Research review paper
Green factory: Plants as bioproduction platforms for recombinant proteins
Jianfeng Xua, Maureen C. Dolana, Giuliana Medranoa, Carole L. Cramera, Pamela J. Weathersb,⁎
aArkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401, United States
bBiology & Biotechnology Department, Worcester Polytechnic Institute, Worcester, MA 01609, United States
a b s t r a c ta r t i c l ei n f o
Available online 7 September 2011
Plant-made recombinant proteins
Plant cell culture
Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only
for human and veterinary medicine, but also for agriculture and industry, now has some commercially avail-
able products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell
suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits.
Effective recombinant strategies are summarized along with descriptions of scalable culture systems and
examples of commercial progress and success.
© 2011 Elsevier Inc. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Classes of recombinant proteins produced by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.Therapeutic proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.Industrial proteins/enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Biopolymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plant expression platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.Whole plants — stable expression systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.Leaf-based . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.Seed-based . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.Whole plants — transient expression system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. In vitro culture systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1.Plant cell suspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2.Hairy roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3. Moss. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.Aquatic plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.1. Duckweed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.2.Microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture scale-up and bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Commercialization status and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Plant-made industrial proteins/enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.Plant-made therapeutic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.Plant-made biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biotechnology Advances 30 (2012) 1171–1184
⁎ Corresponding author at: Dept. Biology & Biotechnology, Worcester Polytechnic Institute, 100 Institute Rd, Worcester, MA 01609, United States. Fax: +1 508 831 5936.
E-mail address: email@example.com (P.J. Weathers).
0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
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ity to use the sun (photosynthesis) and/or very simple media to sup-
port significant biomass and protein accumulation. Their potential
for low-cost production of high quality and bioactive recombinant
protein is well documented (Obembe et al., 2011; Paul and Ma,
2011; Sharma and Sharma,2009). Plantssuccessfully perform the ma-
jority of post-translational modifications important for many complex
eukaryotic proteins and provide tremendous flexibility in bioproduc-
tionplatforms thatdifferentially address productionscale, cost, safety,
and regulatory issues. Plant-based systems provide the spectrum of
production capacity ranging from plant/algal cell bioreactor systems
for lower volume, higher value product to field-grown commodity
crops with potential for metric tons of recombinant protein at highly
competitive costs. Because plants cannot harbor the human and ani-
mal pathogens-of-issue for mammalian cell-based production sys-
tems, they bring significant advantage in increased safety for
patients (Pogue et al., 2010; Xu et al., 2011a). These biosafety advan-
tages also impact commercial aspects; they reduce purification costs
and minimize risks associated with potential production shut-downs,
facility decontamination, and supply limitations leading to unmet
patient/customer demand. Although costs to purify proteins from
plant systems will be comparable to microbial or mammalian cell cul-
ture systems, lower up-front capitalization required to initiate com-
mercial production in plants and potential “economies of scale”
provide key advantages. Applications that directly use whole or mini-
mally-processed plants or plant parts (e.g., seeds) are also being ex-
plored for industrial/bioenergy applications as well as therapeutics
and vaccines to further reduce recombinant protein costs (Boothe
et al.,2010;Hood,2002;Howardetal., 2011). Inmany systems, boost-
ing protein yields remains a challenge for economic feasibility (Paul
and Ma, 2011; Xu et al., 2011a) and key strategies used to boost prod-
uct recovery in plant expression system are summarized in Table 1
(see also Hood et al., 2011, in press; Rybicki, 2010). Plant-based pro-
duction systems involving field-grown GM plants require regulatory
oversight and approvals that are unique to plants compared to other
bioproduction systems (Obembe et al., 2011); regulatory consider-
ations also impact production strategies and costs.
In contrast to other expression systems such as bacterial, mamma-
lian cell and yeast, plant expression systems encompass diverse forms
including whole-plants, suspension cells, hairy roots, moss, duck-
weed, microalgae, etc. (Fig. 1). Each of the platforms has its own
strengths and weaknesses and is often best suited for certain classes
of recombinant proteins based on the market, scale, cost, and up-
stream and downstream processing constraints of the particular pro-
tein product. A multiplicity of plant species can serve as hosts for
plant-based bioproduction that comprise platforms ranging from in
vitro cell and plant tissue cultures to whole plants grown under
glass and in the field. Access to such platform diversity makes it pos-
sible to respond rapidly for expressing novel recombinant proteins,
enables customizing and meeting scale-up needs, and provides op-
portunity for oral-based delivery of proteins. This review highlights
the advantages and challenges associated with each type of plant pro-
duction platform and production strategy, and where appropriate,
will comment on important issues related to regulation. It also high-
lights progress toward product commercialization.
2. Classes of recombinant proteins produced by plants
According to their functions and applications, plant-made recombi-
nantproteinscan be generally categorized into three classes: therapeu-
tic proteins, industrial proteins/enzymes and biopolymers. Examples
of each of these classes are highlighted in Table 2 and discussed briefly
in the following sections.
2.1. Therapeutic proteins
Many proteins of mammalian origin have been expressed in
plants, yielding a product with full function. These include monoclo-
nal antibodies (mAbs), vaccine antigens, therapeutic enzymes, blood
proteins, cytokines, growth factors and growth hormones (see ex-
amples in Table 2) (Davies, 2010; De Muynck et al., 2010; Xu et al.,
2011a). Bioactivity of these proteins often requires protein folding,
disulfide bond formation, subunit assembly, proteolytic cleavage,
and/or glycosylation, highlighting the ability of plants to process
complex human/mammalian proteins. Plant-made antibodies (Plan-
tibodies®) have received considerable interest as they are made at
much lower cost in plants than in mammalian cells without the asso-
ciated risks of potentially harboring animal pathogens (De Muynck
et al., 2010). Plants in fact are currently recognized as the only viable
production platform for the production of secretory antibodies
(sIgAs) (Paul and Ma, 2011). These recombinant therapeutic pro-
teins are produced using a multiplicity of plant-based platforms
that range from cultures cells to field crops with the most common
being tobacco species with protein yields varying dramatically
from 0.01% of total soluble protein (TSP) (Mikschofsky et al., 2009)
and 0.1 μg/L (Kwon et al., 2003), up to 25% of TSP (Vunsh et al.,
2007) and 247 mg/L (McDonald et al., 2005). Several recent reviews
of plant-made therapeutic proteins are available (Davies, 2010;
Obembe et al., 2011; Paul and Ma, 2011; Rybicki, 2010; Sharma and
Sharma, 2009; Tremblay et al., 2010; Weathers et al., 2010; Xu
et al., 2011a), so we will focus here on selective characteristics of
the plant-based systems permitting comparisons with bacterial, fun-
gal and mammalian cell systems.
Plants successfully glycosylate proteins at the signature recogni-
tion motif (N-X-S/T) for N-linked glycosylation. However, subsequent
processing in the Golgi to complex glycans differs from that found in
mammalian cells. Thus, a notable challenge in using plants as hosts
for production of glycosylated therapeutic proteins is that plant-
specific xylose and α-1,3-fucose sugars may be added with a poten-
tial to alter bioactivity or immunogenicity in humans (Doran, 2000;
Gomord et al., 2010). Plants also do not naturally synthesize animal
or human-specific sugars, such as β-1,4-galactose residues or sialic
acid (Gomord et al., 2005; Gomord et al., 2010; Paccalet et al.,
2007). Recent efforts involving RNAi strategies that knock down
fucosyl- and xylosyl-transferases in Nicotiana benthamiana, Arabidop-
sis and Physcomitrella patens and transgenic lines expressing a human
or chimeric β-1,4-galactosyltransferase have been used to engineer
Strategies used to boost recombinant protein yields in plants.
