Biotechnology and
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Plant-made pharmaceuticals:
Leading products and production
platforms
Matthew Paul and Julian K-C. Ma∗
Division of Cellular and Molecular Medicine, St. George’s Hospital Medical School, London, UK
Abstract.
The number of approaches to recombinant protein production
in plants is greater than ever before. Development of these
new and improved technologies as production platforms for
plant-made pharmaceuticals has and will continue to create
new commercial opportunities in the pharmaceutical sector.
However, it is inevitable that no single system will be optimal

C 2011 International Union of Biochemistry and Molecular Biology, Inc.
Volume 58, Number 1, January/February 2011, Pages 58–67 •
E-mail: jma@sgul.ac.uk
1. Introduction
The market for recombinant protein pharmaceuticals is large
and growing rapidly, with nearly all large pharmaceutical com-
panies reporting an increasing revenue share from these prod-
ucts rather than small-molecule drugs [1]. Typically, these phar-
maceuticals are manufactured using mammalian or bacterial
cell-based systems, which are complex to operate and inher-
ently vulnerable to contamination with human pathogens. Pro-
duction facilities compatible with current good manufacturing
practice (cGMP) require huge capital expenditure and involve
considerable financial risk. Plant biotechnology has the poten-
tial to overcome some of these limitations, but several hurdles
remain before this new industry can effectively compete in the
pharmaceutical sector.
Interest in the potential of plants as biofactories for recom-
binant proteins of pharmaceutical relevance was first piqued by
the report of a tobacco line engineered to accumulate a func-
tional murine monoclonal antibody (mAb) [2]. This report built
on previous work with transgenic plants and identified the plant
endomembrane system as a set of compartments in which com-
plex heterologous glycoproteins could correctly fold and assem-
ble. Other examples of recombinant protein pharmaceuticals in
plants followed and early descriptions include human serum
Abbreviations: PMP, plant-made pharmaceutical(s); cGMP, current good manufacturing
practice; ORF, open reading frame; IgG, immunoglobulin G; HIV, human immunodeficiency
virus; CHO, Chinese hamster ovary; UTR, untranslated region.
∗
Address for correspondence: Julian K-C. Ma, PhD, Professor, Division of Cellular and
Molecular Medicine, St. George’s Hospital Medical School, London SW17 ORE, UK;
Tel.: + 44 208 725 5818; e-mail: jma@sgul.ac.uk.
Received 3 December 2010; revised 13 December 2010; accepted 14 December 2010
DOI: 10.1002/bab.6
Published online 30 March 2011 in Wiley Online Library
(wileyonlinelibrary.com)
58
Applied Biochemistry
for the production of all recombinant proteins of interest in
plants due to both the physical characteristics and the
envisaged therapeutic application of each product. Here, we
review a range of promising product/platform pairs
emphasizing synergies during production and in clinical trials.
Keywords: plant-made pharmaceuticals, plant biologic production,
bioprocessing, clinical trials, biosimilars
albumin [3] and hepatitis B surface antigen (HBsAg) [4]. There
are now about 20 plant-made pharmaceuticals (PMPs) in de-
velopment as potential products (Table 1). After 20 years of
research and development (R&D), these candidates are now
starting to make an appearance in the marketplace.
Plant biotechnology for recombinant protein expression
now encompasses a range of different technologies. The first
approaches using transgenic plants have been supplemented
by new techniques, leading to dramatically improved yields and
product consistency. In combination with work to clarify and
mature the regulatory framework surrounding PMPs, these ad-
vances constitute potential for current and future commercial
enterprise in the field. In this review, we will compare plant
production methodologies and discuss the prospects of leading
PMPs with regard to regulatory requirements and commercial
potential.
2. The range of plant production
platforms
Technological development in the field has led to an expansion
in the number of well-developed systems available for the pro-
duction of recombinant pharmaceuticals in plants. Many plant
species are now amenable to genetic manipulation and the de-
tails of each have been reviewed elsewhere [19]. The use of a
certain set of genetic elements in combination with the trans-
gene of interest has allowed high-yield expression in both the
roots and leaves of transgenic plant lines as well as bursts
of transient expression in nontransgenic Nicotiana benthami-
ana plants. In this section, we will highlight the key features
of the main production platforms using products that are in
development.
 Table 1
Plant-made pharmaceuticals, vaccines, and dietary supplement proteins for human use

Indication/
Product Class application Organization Crop Status Reference
Milano
Apo-A1 Therapeutic Cardiovascular SemBioSys Genetics, Safflower Preclinical. Still under http://www.sembiosys.com
protein disease (Calgary, Canada) development as of http://www.sembiosys.com/
03/2010. Docs/ACC.10 Abstract.pdf
Plantechno srl. Transgenic Preclinical. Patent http://www.plantechno.com
(Vicomoscano, rice protection in the USA
Cremona, Italy) (US2010/0168006A1).

