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199
Foreign protein production in plant tissue cultures
Pauline M Doran
Foreign proteins synthesised by plants are now in the
marketplace, and clinical trials for plant-derived therapeutic
proteins are underway. Economic analysis of plant production
systems has helped identify the types of protein that would be
most suitable for manufacture using tissue culture methods.
The major advantages associated with in vitro plant systems
include the ability to manipulate environmental conditions for
better control over protein levels and quality, the rapidity of
production compared with agriculture, and the use of simpler
and cheaper downstream processing schemes for product
recovery from the culture medium.
Addresses
Department of Biotechnology, University of New South Wales, Sydney,
NSW 2052, Australia; e-mail: [email protected]
Current Opinion in Biotechnology 2000, 11:199–204
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
ER
endoplasmic reticulum
GMP
Good Manufacturing Practice
Introduction
Foreign proteins can be synthesised using microbial cell
culture, animal cell culture, plant tissue culture, transgenic
animals and transgenic plants. Choosing the most appropriate method for commercial production requires
case-by-case analysis. A wide range of factors must be considered, including cost of production, market volume, the
efficacy, safety and stability of the product, and whether
the foreign protein has different biochemical or pharmacological properties compared with material from existing
sources. Although most interest during the past 15–20
years has been focused on microbial and animal cell cultures, plants and plant cells are now considered as viable
and competitive expression systems for large-scale protein
production. The development of transgenic plants and
plant viral vectors for foreign protein synthesis has been
reviewed extensively [1•,2–6].
Factors in favour of plant systems as sources of animalderived proteins include: the potential for large-scale,
low-cost biomass production using agriculture; the low risk
of product contamination by mammalian viruses, bloodborne pathogens, oncogenes and bacterial toxins; the
capacity of plant cells to correctly fold and assemble multimeric proteins; low downstream processing requirements
for proteins administered orally in plant food or feed; the
ability to introduce new or multiple transgenes by sexual
crossing of plants; and the avoidance of ethical problems
associated with transgenic animals and the use of animal
materials. There are, however, potential issues of concern
for plant protein production: containment of genetically
modified plants in the environment; allergic reactions to
plant protein glycans and other plant antigens; product
contamination by mycotoxins, pesticides, herbicides and
endogenous plant secondary metabolites; and regulatory
uncertainty, particularly for proteins requiring approval for
human drug use.
An important indicator of the cost competitiveness of
plant-based foreign protein synthesis is the increasing
involvement of industrial groups. Commercial processes
for the production of avidin, β-glucuronidase and aprotinin
from transgenic corn have already been developed
[7,8•,9•]. Plant-derived non-injectable immunotherapeutic
and vaccine proteins are also progressing through human
clinical trials; these include a secretory IgA/G antibody
against oral bacterial colonisation [10•], an edible vaccine
against an Escherichia coli enterotoxin [11•] and an edible
vaccine against hepatitis B [12•]. Details of company activity in selected areas, including plant production of
monoclonal antibodies, vaccines and industrial enzymes,
are provided in recent reviews [2,3,5,13••].
An alternative but less well-developed technology for producing foreign proteins is plant tissue culture. Using this
approach, plant cells in differentiated or dedifferentiated
states are grown axenically in nutrient medium in bioreactors under controlled conditions, with foreign protein
harvested from either the biomass or culture liquid, or a
combination of both. Although plant tissue culture may
not be suitable for all applications of foreign protein synthesis, such as food-based production of edible vaccines, it
offers a number of advantages for the manufacture of proteins that are extracted and purified after synthesis. The
purpose of this review is to outline the potential advantages and limitations associated with plant tissue culture
for foreign protein production, and to summarise recent
developments in the area. The review focuses on the production of proteins of direct commercial value. Application
of foreign proteins in plants for metabolic modulation or to
improve pathogen resistance or nutritional value are discussed elsewhere [14–16].
Recent applications of plant tissue cultures for
production of foreign proteins
Suspended plant cells have been used in several recent
studies as a means of producing a variety of foreign proteins. These include recombinant antibodies and antibody
fragments [17–23], enzymes such as β-glucuronidase [24]
and invertase [25], and proteins of therapeutic value such
as human interleukin (IL)-2 and IL-4 [26], ribosome-inactivating protein [27], ricin [28], and human α1-antitrypsin
[29•,30•]. The most commonly used host species for protein synthesis in suspension cultures is tobacco [17–28],
although rice cell cultures have also been tested [29•,30•].