• Develop strong promoters, double enhanced
promoters, hybrid promoters
• Use inducible promoters
• Engineer better enhancers, activators or repressors
• Optimize 5′- and 3′-UTR (untranslated region);
• Design preferred genetic codon.
• Target nascent proteins to sub-cellular compartments
such as ER
• Co-express with protease inhibitor and protein
co-factor/subunit; co-express antibody with antigen
• Express as fusion to a highly expressed and stable
• Optimize medium composition
• Supplement protein-stabilizing agents
• In situ remove expressed protein
• Select and/or improve bioreactor design
• Select culture strategy (e.g. batch vs. fed-batch vs.
• Evaluate secretion potential
In vitro cell/tissue culture
(for bioreactor system)
(for bioreactor system)
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
more human-like glycosylation machinery in plants (Bakker et al.,
2006; Schahs et al., 2007; Strasser et al., 2008). Furthermore, Agro-in-
filtrated N. benthamiana (plants infiltrated with Agrobacterium tume-
faciens) producing human antibodies, when co-expressed with a
chimaeric human β-1,4-galactosyltransferase resulted in antibodies
with human-like N-glycans (Vezina et al., 2009). Although significant
research has been focused on “humanizing” the glycan of plant-made
glycoproteins, the first human-injected therapeutic with greatest
Examples of the three classes of recombinant proteins being produced using plants.
Classification Recombinant proteins Plant expression platformReferences
Therapeutic proteins Antibody: anti-West Nile virus mAb Hu-E16a,
Guy's 13 SIgA (CaroRx)b
Vaccine: H5N1, H1N1c
Therapeutic enzyme: glucocerebrosidased
Blood protein: human serum albumine
Growth factor: human epidermal growth factor
Growth hormone: human growth hormonef
Oral therapeutic: human intrinsic factorg
Spider silk proteins
Lai et al. (2010), Ma et al. (1998)
Carrot suspension cells
Tobacco hairy roots
Tobacco, potato, Arabidopsis
Tobacco suspension cells
Shoji et al. (2008, 2011)
Aviezer et al. (2009a, 2009b)
Sijmons et al. (1990)
Liu et al. (2009)
Parsons et al. (2010)
Staub et al. (2000)
Fedosov et al. (2003)
Hood et al. (2007, 2011)
Kusnadi et al. (1998)
Woodard et al. (2003)
Hood et al. (1997), Murray et al. (2002)
Pen et al. (1992)
Hood et al. (2003)
Menassa et al. (2004), Scheller et al. (2001), Yang et al. (2005)
Conley et al. (2009)
Ruggiero et al. (2000), Xu et al. (2011b)
Xu et al. (2005)
aRecent on commercial route.
bPhase II clinical trial completed by Planet Biotechnology; approved for use in the EU, but not marketed.
cPhase II and phase I clinical trials for H5N1 and H1N1, respectively, completed by Medicago with positive results.
dPhase III clinical trial completed by Protalix; the first plant-made therapeutic protein entering commercial sector; FDA approval pending.
eFirst complex human protein expressed in plant.
fEarly demonstration of high-yield protein expression in plant chloroplast.
gPhase II clinical trial completed by Cobento Biotech AS; Marketed in the EU.
hSigma Aldrich products come as research enzyme or drug.
iRecently (Feb, 2011) commercialized by Syngenta.
jUnder preclinical development by Medicago and Meristem Therapeutics.
Fig. 1. Various plant cell expression platforms for the production of recombinant proteins.
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
clinical experience (Protalix's taliglucerase; discussed further below)
did not trigger significant antibody production in patients (Aviezer et
al., 2009a, 2009b).
Plant-specific post-translational modifications (PTMs) may pre-
sent an opportunity for not only producing biosimilars, but also com-
plex recombinant proteins with enhanced function and efficacy (Faye
and Gomord, 2010). To this end, continued advances in understand-
ing and manipulating post-translational modifications in plants will
be a key to the full adoption of plant-based therapeutic proteins. In
fact, the “second” generation of plant-made pharmaceutical proteins
is emerging at the research level whereby therapeutic targets are spe-
cifically engineered to enhance the production or utility of plant-
based therapeutics. The goal in these systems is to exploit the biopro-
duction capacity of plants to produce new therapeutic proteins that
integrate novel motifs or fusion to facilitate protein assembly, deliv-
ery, trafficking, protein stability, serum longevity, or protein solubility
in either the production host or the target organism (e.g., Cramer
et al., 2010; Phoolcharoen et al., 2011; Xu et al., 2007, 2010).
2.2. Industrial proteins/enzymes
Industrial proteins are characterized by the fact that they are used
in very large quantities and must therefore be produced at very low
cost (Horn et al., 2004). Transgenic plants provide a viable technology
for producing industrial proteins, in particular enzymes, because of
low cost of agricultural production, stability of protein stored in spe-
cific organs such as seeds, ease and speed of scale-up as well as the
possibility of using crude plant materials directly in industrial pro-
cesses (Hood, 2002). Some plant-made industrial proteins/enzymes
that have received considerable interest are listed in Table 3; most of
them are hydrolases, including glycosidases (e.g., cellulase, α-amylase
and β-glucuronidase (GUS)) and proteases (e.g., trypsin). ProdiGene
Inc (formerly in College Station, TX) was the first company to develop
and commercialize plant-based recombinant proteins/enzymes with
GUS and Avidin being ProdiGene's first two commercialized products
(Basaran and Rodriguez-Cerezo, 2008; Hood, 2002; Sharma and
Sharma, 2009). ProdiGene, a little ahead of itstimetechnically,is unfor-
tunately now out of business.
Corn seed is regarded as an ideal platform for the production of
industrial proteins/enzymes because corn has the largest annual
grainyield and relatively high seed protein content (10%), thus offer-
ing the highest potential recombinant protein yields per hectare
(Christou et al., 2005). Advantages of a seed-based production system
to industrial processes, minimizing handling and enzyme manipulation
and preparation (Boothe et al., 2010; Hood, 2002). Corn-produced en-
zymes such as cellulases and hemicellulases involved in biomass
candidates for commercialization (Obembe et al., 2011). A major hin-
drance to using plant-made industrial proteins/enzymes is regulatory;
very large acreage of transgenic plants will be planted. This will likely
require “deregulation” by federal regulatory agencies for field growth
of those transgenic plants (Horn et al., 2004) and also wider acceptance
by the general public of open field cultivation of transgenic plants for
protein production. Large scale production of GM “biofuel” corn,
which is a major commodity food/feed crop, remains controversial.
However, the regulatory route and public acceptance for crops expres-
sing this class of enzymes should be aided by the fact that these en-
zymes do not act on humans or animals (no substrate) and are
already widespread in the human diet (considered G.R.A.S, generally
recognized as safe). On the other hand, when as projected, global food
tions will need to be developed.