Production platforms for plant-made pharmaceuticals

Insulin (SBS-1000) Therapeutic Diabetes SemBioSys Genetics Safflower Phase I/II completed Q1 [5]
protein 2009.
Glucocerebrosidase Therapeutic Gaucher’s Protalix (Carmiel, Carrot cell Phase III trial complete [6],[7],
(UPLYSO) enzyme disease Israel) culture Sept. 2009. Currently http://www.protalix.com/&
available under the clinicaltrials.gov identifiers
FDA’s Expanded Access NCT00258778, NCT00376168
Program, full licensure
being sought.
Alpha-galactosidase Therapeutic Fabry’s disease Protalix Carrot cell Preclinical See website
(PRX-102) enzyme culture
Acetylcholesterase Therapeutic Biodefense Protalix Carrot cell Phase I (March 2010) See website
(PRX-105) enzyme culture
Antitumor necrosis Antibody Arthritis Protalix Carrot cell Preclinical See website
factor culture
(Pr-anti-TNF)
β-Glucosidase Therapeutic Gaucher’s Plantechno srl Tobacco seeds Preclinical See website
protein disease
TransPharma srl Transgenic Phase I See website
(Trieste, Italy) tobacco
2G12 IgG Antibody HIV Pharma-planta Transgenic Phase I (commencing Q2 http://www.pharma-planta.org
prophylactic consortium tobacco 2009)
Interferon-alpha Cytokine Hepatitis C Biolex Therapeutics Lemna Phase IIb (April http://www.biolex.com & [8]
modified release (Pittsboro, NC, USA) (Duckweed) 2009-ongoing)
(LocteronR)
Recombinant Therapeutic Thrombosis Biolex Therapeutics Lemna Preclinical See website
plasmin (BLX-155) enzyme prophylaxis (Duckweed)
Anti-CD20 mAb Antibody Non-Hodgkin’s Biolex Therapeutics Lemna Preclinical [9]
(BLX-301) lymphomas (Duckweed)
Human serum Therapeutic Maintenance of Agragen (Cincinatti, Flax Preclinical http://www.plantpharma.org/
albumin protein blood OH, USA) 2005/06/state-shouldnt-miss
plasma -opportunities-with-agragen/
pressure

59
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 60
Table 1
Continued

Indication/
Product Class application Organization Crop Status Reference
Lactoferrin Dietary GI infections in Ventria Bioscience (Fort Transgenic FDA GRAS application http://www.ventria.com/ & [10]
infants Collins, CO, USA) rice withdrawn
Meristem therapeutics Transgenic Phase I See website
(Clarmont-Ferrand, maize
France)
Lysozyme Dietary GI infections in Ventria Bioscience Transgenic FDA GRAS application [11]
infants rice withdrawn
(RhinoRx) Antibody Rhinovirus Planet Biotechnology Transgenic Phase II http://www.planetbiotechnology
prophylactic (Hayward, CA, USA) tobacco .com
leaves
Guy’s 13 SIgA Antibody Dental caries Planet Biotechnology Transgenic Phase II complete. http://www.planetbiotechnology
(CaroRx) tobacco Approved for use in the .com
leaves EU, but not marketed.
Collagen Structural Reconstructive Meristem therapeutics Transgenic Preclinical http://web.archive.org/web/
protein surgery maize 20071012232910/www.meristem
-therapeutics.com/rubrique.php3?
id rubrique=64
Therapeutic Cystic fibrosis, Meristem therapeutics Transgenic Phase IIa http://web.archive.org/web/
enzyme pancreatitis maize 20071012232733/www.meristem
-therapeutics.com/rubrique.php3?
id rubrique=38
Human intrinsic Dietary Vitamin B12 Cobento Biotech AS Transgenic Phase II complete. [12],[13]
factor deficiency (Aarhus, Denmark) Arabidopsis Marketed in the EU.
Pandemic and Vaccine Risk of Medicago (Quebec City, ProficiaTM Phase I complete Dec. http://www.medicago.com
seasonal (virus-like influenza Canada) infiltrated 2009. Phase II Q4 2010. Clinical trial NCT00984945
influenza vaccines particle) transmission N. benthamiana
Hepatitis B surface Vaccine Hepatitis B Arntzen group, Arizona Transgenic Phase I [14]
antigen State University potato
Thomas Jefferson Transgenic Phase I [15]
University/Polish lettuce
NAS (Posnan,
Poland)
Rabies glycoprotein Vaccine Rabies Yusibov group, Viral vectors in Phase I [16]
Fraunhofer USA spinach
Various anti-idiotype Vaccine Non-Hodgkin’s Bayer Innovation MagnICON Phase I (Dec. 2009) http://www.bayer-innovation.com
IgG antibodies lymphomas (Halle, Germany) infiltrated [17] clinical trial NCT01022255
N. benthamiana
Norwalk virus capsid Vaccine Norovirus Arntzen group, Arizona Transgenic Phase I [18]
protein vaccine State University potato