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Biochemical engineering
Several stirred bioreactor studies at volumes up to 40 L
have been reported, using batch [22,23], continuous [25]
and two-stage [29•] modes of operation. Hairy root cultures
of transgenic tobacco have also been tested for production
of recombinant IgG1 antibody [18,31], including in a 2 L
sparged bioreactor [18]. In other work, bacterial xylanase
and human placental alkaline phosphatase were produced
using hydroponic culture of transgenic tobacco plants in
sterile, sucrose-containing medium in flasks [32].
Large-scale plant tissue culture versus
agriculture
For bulk manufacture of proteins such as those used in
industrial, analytical and diagnostic applications, most production systems will find it hard to compete with the low
cost of agriculture. A detailed economic evaluation of
β-glucuronidase extraction and purification from transgenic corn yielded a production cost of only US$43 g–1 for
an initial seed protein concentration of 0.015% dry weight,
a product purity of 83%, and an annual production volume
of 137 kg [33••]. Costs of research and development, royalties and sales were not included in the analysis. In
comparison, the cost of producing 100 kg of protein from
transgenic goats’ milk has been estimated at US$105 g–1,
whereas manufacture using mammalian (Chinese hamster
ovary) cell culture costs US$300–3000 g–1 depending on
the product yield and other operating factors [34].
Agricultural production of protein has been reported to be
10–50 times cheaper than E. coli fermentation, even
though product levels in bacteria are higher than those in
plants [35]. Foreign protein production using greenhousecultivated plants is considerably more expensive than with
field-grown crops; recent estimates are US$500–600 g–1
based on operations in a 250 m2 greenhouse and including
heating, labour and consumables for protein extraction and
purification [36].
accumulate protein levels at least an order of magnitude
higher than those achievable in agricultural systems. This
would reduce the costs associated with product recovery to
offset the substantially higher cost of biomass production.
Plant cell cultures provide greater opportunity for manipulation of foreign protein levels, as culture conditions can be
altered much more readily in reactors than in the field. For
example, supplementation of suspended plant cells with
amino acids prior to harvest resulted in a transient threefold
increase in recombinant antibody levels [21], while accumulation of recombinant ricin in tobacco culture medium
was sensitive to the concentration of auxin provided [28]. In
other work, exposure of suspended plant cells to dark/light
cycles was used to control foreign gene expression and protein titre [24]. Whereas these examples demonstrate how
protein production might be enhanced in plant cell cultures, some studies have shown that recombinant protein
levels are significantly lower in plant cell suspensions than
in whole plants or organ cultures such as hairy roots [19,20].
The reasons for this are unknown at present, but could
relate more to post-synthesis product stability rather than to
expression levels per se. Technology to improve foreign protein accumulation in culture systems must be developed if
product levels significantly greater than those achievable in
whole plants are required for economic feasibility.
In vitro cell culture offers intrinsic advantages, or could
even be a necessity, for foreign protein synthesis in certain
situations. Plant tissue culture may be the most suitable
production system when small-to-medium quantities of
specialty, high-price, high-purity proteins are needed, such
as in some therapeutic applications. The time involved for
production using plant cell culture is significantly shorter
than the growth cycle of whole plants; therefore, compared
with the months needed for agriculture, proteins could be
manufactured in days or weeks on a time-scale compatible
with clinical demands. Because bioreactors provide a much
more controlled and reproducible environment than the
open field, regulatory requirements for therapeutic proteins can be met more easily using reactor-grown cells. In
addition, when the protein of interest is secreted into the
medium of plant cell cultures, product recovery and purification could be greatly simplified and much cheaper than
from plant biomass, while eliminating the need to destroy
the cells. However, if the most probable niche for plant cell
cultures is the production of therapeutic proteins, an
important issue is post-translational processing of proteins
and the effects of plant glycans on the human immune system. These factors, together with recent developments
influencing the choice of plant cell culture as a production
vehicle for foreign proteins, are discussed below.
The cost of transgenic corn seed for extraction of foreign
protein has been estimated as only US$0.20 kg–1 [33••].