Differing from those abundant plant biopolymers such as cellulose
and starch, recombinant biopolymers discussed here specifically refer
to those made of proteins such as spider silk proteins, elastin-like
polypeptides (ELPs), collagens and plant gums (Table 2). The proper-
ties and recombinant expression of these biopolymers in plants were
reviewed previously by Scheller and Conrad (2005). Of special inter-
est are the spider silk proteins (also called spidroins) that are modu-
fibrous proteins containing
sequences consisting largely of glycine and alanine (Hinman et al.,
2000; Scheller et al., 2001). Spider silk fibers spun from these spi-
droins are regarded as one of nature's most extraordinary materials
with exceptional flexibility, elasticity, and toughness—three times as
strong as Kevlar and five times as strong as steel (Eisoldt et al.,
2011). Plants offer a more efficient and cheaper production platform
than bacteria for production of recombinant spidroins. The expres-
sion of Nephila clavipes dragline spidroins encoded by synthetic
genes in transgenic tobacco and potato plants has resulted in accu-
mulation of recombinant silk proteins up to a level of 2% of TSP in
the ER (Scheller et al., 2001). In another report, accumulation of drag-
line spidroins in Arabidopsis reached 18% of TSP in seeds (Yang et al.,
2005). Currently, the challenge to the application of such biomaterials
is the lack of appropriate techniques such as “fiber spinning” to con-
vert the raw material into intermediate products (Scheller et al.,
2001). Another interesting biopolymer is the ELPs that are comprised
of the repetitive pentapeptide sequence (VGVPG), which mainly
serves as thermally responsive tags for the non-chromatographic pu-
rification of recombinant proteins (Conley et al., 2009). ELP tags were
Comparisons of different expression systems.
Expression system Commercially viable speciesTime for productiona
Scalability Regulatory compliance
• Stable transgenic plants
• Transient plants
Corn, soy, safflower, rice
Nicotiana sp., lettuce
Unlimited field culture
In vitro cultured plant cells and species
• Hairy roots
• Cell suspension culture
Tobacco BY-2, carrot, rice
• Duckweed (closed system)
Lemna sp., spirodela sp.
20–40 days10,000 LModerate
Limited by water surface area
aThe time required to accumulate maximum amounts of recombinant proteins in a culture system after planting or bioreactor inoculation.
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
found to significantly enhance the production yield of a range of dif-
ferent recombinant proteins in plant leaves (Conley et al., 2011;
Patel et al., 2007). Compared with therapeutic proteins and industrial
enzymes that have shown high potential for commercialization,
plant-made biopolymers still have a long way to go before they are
cost effective to market.
3. Plant expression platforms
The rapid development of plant genetic engineering technologies
has expanded the diversity of well-established plant-based biopro-
duction platforms for recombinant proteins (Paul and Ma, 2011).
These platforms include transgenic plants (using both stable and
transient expression), duckweed, microalgae, and in vitro culture sys-
tems: cell suspensions, hairy roots and moss protonema (Fig. 1). Each
of the platforms has its own strengths and weaknesses, which are de-
scribed here. A generalized comparison of the time needed for pro-
duction, scalability and regulatory compliance of different platforms
is shown in Table 3.
3.1. Whole plants — stable expression systems
To drive expression of foreign genes, many technologies for stable
integration of the transgene into the production plant have been de-
veloped and used over the years. The choices of 1) production host,
2) gene integration site (nuclear vs. plastid), 3) the cellular compart-
ment for recombinant protein accumulation, and 4) the plant tissue
targeted for product expression in establishing stable transgenic
plant lines will depend on a variety of considerations that include
the post-translational modifications required for attaining functional
protein product, the stability of the expressed foreign protein in the
given plant host, desired expression levels of product and the down-
stream purification costs as well as the size and cost constraints of the
product market (see also Hood et al., 2011).
Tobacco has emerged as the leading plant platform for leaf-based
recombinant protein expression (Tremblay et al., 2010). The first
clinical trial of a plant-produced biopharmaceutical, secretory anti-
body variant of Guy's 13, was done with a product expressed in
field grown tobacco leaves by Planet Biotechnology Inc. (Kaiser,
2008; Ma et al., 1998). Tobacco is genetically well studied and easily
manipulated, is classified as a non-food/non-feed crop, produces
high biomass (upwards of 300 tons per acre) and is one of the best
studied platforms to date for expressing recombinant biopharma-
ceuticals. The breeding of low nicotine and low alkaloid tobacco va-
rieties has been used to overcome potential toxicity carryover into
recombinant protein products (Menassa et al., 2001). In addition
to tobacco, many other leafy crops have been used for stable expres-
sion of proteins including lettuce, alfalfa and clover. The use of crops
like alfalfa has additional benefits; it is a perennial that fixes nitro-
gen and displays notable homogeneity of N-glycosylated recombi-
nant proteins (Fischer et al., 2004).
Both nuclear and plastid integration have been used when expressing
recombinant proteins in leaf tissue. The choice of gene integration within
the plant cell is generally dictated by the post-translational requirements
of the target protein. For glycosylated proteins, nuclear integration of the
transgene is needed to enable proper processing of the protein in the
endomembrane system (Gomord et al., 2010). This strategy has been
used in successfully expressing over 100 glycosylated proteins including
antibodies (e.g. Johansen et al., 1999; Ma et al., 1995; Fiedler et al.,
1997), vaccines (e.g. Pogrebnyak et al., 2005) cytokines (e.g. Wang et
al., 2008; Medrano et al., 2010), growth hormones and industrial en-
zymes in plant leaves. Benchabane et al. (2008) and Sharma and
Sharma (2009) have recently reviewed expression of endomembrane-
targeted proteins in plants.
With an average of 100 chloroplasts per plant cell, the plastid pre-
sents an alternative integration site which, in practice, enables thou-
sands of copies of a given transgene to be expressed (Chebolu and
Daniell, 2007; Oey et al., 2009; Ruhlman et al., 2010). Chloroplastic
transformation and expression of chloroplast-targeted genes have
been reviewed in depth in a recent special issue on Chloroplast Bio-
technology in the Plant Biotechnology Journal (June 2011). To date,
over 100 recombinant proteins have been expressed through chloro-
plast transformation including industrial enzymes, antibodies, bio-
pharmaceutical proteins and vaccine antigens as noted in a recent
review by Clarke et al. (2011). While the chloroplast does support
many of the important post-translational modifications desired for
expressing complex proteins, current plastid expression technology
is limited to production of non-glycosylated products.
Among the limitations of targeting recombinant protein expression
ly process upon harvest to ensure product stability and quality. Al-
though plant leaves are advantageous in terms of biomass yield, this
tissue is generally more subject to damage than seeds. There are many
cases of recombinant proteins in leaves having associated stability and
accumulation issues. Product yields in field-grown materials can be
highly variable due to environmental impacts (both biotic and abiotic
stresses) leading to increased consideration of growth in more con-
trolled conditions (e.g., under plastic or in greenhouses), especially for
pharmaceutical applications. In addition to associated instability of the
protein itself, the harvested material has a limited shelf life and there-
fore, must be processed immediately after harvest. It should also be
noted that “at scale”, regulatory-compliant disposal of transgenic bio-
mass waste may have volume and cost implications.