Biotechnology and Applied Biochemistry

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These production platforms are:
(1) Plant cell bioreactors.
(2) Plant tissue expression—leaves (transgenic and transient),
seeds, and exudates.
So far, correlations between the nature of a target recom-
binant protein and the best expression system for that pro-
tein have only been made on the broadest basis. A deeper
understanding of the biochemical nature of proteins that are
critical for different expression strategies is still required.
In addition, the regulatory framework relevant to the differ-
ent expression systems is certainly more advanced in some
cases than in others. Broadly, the relative strengths and weak-
nesses of each class of expression system is summarized in
Table 2.
2.1. PMPs from plant tissues
The first recombinant protein products from plant biotechnol-
ogy to reach the marketplace were not pharmaceuticals but
diagnostic enzymes and reagents. The production and purifi-
cation of egg white avidin and bacterial beta-glucuronidase
for commercial purposes were first reported in 1998 [20],[21],
and both have been marketed at some point through col-
laboration between the former biotech company Prodigene
(College Station, TX, USA) and Sigma–Aldrich (St. Louis, MO,
USA). However, despite active R&D efforts and venture capi-
tal, new nonpharma products entering the marketplace have
remained scarce. Both of these forerunner products were ex-
pressed in a transgenic food crop (maize) in an open-field set-
ting, in order to take advantage of the high protein yields of this
crop and the scalability and economy of conventional agronomic
practices [22].
The use of food crops for recombinant protein produc-
tion has been associated with negative sentiment among con-
sumer groups and the food industry, which has led to the U.S.
Food and Drug Administration (FDA) adopting a “zero toler-
ance” policy on PMPs transgene release and contamination of
food crops in current PMPs draft guidance documents to in-
dustry (www.fda.gov/downloads/Drugs/GuidanceCompliance
RegulatoryInformation/Guidances/ucm124811.pdf). The high-
profile loss of containment experienced by Prodigene has led
the United States Department of Agriculture/Animal and Plant
Health Inspection Service to impose additional requirements
on all future field releases of PMP crops in the USA (a copy of
the current draft environmental risk assessment is available at
http://www.aphis.usda.gov/brs/pdf/complete eis.pdf). These
factors have led to a noticeable shift away from open-field, trans-
genic food crops toward fully contained, nonfood crop systems
for PMPs manufacturing. Unfortunately, this has left the field
outside of the established route to commercialization, defined
by these forerunner nonpharma products.
2.2. Seed-based systems
Cobento Biotech AS (Aarhus, Denmark) has developed a produc-
tion system for recombinant human intrinsic factor (rhIF) based
on the thale cress Arabidopsis thaliana [12],[13]. A. thaliana is
Production platforms for plant-made pharmaceuticals
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a hardy annual weed with a short generation time that can be
readily transformed and grown at high densities in a glasshouse
environment. It is not cultivated for commercial use and has
no sexually compatible weedy relatives (as stated in the risk
assessment lodged with the European Food Safety Author-
ity, http://www.efsa.europa.eu/EFSA/DocumentSet/02 gmo
partii summary gmb12vitamin en.pdf?ssbinary=true), greatly
reducing the potential for dissemination of the transgene. The
yield of rhIF reported in Cobento’s system (70 mg/kg fresh
weight, [13]) can be compared favorably to that reported for the
production of rhIF using a baculovirus system (1–2 mg/L, [23])
and the methylotrophic yeast Pischia pastoris (30–40 mg/L,
[24]). As a widely used model plant, the molecular biology and
development of A. thaliana is extensively characterized and a
well-annotated genome is available. The identification of spe-
cific promoter sequences has resulted in the development of
transgenic lines that restrict the expression of transgenic gene
products to specific tissues or developmental phases. As the
rosette leaves of these plants are small and proportionally poor
sources of biomass, future development of this technology as
a competitive production platform may focus on achieving high
levels of transgenic protein accumulation in the seeds [25].
A unique approach to downstream purification from seeds
has been developed by SemBioSys Genetics (Calgary, Canada).
Seeds of many plant species accumulate large quantities of
triacylglycerides in oil bodies that are stabilized during seed
desiccation by the oleosins, a family of apolipoproteins. Re-
combinant proteins can be designed to associate with oleosin
in vivo either through a direct genetic fusion with an oleosin
open reading frame (ORF) or through a fusion with an ORF en-
coding a single-chain variable fragment (scFv) specific for en-
dogenous oleosin. Oil bodies accumulating recombinant protein
can then be readily separated from other protein-rich subcellu-
lar compartments by centrifugation. This approach has been
used to produce insulin in A. thaliana seeds [5] and produc-
tion for clinical trial has been scaled-up in safflower (Carthamus
tinctorius L.).
A seed-oriented production approach has also been
demonstrated for other plants, including cereals ( maize, rice,
barley, and wheat [26]), legumes (pea and soybean), and the
oilseed crop safflower. Ventria Biosciences (Fort Collins, CO,
USA) has developed human lysozyme [11],[27],[28] and lactofer-
rin [10] as products derived from transgenic rice grains. Through
a combination of technologies including codon optimization for
rice and the use of highly transcribed tissue-specific promoters,
researchers were able to obtain a yield of 5 g/kg dehusked rice
grain for lactoferrin. A high level of lysozyme accumulation in
rice endosperm was achieved by introducing two transgenes
into the plant simultaneously. Each gene was designed to en-
code lysozyme in the context of two distinct sets of cis-elements
and with two separate signal peptides, which have been shown
to influence the routing of the protein product through the se-
cretory pathway in rice [28]. Higher expression levels in these
double transgenic lines may be attributable to greater utiliza-
tion of endoplasmic reticulum subdomains for translation. This
approach may have general relevance to other plant platforms
wherein increased yields are required to achieve cost-effective
61
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Table 2
Production of plant-made antibodies and pharmaceuticals: a comparison of leading approaches