This includes a premium of US$0.09 kg–1 to provide
incentive to farmers and grain handlers, and to cover any
additional costs associated with growing the transgenic
plants and keeping them and their seeds isolated from
other plants and grain products. Although specific cost
analyses for protein production using plant tissue culture
have not been provided in the literature, it would be difficult to produce a kilogram of plant biomass in a reactor at
such a low price. Despite plant culture medium being substantially cheaper than animal culture medium, the cost of
the medium alone for generation of 1 kg of plant cells is
substantially greater than US$0.20, without factoring in
other costs associated with bioreactor operation, such as
labour, utilities, waste treatment, equipment depreciation,
and so on.
Good Manufacturing Practice, process
reproducibility and product quality
Plant cell cultures are likely to be uncompetitive for the
majority of proteins required in the large quantities deliverable by agricultural production, unless they are able to
The regulatory issues relevant to Good Manufacturing
Practice (GMP) for production of therapeutic proteins in
plants have been outlined in recent papers [37,38••].
Present indications are that there is no a priori barrier to
Foreign protein production in plant tissue cultures Doran
the production of therapeutic proteins using plants grown
in open fields, even though this production system is subject to the vagaries of weather, insect activity, soil
variability and other factors that influence product yield
and quality (Z Nikolov, personal communication). If, as
seems likely, regulatory acceptance of the product will
depend mainly on its compliance with purity, efficacy and
biocomparability standards at the end of the production
chain, field-grown protein will be able to satisfy requirements if sufficient safeguards are designed into the process
so that undesirable components are either prevented from
forming or removed during purification. Reactor culture
could make regulatory compliance easier by reducing variation in production conditions and increasing average
batch quality; whether or not this is a crucial advantage will
depend on the properties of the particular system and their
susceptibility to environmental influences. Although variation in the level of protein accumulation has been
reported for transgenic plants in different growing locations [7], lot-to-lot consistency of purified plant vaccine
proteins and conformity with regulatory specifications
have also been demonstrated [39].
Developments in the area of inducible promoters may lead
to future alleviation of problems with environment-related
variability in field-grown plants by separating the biomass
growth and protein synthesis steps. For example, using a
recently patented post-harvest expression system [P1], plant
tissues harvested from the field can be induced to produce
foreign protein during storage in laboratory or GMP facilities. Because conditions during actual product synthesis are
controlled to a greater extent than is possible with constitutive promoters and outdoor plants, better product yield and
less variation in product quality can be expected. Such
improvements in agriculture-based production systems
could reduce the potential benefits associated with plant tissue culture with respect to regulatory compliance.
The ability of plant cells and organs to propagate indefinitely in tissue culture without the need for sexual reproduction
offers a solution to other problems relating to gene segregation and long-term transgene stability in agricultural crops.
Variations in foreign protein expression within and between
successive generations of corn have been reported [7,9•], as
well as loss of fertility and/or pollen transmission of the transgene [7] and loss of the original selection marker [7,9•].
Greater stability may develop with increasing number of
generations [9•]; however, it is probable that breeding programs with continuous selection of elite lines will be
necessary for consistent high-level protein production using
whole plants. This situation could be improved by the use of
perennial host species [36]. In contrast, there are several
reports of stable, long-term foreign protein synthesis in plant
tissue cultures, such as carrot invertase in tobacco suspensions over four years [25] and IgG1 antibody in tobacco hairy
root cultures over 19 months [18]. Gene silencing occurs in
plant tissue cultures as well as in whole plants (EJ Finnegan,
personal communication); however, if transmission of signals
201
for systemic post-transcriptional silencing occurs via plasmodesmata and the vascular system [40], the conditions of
minimal cell–cell contact existing in plant suspensions could
be advantageous for stable foreign protein production. On
the other hand, dedifferentiated plant cells, such as those in
suspension cultures, are subject to a range of genetic modifications through the mechanisms of somaclonal variation.
Further studies are needed to verify whether better longterm stability of protein production can be achieved in vitro
compared with whole-plant systems.
Downstream processing, protein secretion
and stability
Recovery and purification of proteins from plant biomass is
an expensive and technically challenging business, requiring multiple separation steps such as precipitation,
adsorption, chromatography and diafiltration. In a recent
cost analysis of β-glucuronidase production from transgenic
corn seed, milling, protein extraction and purification operations accounted for ~94% of the production cost [33••]. As
the corn itself contributed only 6%, the importance of downstream processing in determining the economic feasibility of
plant protein production is very clear. Costs of protein recovery from seed could be reduced if most of the protein is
localised in a separable component such as the embryo
[8•,41•], or by targeting protein expression to seed oil bodies
for easier extraction and purification [13••].