Recombinant protein expression targeted to plant seeds can over-
come limitations of protein stability and storage associated with leaf
tissue (Boothe et al., 2010; Lau and Sun, 2009; Ma et al., 2003). Several
cereal grains including rice, wheat, barley, soybean and maize are
commonly used as expression hosts (Ramessar et al., 2008a). Proteins
expressed in seeds are generally protected from proteolytic degrada-
tion, and storage upwards of three years at room temperature (longer
with cold storage) resulted in minimal loss of protein activity. The
high protein content of seeds, ranging from 7 to10%, has translated
into high expression for several seed-targeted proteins (e.g. Hood
et al., 1997; Horvath et al., 2000; Woodard et al., 2003; Xie et al.,
2008; Xue et al., 2003; Yang et al., 2006; Yang et al., 2008). Further-
more, maize seeds producing HIV neutralizing antibody 2 G12 (a po-
tential candidate for a topical microbicide), were not only shown to
have cost-effective production, but also simple downstream purifica-
tion processes, which are considered the most significant costs associ-
ated in the production of biopharmaceuticals (Ramessar et al., 2008c).
The potential to use agronomically important food/feed commod-
ity crops and other seed crops for non-food/feed GM-based applica-
tions such as biofuel enzymes or pharmaceuticals raises several
regulatory issues for both regulatory agencies and consumers. Regu-
latory compliance for field growth (e.g., USDA APHIS/EPA) will en-
compass concepts of product segregation and stewardship. Crops
expressing therapeutics or vaccines will likely remain under regulato-
ry oversight for the life of the product and require containment pro-
tocols and documentation at each step in the process. In contrast,
for industrial enzymes which are/will be produced at large scale, pro-
ducers will likely seek deregulation. One of the regulatory consider-
ations for food-based grains is that while there is the need to
segregate the bioproduct source grain from the food/feed supply
chain, they do have a G.R.A.S. status from the US Food and Drug Ad-
ministration (FDA) which may reduce downstream hurdles linked
with pharmaceutical use of these plant-based products (Ramessar
et al., 2008b, 2009).
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
While the first plant-derived commercialized product was pro-
duced in maize (Hood et al., 1997; Witcher et al., 1998), oil-seeds
are emerging as a promising platform for recombinant protein pro-
duction due to their inherently low associated proteolytic activity
and simplified protein isolation via oil body separations. Among the
leading companies in this plant biotechnology sector is SemBioSys
Genetics (Calgary, Canada), which has developed the oleosin-fusion
platform, in which the target recombinant protein is produced as a fu-
sion with oleosin and accumulates in safflower oilbodies (www.
sembiosys.com/). Application of this technology has highlighted the
development of an exceptionally low cost human insulin biosimilar
for improved accessibility and global distribution of insulin in coun-
tering the global diabetes epidemic. Over the past several years, a
number of other seed-based platforms have emerged, including Ven-
tria Biosciences and Meristem Therapeutics, which have invested in
field-grown rice for the production of the human proteins lactoferrin
and lysozyme (www.ventria.com/). ORF Genetics Ltd., based in Ice-
land, has targeted barley grain as the expression site for a number
of human cytokines and growth factors (www.orfgenetics.com/).
3.2. Whole plants — transient expression system
Transient expression systems, mediated by recombinant viral or
plant binary vectors, are being increasingly used for the expression of
biopharmaceuticals in plants due to speed, highprotein yields, and reg-
ulatory considerations. During this process, foreign genes are typically
introduced into leaf tissue of intact plants (generally non-transgenic)
by vacuum infiltration of engineered Agrobacterium (containing
gene(s) of interest within T-DNA with or without additional virus-der-
ived components). Recombinant protein production (based on extra-
chromosomal gene expression within the plant cell) is initiated in the
leaves within 24 h and continues for several days (Agro-mediated) to
several weeks (viral-mediated) depending on vector and protein.
While chromosomal integration is possible, it occurs at considerably
lower frequency in comparison to the number of cells transiently
expressing the desired gene (Circelli et al., 2010; Sheludko, 2008). In
these transgenic plants are grown only in contained environments, e.g.,
greenhouses. This system is widely used for assessing gene expression
constructs in plants and to validate activity of new recombinant pro-
teins (Circelli et al., 2010; Daniell et al., 2009; Dhillon et al., 2009;
Lombardi et al., 2009; Pogue et al., 2010; Tremblay et al., 2010,
2011; Voinnet et al., 2003; Whaley et al., 2011). Biomass production
for transient systems typically involves non-transgenic plants (gen-
erally Nicotiana benthamiana or other Nicotiana species) often
grown in contained environments, e.g., greenhouses, prior to infil-
tration, which is a highly automated process at production scales.
In addition to rapid transgene assessment and production runs and
high product yields, these systems also provide the ability to easily
co-express several genes at the same time (e.g., Huang et al., 2010;
Medrano et al., 2009). However, transiently expressed products re-
quire sizable greenhouse space and infiltration infrastructure, imme-
diate processing of harvested leaf biomass, and, if using the product
in humans or animal, an additional purification step to remove endo-
toxins derived from the infiltrated Agrobacterium.
Transient expression is typically carried out in leaves of N.
benthamiana, but also has been demonstrated to work in other plants
including potato, green pea, Arabidopsis, lettuce, and other Nicotiana
species such as N. debneyi, N. excelsior, N. exigua, N. maritime, and N.
simulans (Bhaskar et al., 2009; Green et al., 2009; Kim et al., 2009;
Negrouk et al., 2005; Sheludko et al., 2007; Sindarovska et al., 2008,
2010). There are two basic approaches for plant transient expression
based on the mechanism by which the transgene is transferred into
the plant cell: viral-mediated or plant binary vector-based (Fischer
et al., 1999; Matoba et al., 2011). In a transient expression system
using “conventional” plant expression vectors, the accumulation of
recombinant proteins in plants occurs within a shorter time, typically
2–4 days post-infiltration, with recoveries typically ranging from ~0.1
to 180 μg/g fresh leaf depending on the gene of interest (Condori
et al., 2009; Cramer et al., 1999, 2010; Liu et al., 2008; Medrano
et al., 2009, 2010). Recently, transient-expressed xylanase targeted
to the endomembrane system was reported with product recoveries
as high as 1 mg/g fresh leaf (Llop-Tous et al., 2011). A direct compar-
ison of stable versus transient expression of identical transgenes sug-
gests yields of 4 to 20-fold higher for the transient systems (using
de35S promoter) on a leaf fresh weight basis (Medrano et al., 2009).
Plant viral vectors also use the same Agro-infiltration system to in-
generally produces higher levels of recombinant protein with yields
reported to be as high as 5 mg/g of fresh leaf for GFP (Marillonnet
et al., 2004). However, recombinant protein expression by this process
takes up to 14 days, which can present issues for proteins prone to pro-
tease degradation and instability. Geneware (Kentucky BioProcessing
LLC; originally developed by Large Scale Biology Corp.), MagnIcon
versity) are some examples of effective viral vector expression systems
(Gleba et al., 2005, 2007; Huang et al., 2009a; Mason et al., 2002).