N. benthamiana
Open-field tobacco Glasshouse tobacco Rhizosecretion transient expression Cell culture
Typical model PMP Antibodies, Antibodies, Small proteins Antibodies, Veterinary vaccines,
or product antigens antigens (CV-N), antibodies antigens glucocerebrosidase (UPLYSO)
Yield (range) ++ ++ + +++ +++
(15–50 mg/kg leaf (15–50 mg/kg leaf (0.25–3 μg/mL/ (0.5–4 g/kg leaf
fresh weight) fresh weight) 24 H) fresh weight)
Scalability +++ + ++ ++ +
Consistency + ++ ++ ++ +++
Downstream + + +++ + ++
purification burden
Regulatory + ++ ++ + +++
development

production by reducing postharvesting (downstream) purifica-
tion costs.
2.3. Production in transgenic green tissues
Recombinant antibodies have unlocked the potential of mAbs
in immunotherapy by increasing both repertoire and affinity
while reducing the immunogenicity of nonhuman antibodies.
The high cost of production associated with the production of
these drugs in mammalian cell bioreactors combined with high
therapeutic doses required frequently limited access to these
treatments to a select group. The production of several classes
of antibodies and antibody-based molecules has been reported
in plants (reviewed recently [29]).
Planet Biotechnology (Hayward, CA, USA) has progressed
the development of CaroRXTM , a secretory immunoglobulin A
(SIgA) designed to block colonization of the oral cavity by
Streptococcus mutans through binding to the bacterial sur-
face adhesin (an archive of products in development is avail-
able at http://web.archive.org/web/20080531015736/http://
www.planetbiotechnology.com/products.html). SIgA is a het-
erodecameric complex consisting of four heavy chains, four light
chains, the J chain, and the secretory component of the poly-
immunoglobulin receptor. The development of a transgenic to-
bacco (Nicotiana tabacum) line expressing each of the four com-
ponent protein chains was achieved through conventional plant
breeding techniques. Despite lengthy generation times and the
need to screen many progeny, the use of transgenic lines bred
in this manner remains a robust approach for the production
of heteromultimeric complexes in plants. In this example, an-
tibody was produced from leaf matter grown in an open-field
site in Kentucky. The scalability of plant production in such an
environment, coupled with the application of conventional agri-
cultural machinery for planting, husbandry, and harvesting, is
an attractive economic argument for such an approach. How-
ever, the need to address tightening regulatory oversight, even
in case of a nonfood crop such as tobacco, may impact both the
business case and sustainability of open-field production.
The Pharma-Planta consortium (http://www.pharma
-planta.org), a European Union (EU)–funded collaborative re-
search project designed to define procedures and methods for
the production of pharmaceutical proteins in plants, is aiming
to deliver a second plant-derived mAb to clinical trials. 2G12 im-
munoglobulin G (IgG) was identified from human sera as a po-
tent, broadly neutralizing antibody against many human immun-
odeficiency virus (HIV) isolates [30]. A contained glasshouse
scenario was envisaged for production of 2G12 in transgenic
tobacco plants. The cost of biomass produced in such facilities
will always exceed that of open-field crops due to high start-
up costs, labor, and running costs. However, there are several
key advantages. Growing plants in a glasshouse under contain-
ment eliminates both potential gene flow through the escape of
pollen and release of the product into the environment, facilitat-
ing compliance with environmental regulations. Close control of
environmental factors can be used to optimize PMP yield in mod-
ern greenhouses, and temperature has been shown to affect the
yield of a murine IgG and the HIV microbicide cyanovirin-N (CV-
N) in transgenic tobacco [31]. The glasshouse environment also
limits the potential for physical damage to the crop, which has
also been linked to yield variability in IgG and CV-N tobacco
plants (unpublished data from our group). Both these factors
may improve batch-to-batch consistency after downstream pro-
cessing, which is a core principle of cGMP, an internationally
harmonized set of standards for the production of therapeutics.
Glasshouse-grown transgenic plants therefore represent
a technically feasible production system for PMP. However,
limited yields have hampered the adoption of such an envi-
ronment for commercial production purposes. Increasing PMP
yield decreases both the cost of plant cultivation and the burden
on downstream processing. High-level transient expression of
PMP transcripts can be achieved through the use of Agrobac-
terium leaf infiltration technique in combination with optimized
expression vectors. Medicago (Quebec City, Quebec, Canada)
has based a production methodology for PMPs including anti-
bodies and influenza vaccines (seasonal and pandemic) around
its proprietary ProficiaTM technologies. ProficiaTM incorporates
plasmid vectors based on highly transcribed cis-regulatory ele-
ments, including a range of promoter sequences isolated from
photosynthetic genes. These vectors are designed to include
multiple expression cassettes, allowing for the coexpression
of separate protein products, which may represent a subunit
of a multimeric PMP, an inhibitor of posttranscriptional gene