Because plant nutrient media are relatively simple solutions with no added proteins, if a foreign protein is
produced in tissue culture and secreted into the medium
rather than stored inside the cells, product recovery and
purification could be carried out in the absence of large
quantities of contaminating proteins. Economic analysis of
protein recovery from spent plant culture medium has not
been reported in the literature. Yet, significant cost advantages may be derived from this type of production system,
despite the protein being diluted to low concentrations in
the relatively large liquid volume. Extracellular protein
secretion is effected in plant cells using signal peptides
that direct transport of peptides into the lumen of the
endoplasmic reticulum (ER). After folding and assembly
in the ER, unless the protein is carrying a signal that targets it for retention in the ER or transport to another
organelle such as the vacuole, proteins leaving the ER
enter the default secretory pathway to the Golgi apparatus
and finally to the extracellular space [42].
Whether or not secretion is the best strategy for protein accumulation depends on the relative stability of the protein in
the extracellular environment. For some recombinant proteins, highest accumulation is achieved by retention in the
ER. For example, carboxy-terminal fusion of the KDEL signal peptide to single-chain antibody variable-region
fragments (scFvs) and subsequent ER retention has been
found to increase antibody levels by a factor of up to 10–100
compared with either extracellular secretion or expression in
the cytosol [1•,20,43]. There is also evidence that secreted
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Biochemical engineering
0.30
3.0
0.25
2.5
0.20
2.0
0.15
1.5
0.10
1.0
0.05
0.5
0.00
0
10
20
30
40
50
Antibody in the medium (µg mL-1)
Antibody in the biomass (mg)
Figure 1
Expression and secretion of assembled IgG1
antibody during culture of transgenic tobacco
hairy roots. (䊉) Antibody in the biomass; (䊊)
measured antibody concentration in the
medium. The error bars indicate standard
errors from triplicate cultures; the initial
medium volume was 50 mL. The broken line
indicates the medium antibody concentration
calculated by mass balance, assuming that all
antibody secreted from the biomass
accumulates in the medium. The difference
between the calculated and measured
antibody levels in the medium indicates the
extent of antibody degradation. Data adapted
from [19].
0.0
60
Time (days)
Current Opinion in Biotechnology
full-length antibodies and antibody heavy chains are degraded in suspended plant cell and hairy root cultures [17–19]. As
indicated in Figure 1, a mass balance of assembled antibody
during culture of transgenic tobacco hairy roots shows the
extent to which product is lost from the system. Use of protein stabilising agents such as polyvinylpyrrolidone (PVP)
improved antibody titres in the culture fluid by up to 35-fold
[17,18], suggesting that the medium is the site of antibody
degradation. Application of bacitracin, a broad-spectrum protease inhibitor, did not prevent antibody loss [19]. Further
work is required to identify the mechanisms of extracellular
antibody instability in plant culture medium. In other studies, degradation of human α1-antitrypsin in the medium of
transgenic rice cultures was attributed to protease release
from disrupted cells; adjusting the medium osmotic pressure
inhibited cell disruption and improved the active protein
titre [30•]. Although secreted product levels of up to 3% of
total secreted protein have been reported in plant cultures
[32], in some systems, concentrations of foreign protein are
very low relative to other secreted proteins, for example,
0.05% of total medium protein [28].
For foreign proteins secreted into the apoplastic spaces of
organs such as leaves and roots, methods are available for
extraction of the protein-rich intercellular fluid without
homogenisation of the biomass. Based on vacuum infiltration of the tissue with buffer followed by mild
centrifugation [32,44], these methods have been demonstrated mainly at the laboratory scale. Whether such
techniques can be scaled up readily to handle high throughputs of biomass is unclear, although large-scale application
for purification of human α-galactosidase A from plant
leaves has been reported [39]. Protein harvesting from the
apoplast might also be feasible for in vitro systems such as
hairy root cultures. Significant enrichment of recombinant
protein can be achieved using this approach: intercellular
fluids in which the foreign protein accounted for 30% of the
total protein present have been recovered [45].
Glycosylation
There are several differences in the biosynthesis and structure of protein glycans between plants and mammals
[46,47••]. Although differences in glycosylation may not
alter the activity of a protein, other properties, such as folding, stability, solubility, susceptibility to proteases, blood
clearance rate and antigenicity, can be affected profoundly
[48,49]. Addition in the ER of high-mannose glycans at
specific asparagine (N) sites on proteins is identical in
mammalian and plant cells; however, subsequent trimming of the sugar residues in the ER and Golgi generate
complex N-glycans with very different structures and properties. Plant glycoproteins exhibit a high degree of
heterogeneity of N-glycosylation, suggesting that many
variables can affect the processes involved [46,47••].