Alpha-trichosanthin and human lysosomal acid lipase have been
expressed using Geneware technology (Kumagai et al., 1993). MagnI-
con has been used to express hepatitis B core antigen (Huang et al.,
2006, 2008), Norwalk virus-like particles (Santi et al., 2008), and Yersi-
nia pestis antigens F1 and V (Santi et al., 2006). Geminivirus technology
6D8 against Ebola virus GP1 (Wilson et al., 2000), hepatitis B core anti-
gen (HBc) and Norwalk virus capsid protein (Huang et al., 2009b). The
Launch vector system includes a combination of a plant viral vector ge-
nome and a binary plasmid of Agrobacterium in which several proteins
including influenza targets have been expressed with recoveries of
60 μg/g fresh leaf for recombinant H5HA-I (Musiychuk et al., 2007;
Shoji et al., 2009; Yusibov and Mamedov, 2010). Additional examples
and yield information of viral vector-mediated bioproduction are sum-
marized elsewhere (Daniell et al., 2009; Sheludko, 2008). Examples of
by Yusibov et al. (2011).
Recombinant protein production using transient expression is now
being mobilized to large scale by industry. For example, the Fraunhofer
Center for Molecular Biotechnology (Newark, DE) (http://www.
fraunhofer.org) has developed a scalable, automated plant-based facto-
ry using plant viral vector technology to efficiently produce large
ada) (http://www.medicago.com) has a cGMP manufacturing facility
using a transient expression system to produce vaccines for Phase II
clinical trials. Icon Genetics (Bayer; Halle, Germany) (http://www.
icongenetics.com) also has a GMP manufacturing facility. Texas A&M
(College Station, TX) and Kentucky BioProcessing LLC (Owensboro,
ing facility. Terrasphere (http://terraspheresystems.com/) has devel-
oped a high-density vertical hydroponic cultivation system that can
be used year round to produce large quantities of transient plants and
their products. The production of large quantities of recombinant pro-
tein offered by transient plant expression systems, coupled with use
of current technology to increase yields and many technical promising
solutions seem to favorably compare with mammalian or insect cell-
based systems in quality, cost, and scale.
3.3. In vitro culture systems
In vitro culture systems are characterized by the fact that plant
biomass is cultured in confined bioreactors under sterile conditions
for large-scale production of recombinant proteins. Plant suspension
cells, hairy roots and moss fall into this category.
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
3.3.1. Plant cell suspensions
Like microorganisms, undifferentiated clusters of plant cells (callus)
can be dispersed and propagated in a liquid medium to generate stable
cell suspension cultures, which can retain the same production capacity
as whole plants. Increased concerns about regulatory compliance and
product safety of mammalian systems recently have renewed interest in
plant cell cultures as an alternative production platform for complex
pharmaceutical proteins (Huang and McDonald, 2009; Shih and Doran,
dustry with added benefits of complex protein processing compared to
commercial therapeutic protein for human infusion, glucocerebrosidase,
is being produced in carrot cells by Protalix Biotherapeutics, Inc. (Israel;
http://www.protalix.com), although proof-of-concept was originally
established previously using tobacco plants (Cramer et al., 1999; Radin
et al., 1999).
Plant cell suspension culture combines the advantages of whole-
plants with those of microbial cultures (reviewed in Hellwig et al.,
2004; Huang and McDonald, 2009; Xu et al., 2011a). Although plant
cell cultures do not share the perspectives of unlimited scalability of
field-cultivated whole plants, culture of plant cells in a sterile environ-
ment allows for precise control over growth conditions, batch-to-batch
product consistency, and a production process aligned with current
pects of both production and product reduces the regulatory burden
with established biopharmaceutical production systems potentially aid-
ing both regulatory and industry acceptance. The reduced regulatory
costs maypartlyoutweigh the currentlylowerproductivities of this pro-
duction platform along with its potentially higher capital costs. A wide
array of biologically active recombinant proteins have been successfully
expressed in plant cells, particularly in BY-2 (Bright Yellow-2) and NT-1
nization (Hellwig et al., 2004; Ozawa and Takaiwa, 2010; Su and Lee,
2007; Tremblay et al., 2010).
One bottleneck in exploiting plant cell suspension culture for com-
mercial purposes has been low productivity. Exceptions are expres-
sion of human proteins such as α1-antitrypsin, interleukin-12 and
hGM-CSF in rice cells using a sucrose-inducible RAmy3D promoter
(Huang et al., 2001; McDonald et al., 2005; Trexler et al., 2005; Shin
et al., 2003, 2010), where up to 247 mg/L was achieved (McDonald
et al., 2005). However, the growth rates and characteristics of rice
cell lines are inferior to those of tobacco BY-2 and NT-1 cell lines
(Hellwig et al., 2004) and the viability of rice cells is significantly de-
creased when cultivated in a sucrose-starvation medium (Huang and
McDonald, 2009). To attain a reasonable profit margin, the productiv-
ity of plant cell cultures needs to increase 10–50-fold, and this re-
quires a systematic strategy to maximize the efficiency of all stages
of the production pipeline from gene expression to cell culture, pro-
cess development, and finally downstream protein purification
(Weathers et al., 2010; Xu et al., 2011a).
for a significant portion of the preclinical and clinical pipeline, plant
cells and tissues are emerging asa more compliant alternative “factory”
(Xu et al., 2011a). Currently, two biotechnology companies, Protalix
BioTherapeutics, Inc. (Israel; http://www.protalix.com) and Dow
AgroSciences (Indianapolis, IN; http://www.dowagro.com/) have fo-
cused on the development and commercialization of pharmaceutical
proteins expressed by proprietary plant cultures, named ProCellEx™
and Concert™ Plant-Cell-Produced System, respectively. The commer-
cial successes of these companies will undeniably define a new era in
the biopharmaceutical industry that promises to provide a growth op-
portunity for plant-produced products.
3.3.2. Hairy roots
Hairyrootsare generatedby infectionofplantswithAgrobacterium
rhizogenes that harbors a large root-inducing (Ri) plasmid (Shanks
and Morgan, 1999). Integration of T-DNA carried on the Ri plasmid
into the plant genome results in differentiation and growth at the in-
fection site of neoplastic roots, called hairy roots (Guillon et al.,
2006b; Shanks and Morgan, 1999). These induced root tissues can be
grown indefinitely in vitro and have become a viable alternative pro-
duction platform for heterologous proteins as well as other plant me-
tabolites (Giri and Narasu, 2000; Guillon et al., 2006a).
Similar to suspension cells, hairy roots can be axenically cultured
in a controlled environment suitable for the production of high-value
pharmaceutical proteins under cGMP requirements. In addition, the
possible extracellular secretion of expressed proteins from cultured
hairy roots, or rhizosecretion (Gaume et al., 2003), offers a simplified
method for the recovery of foreign proteins from an inexpensive and
well-defined medium. The advantages of rapidly growing hairy roots
over suspension cells include long-term genotype and phenotype sta-
bility, efficient productivity and the ability of hairy roots to grow on
hormone-free medium. Since the first plant recombinant protein, a
full-length murine IgG1, was successfully produced in hairy root cul-
ture N10 years ago (Wongsamuth and Doran, 1997), nearly 20 recom-
binant proteins, including reporter proteins (e.g. GUS and GFP) (Lee
et al., 2007; Medina-Bolivar and Cramer, 2004), enzymes (Gaume et
al., 2003; Woods et al., 2008), antibodies (Martinez et al., 2005;
Sharp and Doran, 2001), antigens (Ko et al., 2006; Kumar et al.,
2006; Rukavtsova et al., 2007), growth factors (Komarnytsky et al.,
2006; Parsons et al., 2010), and immunomodulators such as ricin-B
(the non-toxic lectin subunit of ricin as antigen carrier) (Medina-
Bolivar et al., 2003) and interleukin-12 (Liu et al., 2008; Liu et al.,
2009) have been reported to be expressed by hairy roots with protein
yields up to 3.3% of TSP (Woods et al., 2008).