62 Biotechnology and Applied Biochemistry


silencing, or an enzyme involved in a glycosylation pathway
[32]. Using this system, the bioaccumulation of a mAb in N.
benthamiana was reported to approach 25% total soluble pro-
tein or 1.5 g/kg fresh leaf weight (g/kg flw) [32], a figure that
exceeds that of most transgenic plant lines expressing mAbs.
Medicago has recently committed to develop production facili-
ties for influenza vaccines using this system in both Canada and
France.
Further efforts to improve expression levels after Agrobac-
terium leaf infiltration have focused on the use of plasmids
designed to use sequences derived from plant viruses. These
sequences may take the form of simple translational enhancers,
such as the cowpea mosaic virus 5 and 3 untranslated regions
(UTRs) that form part of the pEAQ-HT series of vectors [33],
or they may include viral genes or even entire recombinant
genomes based within plasmid vectors. A vector system based
on bean yellow dwarf virus, a member of the single-stranded
DNA virus family Geminiviridae, utilizes two viral ORFs and two
viral UTRs to direct the synthesis and activity of a viral replicase
in plants [34],[35]. DNA copies of the expression cassette con-
sisting of a constitutive promoter, transgene ORF, and a termina-
tor produced by this replicase then act to maximize transcription
of the desired gene product. This system has been employed
on a laboratory scale in N. benthamiana leaves to produce an
Ebola-directed mAb (0.5 g/kg flw [35]) and virus-like particles
consisting of either the hepatitis C core antigen or norovirus
capsid protein (0.8 and 0.53 g/kg flw, respectively; [34]).
ICON Genetics (Halle, Germany), a wholly owned sub-
sidiary of Bayer Innovation (Dusseldorf, Germany), is devel-
oping a separate set of vectors for the commercial production
of patient-specific antibodies as idiotype vaccines for the treat-
ment of non-Hodgkin’s lymphomas (NHLs) [36]. In this system,
the leaves of nontransgenic N. benthamiana plants are infil-
trated with a mixed Agrobacterium culture capable of directing
the expression of an antibody idiotype isolated from the pa-
tient’s lymphoma in the context of either the tobacco mosaic
virus or potato virus X minigenome. Two viral genomes are rou-
tinely used to avoid segregation of one transgene as the virus
replicates. Both minigenomes include an RNA-dependent RNA
polymerase (RDRP) and are thought to boost protein yields
through a mechanism based on RDRP-directed transcript am-
plification as well as virus-directed cell-to-cell movement [37].
Yields of antibody (IgG1) in the latest version of this system
were reported to range from 0.5 to 4.8 g/kg flw [17]. The au-
thors estimate that 225 mg of product would suffice for quality
control and therapy of a single patient, and using the proposed
system, this quantity could be produced within 2 weeks of the
completion of molecular cloning. Other demonstrations of this
technology have reported yields of the fluorophore green fluo-
rescent protein in excess of 4 g/kg flw [38].
Given the advantages of speed and scalability inherent
to transient expression approaches, these yields can be com-
pared favorably to reported mammalian cell culture IgG yields of
10 g/L using an immortalized human retina cells (PER.C6 R ; Cru-
cell, Leiden, the Netherlands) or Chinese hamster ovary (CHO)
cells [39]. Although the high yield of transient expression sys-
tems greatly reduces the burden on downstream processing,
the concomitant introduction of the Gram-negative bacterium
Production platforms for plant-made pharmaceuticals
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Agrobacterium tumefaciens into the biomass increases the risk
of bacterial endotoxin contamination. Optimization of down-
stream processing procedures may reduce the release of endo-
toxin from bacterial cell membranes, but costly lipopolysaccha-
ride monitoring and removal is likely to be required for products
produced in this fashion to meet cGMP guidelines.
2.4. Plastids
In addition to the insertion of transgenes into the plant nucleus
and their transcription from either episomes or as integrated el-
ements, the introduction of transgenes into the plastid genome
has also been used to express high levels of recombinant pro-
teins in plant leaf tissue. Typically, this is achieved through the
microbombardment of leaf sections with gold particles coated
with linear DNA fragments designed to integrate into the plas-
tid genome via homologous recombination (HR). This approach
tends to yield high levels of protein accumulation due to the
high multiplicity of the transgene after segregation. In addition,
transplastomic gene expression is not limited by epigenetic ef-
fects often observed at chromosomal loci such as transcriptional
gene silencing and the positional effects of random transgene
insertion. Plastids lack posttranscriptional modification path-
ways, characteristic of the eukaryote secretory pathway, princi-
pally glycosylation, which may render them unsuitable for the
production of PMPs that rely on such modifications for stability
or functionality.
Currently, there are no companies actively seeking to ex-
ploit plastid-based technologies, despite numerous reports in
the scientific literature of chloroplast expression of proteins with
pharmaceutical potential, such as antigens and antibiotics (for
a recent review, see [40]). Chlorogen (St. Louis, MO, USA), now
a defunct biotech start-up, acquired or licensed an extensive
portfolio of Intellectual Property relating to chloroplast trans-
formation. The sale of this company’s assets may allow a new
player to attempt to commercialize this process in the future.
2.5. Plant bioreactors
The majority of recombinant protein pharmaceuticals currently
available are produced in bioreactors using mammalian, insect,
or microbial cells. Plant cell bioreactors maintain several key
advantages of plant-based production over conventional sys-
tems: they do not harbor human-trophic pathogens, and they
are typically cheaper to operate and scale-up due to the robust
nature and simple growth medium requirements of plant cells
[41]. These systems are not subject to several perceived disad-
vantages of whole plant PMPs production as there is a greatly
reduced potential for gene flow and contamination to the envi-
ronment and food chain, and a greater compatibility with cGMP.
Importantly, the downstream processing of product may be sim-
plified by the comparative lack of secondary metabolics, fibers,
and oils or waxes compared with production in whole plants.
Challenges remain over the yield of many products in plant cell
bioreactors, with reported yields for recombinant antibodies
falling far short of those reported in mammalian (CHO) cell sys-
tems. Recent research has suggested that this disparity may be
at least partly due to the comparative lack of maturity of plant
63

