficult than the development of generic small-molecule
pharmaceuticals. Identical replication of a therapeutic protein
cannot reasonably be achieved, so biosimilars will likely have to
undergo clinical trials to prove their safety and efficacy (Ledford,
2010). Despite these complications, the approval of biosimilars
is on the rise, with 14 drugs being approved in Europe in 2010,
and a regulatory pathway for biosimilar approval has recently
been created in the United States (Walsh, 2010). Biosimilars,
such as the prGCD produced by Protalix Biotherapeutics, offer
an opening for plant cell culture production technologies, which
could allow for decreased production costs over traditional bio-
pharmaceutical production systems. Also, the FDA’s request for
supply of Protalix Biotherapeutics’ prGCD to patients with Gau-
cher’s disease before approval signals the acceptance of plant
cell-based production systems (Ratner, 2010). To compete with
existing biologic production systems, the benefits of plant cell
culture, such as safety, controlled culture conditions and protein
secretion, must be promoted and exploited. In addition, func-
tional product yields can be improved by manipulating the plant
cell host to produce proteins with human-like glycosylation pat-
terns, creating low protease cell culture lines and ⁄ or overcom-
ing gene silencing. Optimization of viral and inducible gene
expression systems and improving product secretion and stabil-
ity in the culture media will help to decrease production costs
associated with plant cell culture systems, making them more
commercially attractive for the production of biosimilars and
other biologics.
Acknowledgements
The authors would like to acknowledge the support of grants
from the National Science Foundation (CBET 0730779) and the
ª 2011 The Authors

National Institutes of Health (GM070852). In addition, S.A.W.
would like to acknowledge the support of the National Science
Foundation-sponsored Institute for Cellular Engineering IGERT
program DGE-0654128.
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