There are, however, challenges for large scale culture of hairy roots
unique physiological characteristics. Improved bioreactor designs, e.g.
the mist reactor, originally developed by Weathers and Giles (1988),
that offerlowhydrodynamic stress, yetachievehighvolumetric oxygen
transfer are needed for culture of hairy roots (Choi et al., 2006; Kim and
Yoo, 1993; Liu et al., 2009; Sivakumar et al., 2010). A Swiss biotechnol-
ogy company, ROOTec Bioactives Ltd (http://www.rootec.com/), is cur-
rently focusing on commercialization of plant-derived products with
hairy roots grown in a mist bioreactor. Hairy roots appear to be an at-
tractive in vitro expression system with several advantages compared
to field-grown plants and suspension cultured cells. Future research
should be focused on establishing effective and economical bioreactors
for industrial production.
The moss, P. patens, with its fully sequenced genome (http://www.
cosmoss.org/) is also providing a promisingplatformfor producingre-
combinant products. Knockout strains lacking xylosylation and fuco-
sylation activity have been produced so that recombinant proteins
can be humanized (Koprivova et al., 2004), and galactosylation has
been obtained by adding human glactosyltransferase to the xylosyl
and fucosyltranferase locus (Huether et al., 2005). Using the moss
platform, a variety of different engineered proteins have now been
produced including IgG1 and IgG4 antibodies, human VEGF, erythro-
poietin, and factor H (Buttner-Mainik et al., 2011; Decker and Reski,
2008). Secretion into the culture medium has also been achieved
and found to be most effective when P. patens endogenous signal se-
quences are used instead of human sequences (Schaaf et al., 2005).
The haploid juvenile gametophyte (protonema) stage of the plant
is morphologically similar to filamentous green algae and readily cul-
tured in liquid media in photobioreactors. By controlling pH or am-
monium tartrate levels in the medium, the moss can be maintained
indefinitely in the protonema stage, thereby preventing development
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
to the leafy mature gametophyte (Hohe et al., 2002). P. patens sus-
pensions also seem quite resistant to sheer stress; growth is not
inhibited even at 400–500 rpm in stirred tank reactors (Hohe et al.,
2002). Greenovation GmbH has now developed both process technol-
ogy and proprietary strains of P. patens for contract production of de-
sired proteins up to 200 L using tubular or disposable rocker bag
3.4. Aquatic plants
An aquatic higher plant, duckweed, is another promising biopro-
duction platform for recombinant proteins. Duckweed is the common
name for Lemnaceae, a monocot plant family consisting of four major
genera: Lemna, Spirodela, Wolffia and Wolffiella. As the world's smal-
lest, fastest-growing and simplest flowering plant (Zhang et al.,
2010), duckweed seems ideal for the production of recombinant pro-
teins (Stomp, 2005; Yamamoto et al., 2001). It is safe, fast-growing
(doubling time as short as 36 h), easy to grow and harvest, and has
a high protein content (up to 45% dry weight) (Stomp, 2005). Duck-
weed is capable of producing complex proteins that are not easily
made by bacteria and yeast, or made only at great cost over a long pe-
riod of time by mammalian cells. Moreover, duckweed is edible and
thus offers an attractive system for oral vaccines (Popov et al., 2006;
Rival et al., 2008).
Similar to other higher plants, duckweed can be transformed
using either biolistics or A. tumefaciens. Resulting transgenic lines
are stable for both gene insertion and expression. More than 20 ther-
apeutic proteins including plasminogen (Spencer et al., 2010), aproti-
nin (Rival et al., 2008), monoclonal antibody (Cox et al., 2006), avian
influenza H5N1 hemagglutinin (Guo et al., 2009) and interferon α2
(De Leede et al., 2008) have been produced in duckweeds with ex-
pression levels up to 7% of TSP (Stomp, 2005). A reporter protein
gene, GFP, expressed in Spirodela oligorrhiza reached a protein yield
of more than 25% of TSP, making it among the highest expressing sys-
tems for nuclear transformation in a higher plant (Vunsh et al., 2007).
Biolex, Inc., based in Pittsboro, North Carolina (www.biolex.com),
is the major biotechnology company developing the duckweed (Lem-
na)-based expression (LEX) system for producing pharmaceutical
proteins. Currently, the leading product of Biolex is a controlled-re-
lease interferon α2b (Locteron®) for the treatment of hepatitis C,
which offers tolerability and dosing advantages over PEGylated inter-
ferons currently on the market. Two other products that are in pre-
clinical development are a human plasmin (BLX-155) that is an
anticoagulant, and an anti-CD20 mAb (BLX-301) (Paul and Ma,
2011). For the duckweed platform to be economically viable for in-
dustrial production, several issues need to be addressed including im-
proved protein expression levels, human glycosylation, more
genomic information on duckweed and duckweed reproduction,
and methods for scale-up to commercial levels in both closed and
open systems (Stomp, 2005).
Microalgae also offer potential for large-scale and cost-effective
production of recombinant proteins. They integrate the merits of mi-
crobes including rapid growth and ease of culture with those of
higher plants in performing post-translational modification and pho-
tosynthesis (Franklin and Mayfield, 2005; Potvin and Zhang, 2010;
Specht et al., 2010). Use of transgenic microalgae for production of re-
combinant proteins has lagged behind other microorganisms, such as
bacteria and yeasts (Specht et al., 2010), but recently there has been a
surge of interest in microalgae as a viable bioproduction system
(Rasala et al., 2010). The majority of current work is performed
with the well-characterized green unicellular alga, Chlamydomonas
reinhardtii (Potvin and Zhang, 2010; Rosenberg et al., 2008; Rasala
et al., 2010). This alga has a rapid growth rate (doubling time of
≈10 h), supports easy nuclear and chloroplast transformation, and
can be grown either photoautotrophically or with acetate as a carbon
source. A variety of recombinant proteins have been successfully
expressed in microalgae as documented in a number of reviews
(Cadoret et al., 2008; Franklin and Mayfield, 2004, 2005; Leon-Ba-
nares et al., 2004; Mayfield et al., 2007; Potvin and Zhang, 2010;
Specht et al., 2010; Walker et al., 2005).