cell systems with regards to media composition, fermenter de-
sign, and cell line engineering [42],[43].
Two companies are actively involved in PMPs pro-
duction from cell culture-based systems. Dow Agrosciences
(Indianapolis, IN, USA) have developed a plant cell biore-
actor system (ConcertTM ) for the production of veterinary
vaccines. The lead product, a subunit vaccine against
Newcastle disease in poultry, has been produced using
transformed N. tabacum-1 (NT-1) cells cultivated in a tra-
ditional nondisposable bioreactor (US patent application
US2008/0076177, Cardineau et al.). Despite receiving regula-
tory approval in 2006 (the corresponding press release is avail-
able at http://web.archive.org/web/20080123120809/http://
www.dowagro.com/animalhealth/resources/firstlic.htm), no
attempt to commercialize this product has yet been made,
although a second vaccine employing this technology is still
under development (http://www.dowagro.com/animalhealth/
resources/news/20070827b.htm). Comparatively low product
yield [8 μg/mL in culture medium, (US patent application
US2008/0076177, Cardineau et al.)] may be a limiting factor
to commercial production with this system.
Protalix Biotherapeutics (Carmiel, Israel) has employed
a system using carrot cells and disposable, highly scalable
polyethelene bioreactors to produce three candidate products.
The production of the lead candidate, human glucocerebrosi-
dase (GCD), has been described [6]. Recombinant GCD puri-
fied from CHO cells (Genzyme’s Cerezyme R ; Genzyme, Cam-
bridge, MA, USA) is currently used as an effective treatment
for Gaucher’s disease, a lysosomal storage disorder primarily
affecting macrophages. The use of Cerezyme R is limited by the
high cost associated with production and the postproduction
enzymatic modification of glycan residues necessary for effec-
tive macrophage uptake. Protalix has made significant modifica-
tions to the protein to alter its subcellular accumulation within
the cells, including the addition of a C-terminal vacuolar sorting
signal from tobacco chitinase [44]. Subsequent glycoanalysis of
plant recombinant GCD (prGCD) revealed the presence of pauci-
mannosidic glycans suitable for macrophage uptake [6], hence
eliminating the requirement for further processing to expose
these ligands.
Moss is adaptable for growth in culture vessels and
has been developed as an expression platform for PMPs
by Greenovation GmbH (Heilbronn, Germany; http://www
.greenovation.com) [45]. Moss protonema cultivated in bioreac-
tors is capable of photosynthesis, further reducing the nutrient
requirements in the culture medium. In Greenovation GmbH’s
system, the recombinant protein product is designed to be se-
creted from the protonema and harvested from the medium.
Moss is an unusual plant as it has been shown to undergo HR
at nuclear loci at a significant frequency [46]. Combined with
excellent genomic resources [47], this trait allows researchers
to design transgenes to integrate into specific regions of the
moss genome. This approach may reduce variability in expres-
sion levels between transgenic lines, thus facilitating a rapid
and predictable scale-up of production. Second, knowledge of
the integration site of the transgene may be advantageous to
any application for IP protection and regulatory approval. Fi-
nally, targeted mutation of the moss genome using HR can be
64
14708744, 2011, 1, Downloaded from https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.6 by INASP/HINARI - INDONESIA, Wiley Online Library on [28/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
used to create moss lines with improved protein processing
characteristics, for example, engineered glycosylation patterns
[48].
Lemna species, collectively known as duckweed, are
protein-rich, clonal, and rapidly growing aquatic plants that
are adaptable as PMP production systems [49]. Using Lemna
as an expression platform, Biolex Therapeutics (Pittsboro,
NC, USA) has focused on developing products with superior
clinical efficacy than existing related drugs on the market.
BLX-301, a CD20-specific antibody with potential to treat
NHLs, is produced in the context of a regulated glycosylation
pathway achieved through the use of RNA interference.
Antibodies produced in this fashion were shown to bear a
more homogeneous population of glycans that correlated with
improved antibody effector functions [9]. A second product,
human plasmin (BLX-301), has been under investigation as an
anticoagulant for over 50 years. However, owing to difficulties
encountered during the purification of active plasmin for
blood sera and later from nonplant recombinant sources, and
the relative tractability of enzymes upstream in the plasmin
pathway, no therapeutic version has yet been developed.
Recently, the therapeutic potential of two deleted versions
of plasmin expressed in Escherichia coli was demonstrated
[50],[51]. In contrast, full-length human plasmin accumulated
in an active conformation and at high yields in the Lemna
production system, and results for preclinical trials are available
(http://www.biolex.com/pdfs/Biolex%20Press%20Release%
20-%20SIR%20Presentation%2031808.pdf). It is proposed
that the domains retained in the Lemna product may enhance
efficacy by increasing affinity for fibrin, the major component of
blood clots. Lemna-derived plasmin may also benefit from an
improved safety profile as it is correctly inhibited by endogenous
plasmin inhibitors. The development of the company’s lead
product, interferon-alpha-2b in a novel modified-release formu-
lation (Locteron R ), is now supported by significant venture cap-
ital (Biolex press release available at http://www.biolex.com/
pdfs/Biolex%20Series%20D%20-%20October%206%202008
.pdf). This investment, the fourth injection of funds into the
company, underlines the confidence of the biotech sector in
Biolex’s technology and product portfolio.
2.6. Plant exudates
The collection of recombinant proteins from the space around
the tissues of transgenic plants is an attractive modality for
PMP production. Two approaches have been described: the col-
lection of apoplastic fluids either as tobacco leaf guttation flu-
ids [52], or via vacuum collection [53], and of root exudates
(rhizosecretion) [54],[55]. Compared with homogenized plant
tissue, these fluids are excellent feedstocks for downstream
purification as they do not require expensive and lossy initial
processing steps such as mechanical disruption and extract clar-
ification. Furthermore, as the plant tissue is not disrupted during
harvesting, the feedstock is not contaminated with intracellular
proteases or degraded fragments of the product, and continu-
ous production can be envisaged. These approaches also retain
many advantages of using whole plants: genetic stability [56],
photoautotrophy, and scalability. Despite these advantages, the
Biotechnology and Applied Biochemistry