Both nuclear and chloroplast genomes of microalgae have been
successfully transformed for expressing recombinant proteins. How-
ever, nuclear transformants generally failed to accumulate recombi-
nant proteins to the levels observed in chloroplasts, most likely due
to nuclear silencing mechanisms (Specht et al., 2010). Thus, only
the chloroplast system is currently regarded as feasible for commer-
cial production (Potvin and Zhang, 2010). A variety of functional re-
combinant proteins including an antibody (Mayfield and Franklin,
2005), a vaccine (Streatfield, 2006; Surzycki et al., 2009), a growth
factor (Rasala et al., 2010), a blood protein (Manuell et al., 2007),
and an industrial enzyme phytases (Yoon et al., 2011), have been suc-
cessfully expressed in the chloroplast of C. reinhardtii. The disadvan-
tage of chloroplast transformation is that the expressed proteins
lack posttranslational modifications such as glycosylation (Franklin
and Mayfield, 2005). Although problematic for some products, in an-
tibody production, this can provide a benefit because lack of glycosyl-
ation prevents antibodies from activating the immune system (Dance,
2010; Tran et al., 2009).
Recombinant protein production with microalgae is still in its in-
fancy. The development of economically viable microalgal expression
systems is currently hindered by a lack of effective and consistent
transformation methods for a wider variety of species, low (nuclear
expression) or inconsistent (chloroplast expression) recombinant
protein yields, as well as the lack of production systems optimized
for large-scale growth and harvesting under photoautotrophic condi-
tions (Specht et al., 2010). Systematic and concerted research efforts
that are both biological- and engineering-based such as optimization
of promoters, regulatory elements and codon usage as well as devel-
opment of improved photobioreactor culture systems will be critical
to the success of the microalgal production platform.
4. Culture scale-up and bioreactors
While the scale of field-grown plants can be enlarged almost in-
definitely at low cost, scale-up of the elite genotype is the critical
first step involving in vitro cultured plant cell and tissue systems.
Large scale culture of these in vitro cultures, especially to obtain
small plantlets (micropropagation) is still a major challenge. There
are two main categories of bioreactors typically used to culture plants
cells, tissues, and organs: liquid-phase and gas-phase reactors (Kim
et al., 2002). Liquid-phase reactors are the most commonly used,
but delivery of adequate oxygen to submerged cells or tissues re-
mains a challenge. Most plant cells and tissues are susceptible to
shear stress so, unlike microbial cells, aggressive agitation is not par-
ticularly beneficial. As a result, gas-phase reactors were also devel-
oped to enhance O2delivery, but with minimal shear. Since gasses
are very insoluble in liquids, gas-phase reactors expose the biomass
to air or a gas mixture, and nutrients are delivered as gas-infused
droplets. Hybrid versions of both types of reactor also exist wherein
the headspace gas spans a large area of relatively shallow liquid to en-
hance gas exchange to the submerged cells or tissues. Because there
are a number of recent reviews on this topic (Ducos et al., 2010;
Huang and McDonald, 2009; Weathers et al., 2010, 2011; Xu et al.,
2009), only a brief summary is provided here and the reader is direct-
ed to those for details.
Bioreactors typically used for larger scale culture of plant cells and
tissues include stirred tank, airlift, bubble column, temporary immer-
sion, balloon-type bubble, ebb and flow, mist reactor, and the rotating
drum. Typical micropropagation uses a very labor- and cost-intensive
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
process involving small culture boxes with semi-solid medium. De-
sign and use of different reactor types and scale-up have been devel-
oped for micropropagation and other plant production systems
including cell suspensions, microalgae, hairy roots, moss protonema,
duckweed and other aquatic small plants, and even co-culture of mul-
tiple species. There are also many plant production systems being de-
veloped that instead use disposable (single use) bioreactors for
culture of plant cells and tissues. These usually employ plastic bags
of various designs including as a liner inside another vessel, a hanging
bag, the horizontal wave and wave and undertow bioreactors, the
slug bubble, the temporary immersion system (Box-In-Bag; RITA),
and a mist bag reactor. These newer disposable and issues regarding
their scale-up are described in greater detailed in recent reviews
(Eibl et al., 2010; Eibl and Eibl, 2008; Weathers et al., 2010, 2011).
5. Commercialization status and outlook
Plants and their cells or organs are essentially a production factory
for recombinant proteins. They provide both economic and safety ad-
vantages over conventional production systems. It was estimated that
with 1% (w/w) of protein expression in corn on the dry weight base
as well as 50% of protein recovery during purification, the cost of
plant-produced proteins is 10 to 50- fold lower than that of micro-
bial systems, and up to 1000-fold lower than that of mammalian
cell culture systems (Chen et al., 2005; Hood, 2002; Twyman et
al., 2003), implying the great potential of transgenic plants as an
economically competent production platform. The molecular farm-
ing industry has grown rapidly in the past 15 years. While some
plant-made industrial proteins/enzymes have been commercial-
ized, many plant-made pharmaceuticals are in various develop-
mental stages. Examples of some successful plant-produced
products, either on the market or in commercial development, are
listed in recent reviews (Obembe et al., 2011; Paul and Ma, 2011;
Sharma and Sharma, 2009), or on the Molecular Farming website
last updated in 2010 (http://www.molecularfarming.com/PMPs-
and-PMIPs.html). The major companies involved in research and
development of plant-produced recombinant proteins include: Biolex
Therapeutics Inc. (Pittsboro, NC; http://www.biolex.com), BioStrategies
LC (Jonesboro, AR; http://www.biostrategies-lc.com), Dow AgroSciences
(Indianapolis, IN; http://www.dowagro.com), Fraunhofer Center for Mo-
lecular Biotechnology (Newark, DE; http://www.fraunhofer.org), Ken-
tucky BioProcessing LLC (Owensboro, KY; http://www.kbpllc.com/),
Medicago Inc. (Québec, Canada; http://www2.medicargo.com/en), Meri-
stem TherapeuticsLLC (Cambridge,
therapeutics.com), Protalix Biotherapeutics (Carmiel, Israel; http://
www.protalix.com), Planet Biotechnology (Hayward, CA; http://www.
planetbiotechnology.com), SemBioSys (Calgary, Canada; http://www.
sembiosys.com) and Ventria Bioscience (Fort Collins, CO; http://www.
5.1. Plant-made industrial proteins/enzymes
Plant-made industrial proteins/enzymes have spearheaded com-
mercialization over the other two classes of recombinant proteins;
this progress is mainly credited to ProdiGene's pioneering work on
the development and commercialization of hydrolases including
GUS, avidin and trypsin (TrypZean™) and aprotonin (AproliZean™)
in the late 1990s (Basaran and Rodriguez-Cerezo, 2008; Hood, 2002;
Hood et al., 1997; Sharma and Sharma, 2009; Witcher et al., 1998).