vacuum collection of alpha-galactosidase A from plant leaves as
demonstrated by the former biotech company Biosource Tech-
nologies remains the only reported large-scale application of a
nondestructive harvesting approach [57].
Because of the porosity of plant cell walls, the accumu-
lation of recombinant proteins in exudates is influenced by the
effective molecular radius of the molecule. These systems may
therefore be most suited to the production of smaller proteins,
or those with compact structures, such as antibodies [54],[58].
Noncovalent interactions between the PMPs and molecules
present in plant tissues may also prevent efficient harvesting
through washing with a physiological buffer. High ionic strength
buffers have typically been used to disrupt these interactions
and release PMPs from suspension cultured plant cells [59],
but it remains to be shown whether this approach is viable in
rhizosecretion models under continuous production.
Product yield has remained a major drawback for systems
relying on plant tissue exudates. Guttation of secreted alkaline
phosphatase (SEAP) from transgenic tobacco leaves was quan-
tified as 0.15–1.1 μg/g leaf dry weight per day [52]. The authors
state that no attempt was made to optimize yield in this system;
nevertheless, it is difficult to envisage a cost-effective engineer-
ing solution to collect this material. Efforts to increase rhizose-
cretion yield have included the use of root-specific promoters
[60], induction of hairy roots [61], co-expression of secreted
protease inhibitors [62], and the addition of plant growth reg-
ulators [54]. In the last example, the maximum rate of rhizose-
cretion of a murine IgG and cyanovirin-N (CV-N) was reported
as 58 and 766 μg/g root dry weight/24 H, respectively. These
yields represent a fivefold increase over previously reported
rates [63],[64]. Establishing a suitable engineering solution for
the collection of hydroponic fluid on a large scale, either through
the use of recirculating nutrient film technique trays or batch
production in sterile containers, is the next step in developing
a rhizosecretion-based PMPs production system.
2.7. Clinical trials
As with all novel drugs, PMPs must pass through a series of
clinical trials in order to gain regulatory approval for marketing.
The precise path of each PMP through clinical trials is subject to
the nature of the drug; for example, plant-produced versions of
biologic pharmaceuticals already on the market (“biosimilars”)
or drugs designed to treat orphan or rare diseases may enjoy a
shorter passage through the approval process. Such drugs may
therefore be attractive candidates for development as PMPs.
It is clear, however, that the production process of candidate
PMPs must be designed from the very beginning to cope
with the regulatory requirements for clinical trials. Guidance
for the production of recombinant protein pharmaceuticals
specifically from transgenic plants has now been adopted
in the EU (EMEA/CHMP/BWP/48316/2006). A more general
set of guidelines is under draft in the USA (FDA CVM GFI
#153, www.fda.gov/downloads/Drugs/GuidanceCompliance
RegulatoryInformation/Guidances/ucm124811.pdf) that also
covers some aspects of transient expression systems. In the
case of transgenic plants, these guidelines establish require-
ments for host plant characterization and seed banking. Spec-
Production platforms for plant-made pharmaceuticals
14708744, 2011, 1, Downloaded from https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.6 by INASP/HINARI - INDONESIA, Wiley Online Library on [28/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ifications for PMPs characterization must be established on a
case-by-case basis but should include a suitable comparison
with the natural counterpart where feasible and appropriate.
The analysis of plant-specific modifications, such as spe-
cific complex glycosylation patterns and other posttranslational
modifications, is an important component of PMP specifica-
tions. Considerable concern has centered on the induction of
inappropriate immune responses to parenterally administered
PMPs triggered by plant-specific glycoforms. Production using
seed crops, or in systems wherein the transgene product is
effectively retained in the early secretory pathway, may be pre-
ferred in order to limit the addition of these moieties. How-
ever, there is little evidence that these reactions, when they
do occur, are clinically significant [65]. Indeed, in the results
of a phase I clinical trial of prGCD (Protalix Biotherapeutics
trial reference NCT00258778 and [7]), no specific antibodies
were raised to prGCD, which bears a proportion of glycans with
plant-specific monosaccharides and sugar linkages. In the case
of this drug, the orphan disease status of the indication in
combination with the high cost of existing therapies has al-
lowed Protalix to apply for a “fast track” to FDA approval (a de-
scription of the process is available here http://www.fda.gov/
AboutFDA/CentersOffices/CBER/ucm122932.htm), which in-
cludes enhanced regulatory oversight and rolling reviews of
the new drug application (NDA). The clinical use of prGCD has
also been accelerated by the apparent contamination of the ex-
isting CHO cell-derived product (Cerezyme R , Genzyme) with a
potentially pathogenic calcivirus (Genzyme press release avail-
able at http://www.genzyme.com/corp/media/GENZ%20PR
-061609.asp). In contrast, most plant systems offer limited or
no exposure to human-trophic viral adventitious agents.
Glycoprotein pharmaceuticals from all production sys-
tems are routinely analyzed for the homogeneity and nature of
their associated carbohydrates [66], including those produced
using mammalian cell systems, as these moieties can influence
the potency and pharmacokinetic profile of the drug. Various
plant systems including those based on Nicotiana spp. and
Lemna have been developed with altered glycosylation path-
ways [9],[67],[68], opening up the possibility of glycoengineer-
ing PMPs for improved therapeutic effect. This has particular rel-
evance to antibodies, such as Bayer Innovation’s anti-idiotype
NHL vaccines, which are currently undergoing phase 1 clini-
cal trials (clinicaltrials.gov identifier NCT01022255). It will be
interesting to compare the results of this trial against a previ-
ous iteration of this technology, which utilized plant-produced
scFvs [36].
Insulin produced using the safflower seed oil body sys-
tem (SBS-1000, Sembiosys Genetics) has progressed through
a phase I/II trial involving 23 healthy volunteers. The human
arm of the study aimed to establish bioequivalence between
SBS-1000 and Humulin-R R (Eli Lilly, Indianapolis, IN, USA), a
widely used off-patent insulin product produced in bacterial fer-
mentors. In each group, the total glucose infusion required to
maintain a euglycemic clamp and insulin concentration in the
serum of healthy volunteers was found to be within an equiva-
lence range. Adverse reactions to the product were also within
an acceptable profile for the administration of recombinant hu-
man insulin. This demonstration of bioequivalence will likely
65

reduce the requirement for further clinical efficacy data in or-
der to obtain a license from the relevant agencies. Guidance
on this process has been issued in the EU (CHMP/437/2004),
and in the USA certain biosimilar products have been approved
for an abbreviated NDA.However, as biosimilar products such
as SBS-1000 lack demonstrable clinical advantages over rival
generics, the economic advantages of the plant production sys-
tem must be sufficient on their own. In contrast, a phase I trial of
interferon-alpha from Lemna (Locteron R , Biolex Therapeutics
[8]) aimed to demonstrate a superior pharmacokinetic profile
when compared with Intron A (Merck, Whitehouse Station, NJ,
USA) through the use of a modified-release formulation, and
will therefore require full approval as a new drug. Nevertheless,
formulating PMPs to achieve superior clinical efficacy may be
the key to market penetration.
Subunit vaccines delivered as unprocessed or minimally
processed plant biomass preparations have also entered phase
I clinical trials. These include HBsAg from potato [14] and let-
tuce [15], rabies glycoprotein from spinach [16], enterotoxigenic
E. coli heat-labile toxin B subunit from corn [69] and potato [70],
and norovirus nucleocapsid from potato [18]. Each preparation
was well tolerated. The efficacy of this approach will be largely
determined by the relevant stability of the antigen in the gut,
the development of suitable adjuvants, and methods to ensure
consistent dosing.
3. Conclusions
In this review, we have covered only some of the approaches
described to generate recombinant proteins from plants. The
myriad of plant production systems available offers unrivalled
flexibility to create financially and technically viable approaches
for the production of PMPs, but the lack of focus on a particular
technology has doubtlessly delayed the application of plant
biotechnology as a whole to this field. Thus, the pathway to
commercialization for PMPs remains unclear. It is likely that
the first wave of human therapeutics from plants will employ
cell culture approaches, by virtue of compatibility with existing
regulatory frameworks.
Three broad classes of drug have received particular at-
tention from the PMP community. The first class is therapeu-
tic antibodies and antibody-derived proteins. Antibody PMPs
manufacture is now supported by a well-developed set of tech-
nologies, which can offer excellent yields and unique technical
advantages. Indeed, plants remain the only viable production
platform for the manufacture of SIgA molecules. Antibodies pro-
duced in plants have been approved for topical use in the EU
(CaroRXTM , Planet Biotechnology) and are progressing through
clinical trials for systemic use in the USA (NHL scFvs). Trans-
genic tobacco, in either an open-field or contained glasshouse
environment, offers an inexpensive manufacturing route that is
currently employed in the production of antibodies for topical
use (e.g., CaroRXTM and mAb 2G12) as well as an antibody used
in downstream processing of a hepatitis B vaccine (anti-HbsAg;
CIBR, Cuba; [71]). The involvement of the EU Pharma-Planta con-
sortium in bringing mAb 2G12 to the brink of a phase I clinical
66
14708744, 2011, 1, Downloaded from https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.6 by INASP/HINARI - INDONESIA, Wiley Online Library on [28/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
trial has left clear regulatory guidelines in place for greenhouse
production of PMPs from whole plant biomass.
The second group is subunit vaccine antigens. Noninva-
sive immunization by oral delivery is an appealing route of im-
munization, and plant expression hosts have linked with oral
delivery since the inception of the technology. Several “edible
vaccines” have progressed to phase I clinical trials, although
important issues regarding dose standardization and contam-
ination of the food chain, as well as the efficacy of oral vac-
cination, remain to be addressed. Plant-produced oral vaccine
antigens may find greatest relevance as the boosting compo-
nents of heterologous prime-boost strategies, as demonstrated
for measles and hepatitis B vaccines [14],[72]. The rapid pro-
duction of vaccine antigens in transiently transfected leaves of
N. benthamiana provides a further potential advantage of plant
production for vaccine antigens. This approach is exemplified
by the seasonal and pandemic influenza vaccines currently in
clinical trials sponsored by Medicago.
The third class of PMPs is therapeutic enzymes. At least
initially, PMPs in this class are likely to be “biosimilars” such as
insulin (SBS-100, SemBioSys Genetics), glucocerebroside (UP-
LYSO, Protalix), and interferon-alpha (Locteron R , Biolex Thera-
peutics). These products benefit from a potentially abbreviated
review process and streamlined product development. The use
of PMP technologies may allow the marketing of biosimilar ver-
sions of drugs with extant patent protection in other production
systems. However, in view of the vast manufacturing capacity
already in place in the generics sector, PMPs must also rely
on the advantages of a plant production platform to compete
effectively.
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