All of these products are produced in transgenic corn and sold by Sig-
ma-Aldrich (St Louis, MO) (Sharma and Sharma, 2009). The commercial
success of ProdiGene's products was followed by many efforts to de-
velop and commercialize other plant-made enzymes, in particular,
xylanase and ligninase (Hood et al., 2007, 2011; Sticklen, 2007),
α-amylase (Pen et al., 1992), as well as an oxido-reductase fungal
as cellulase, hemicellulase,
enzyme, laccase (Hirai et al., 2008; Hood et al., 2003). For example,
an Arkansas-based small company, Infinite Enzymes (Jonesboro,
AR; http://www.infiniteenzymes.com/index.htm) is engaged in de-
veloping a cost-effective corn seed production system for cellulases
for cellulosic ethanol production. However, few products entered
the marketplace until 2011 when a transgenic variety of corn expres-
sing α-amylase was approved (deregulated for field use) by the
USDA. This α-amylase-producing crop was developed by Base-
l-based Syngenta and marketed as Enogen (http://www2.syngenta.
com/en/index.html). Unfortunately, it has sparked worries not only
from anti-GM organizations, but also from some biotechnology sup-
porters because of environmental and human health questions
5.2. Plant-made therapeutic proteins
Because of the safety and economic concerns of using mammalian
cell cultures, plant-made therapeutic proteins are receiving renewed
interest by pharmaceutical companies. In fact, several plant-made
mammalian proteins such as human lactoferrin and lysozyme (Ven-
tria Bioscience), human aprotonin (ProdiGene), mammalian gastric
lipases (Meristem Therapeutics), and human intrinsic factor (Cobento
Biotechnology, Aarhus, Denmark) are now on the market (Obembe
et al., 2011; Paul and Ma, 2011; Sharma and Sharma, 2009). However,
they are regarded as either nutraceutical or diagnostic reagents rather
than pharmaceuticals. As of July 2011, there is no plant-made thera-
peutic protein approved for human systemic use by the US FDA.
Antibodies and vaccines are the two major classes of plant-made
therapeutic proteins that are under commercial development. Planet
Biotechnology took the lead in developing and commercializing Plan-
tibodies produced in tobacco; those being tested in clinical trials in-
clude CaroRX (for dental caries), DoxoRX (for side-effects of cancer
therapy), RhinoRX (for Rhinovirus prophylactic) and an IgG
(ICAM1) (for common cold) (Obembe et al., 2011). In addition, a
2G12 IgG used as prophylactic treatment for HIV is in Phase I clinical
trials by the Pharma-Planta Consortium (Paul and Ma, 2011), and a
plant-made scFv monoclonal antibody, used in downstream proces-
sing of a hepatitis B vaccine, has been commercialized in Cuba
(Pujol et al., 2005).
Subunit vaccine antigens produced in plants have received the most
attention in the molecular farming community. In 2006, Dow AgroS-
ciences received the world's first regulatory approval by the USDA for a
tobacco cell-based vaccine against Newcastle disease virus, which served
as a landmark for production of therapeutic (veterinary) vaccines in
plants. One of the unique features of plant-produced vaccines is that
plants not only serve as the production “bioreactor” but also as the deliv-
Several oral vaccines have been subjected to Phase I clinical trials, e.g., a
ing oral vaccines lie in the difficulty in dose standardization, concerns
linked to potential contamination of the food chain, and the low efficacy
of the oral route for immunization. However, the lower production and
delivery costs of these technologies may be essential for bringing these
rapidly, somewhat due to the recent outbreak of avian and swine flu,
which led to the development of seasonal and pandemic influenza vac-
cines. Such vaccines are currently in pre-clinical and clinical trials spon-
sored by Medicago Inc, The Texas Plant-Expressed Vaccine Consortium
and the Fraunhofer Center for Molecular Biotechnology. In addition, a re-
search group led by Charles J. Arntzen (a pioneer in plant-made edible
vaccines) at Arizona State University is engaged in developing several
plant-based vaccines including Hepatitis B surface antigen (Phase I in let-
of Escherichia coli and the capsid protein of Norwalk virus (all are Phase I
in potato) (Obembe et al., 2011; Paul and Ma, 2011).
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
In addition to antibodies and vaccines, there is special interest in
plant-produced therapeutic enzymes. The most encouraging example
is a plant (carrot) cell-produced recombinant glucocerebrosidase
(prGCD) used as enzyme replacement therapy for the treatment of
Gaucher disease (Shaaltiel et al., 2007). This prGCD was developed
by Protalix and will be marketed by Pfizer with the commercial
name of taliglucerase alpha (Uplyso™). It was awarded temporary
authorization for use in France in 2010, and thereby became the
first plant cell-made human pharmaceutical to enter the commercial
market. Phases I–III clinical trials were accepted by the FDA and
final approval is currently pending in the USA. Patient acceptance of
taliglucerase alpha was enhanced by supply and safety concerns
over Genzyme's CHO-based glucocerebrosidase, Cerezyme®. Produc-
tion of Cerezyme was shut down several times in 2010 by the FDA
due to viral contamination and follow-on production issues leading
to drug shortages. Against this backdrop, the availability of Protalix's
taliglucerase alpha highlighted the key advantages in safety and
cost of the plant-derived enzyme. In addition, commercial partner-
ship with Pfizer, the largest pharmaceutical company in the world,
suggests that plant-based bioproduction systems are being recog-
nized as competitive in this arena. Other plant cell-produced enzymes
in preclinical and clinical development by Protalix include a human
acetylcholinesterase (PRX-105) for several therapeutic and prophy-
lactic indications, α-galactosidase (PRX-102) for the treatment of
Fabry disease, and a pr-antiTNF, a biosimilar version of etanercept
(Enbrel™) for the treatment of rheumatoid arthritis.
5.3. Plant-made biopolymers
The commercial development of plant-made biopolymers lags far
behind the above two classes of recombinant proteins. Collagen, a
type of human protein that is fibrous and connects and supports
other bodily tissues, is under preclinical development by Medicago
Inc. (produced in alfalfa) and Meristem Therapeutics (produced in to-
bacco and corn) (Merle et al., 2002; Paul and Ma, 2011; Ruggiero
et al., 2000).
Plant-based bioproduction of proteins has evolved into a complex
arena supporting diverse production platforms that offer advantages
in scale, cost, safety, and regulatory acceptability. A comparison of
the advantages and remaining challenges of the main plant produc-
tion platforms is summarized in Fig. 2. As the field matures, there is
greater differentiation between industrial and pharmaceutical appli-
cation with the industrial enzymes targeting field-based production
primarily in seed/grain crops driven by scale and cost. In contrast,
therapeutic proteins are moving toward greater containment and
consistency through bioreactor-based systems or transient expres-
sion. It appears that plant-produced biologicals, long held as one of
the golden prospects of biotechnology, have finally come into their
own with commercialization of major plant-produced recombinant
proteins. With the exception of several plant-made enzymes that
were commercialized early, most plant-made pharmaceuticals only
recently entered the marketplace. Specifically, the human therapeu-
tic, taliglucerase alpha by Pfizer (Protalix technology), was the first
to enter the commercial market. Others at various stages of clinical
trials are anticipated to appear in the market over the next few
years. Significant progress has been made in the engineering of re-
combinant proteins for production by in vitro plant cell and tissue
cultures. Advances in bioreactor design and implementation have
further enhanced this potential, especially for growing more com-
plex 3-D cultures like hairy roots. Many early challenges related
to low protein yields and non-human glycosylation for therapeutic
Fig. 2. Comparison of benefits and challenges associated with the three major types of plant production platforms. Because they are common to all plant platforms, the advantages
of plants over other production systems, e.g., eukaryotic protein processing, lack of human pathogens, scalability options, are not listed here.
J. Xu et al. / Biotechnology Advances 30 (2012) 1171–1184
application have been overcome for select proteins and the field
continues to develop innovative solutions to enhance the competi-
tiveness and economic impact of plant-based systems. The green
phyto-pharm is thus, emerging as an alternative and important
source of safe and cost effective therapeutics as well as industrial
This work was supported in part by the Worcester Polytechnic In-
stitute and 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
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