The xylosyl and fucosyl residues on plant N-linked complex
glycans have been demonstrated to be the key epitopes
responsible for the allergenicity of plant glycoproteins in
humans [50]. The possibility that plant-derived therapeutic
proteins could elicit an immune or sensitisation response in
patients is of concern, although clinical application might still
be possible depending on the dose requirements and blood
clearance rate [37]. Glycosylation differences are not an issue
for non-glycosylated proteins and peptides, such as those
used as antigens in some vaccine applications, and may only
be a problem for therapeutic glycoproteins administered to
patients by injection. Plant-derived antibodies given orally
have been shown in human trials not to elicit abnormally
high titres of serum IgG, IgA or IgM antibodies capable of
binding to the foreign protein [10•]. This result is consistent
Foreign protein production in plant tissue cultures Doran
203
with our repeated exposure to plant oligosaccharides in
foods, and the lack of novelty of plant glycans to the mucosal immune system.
2.
Larrick JW, Yu L, Chen J, Jaiswal S, Wycoff K: Production of
antibodies in transgenic plants. Res Immunol 1998, 149:603-608.
3.
Fischer R, Liao Y-C, Hoffmann K, Schillberg S, Emans N: Molecular
farming of recombinant antibodies in plants. Biol Chem 1999,
380:825-839.
Strategies to avoid the formation of immunogenic plant glycans may be necessary for plant proteins administered
systemically. Two possible approaches include the retention of recombinant glycoproteins in the ER to prevent
plant-specific modifications in the Golgi, and the modification of Golgi activity by removing or adding specific
enzymes [46]. For application in plant cell cultures, the first
strategy prevents extracellular protein secretion and puts
the associated cost benefits for downstream processing out
of reach; the second may be possible in the future as our
understanding of plant glycosylation improves. Gross modifications, such as complete inhibition of glycosylation or
the removal of glycosylation sites from peptide chains, can
result in protein folding defects, higher levels of aggregation, and increased protease susceptibility [49,51]. Just as
culture conditions such as pH, hormone levels, ammonium
ion concentration and reactor configuration have been
shown to affect N-linked glycosylation in mammalian cell
systems [48,51], it is also possible that some control over
plant glycan composition and structure may be achieved in
plant tissue culture by manipulation of environmental variables. As yet, however, this has not been demonstrated.
4.
Hammond J, McGarvey P, Yusibov V (Eds): Plant Biotechnology: New
Products and Applications. Berlin: Springer-Verlag; 1999.
5.
Hood EE, Jilka JM: Plant-based production of xenogenic proteins.
Curr Opin Biotechnol 1999, 10:382-386.
6.
Ma JK-C, Vine ND: Plant expression systems for the production of
vaccines. In Defense of Mucosal Surfaces: Pathogenesis, Immunity
and Vaccines. Edited by Kraehenbuhl J-P, Neutra MR. Berlin: SpringerVerlag; 1999:275-292.
7.
Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M,
Flynn P, Register J, Marshall L, Bond D et al.: Commercial production
of avidin from transgenic maize: characterization of transformant,
production, processing, extraction and purification. Mol Breed
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Conclusions
Plant cell and root cultures have been demonstrated to produce a wide range of recombinant proteins. The role of plant
tissue culture as a means for commercial protein production
is not clear at present, but is likely to depend on the ability
to express high levels of proteins in vitro by manipulating
culture conditions. Extracellular localisation and the stability
of secreted proteins in the medium of plant cultures may be
important keys to economic feasibility, allowing easier product recovery without destroying the biomass. Because the
controlled environment in plant cultures offers advantages
for GMP and regulatory compliance, a potential niche for
reactor-scale plant tissue culture is the rapid production of
low-to-medium volume therapeutic proteins. Technology for
modifying the glycans of plant glycoproteins may be
required before plant systems can be fully exploited for the
manufacture of injectable drugs and vaccines.
Acknowledgements
The author’s research was supported by the Australian Research Council (ARC).
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Miele L: Plants as bioreactors for biopharmaceuticals: regulatory
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38. Russell DA: Feasibility of antibody production in plants for human
•• therapeutic use. In Plant Biotechnology: New Products and
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Discusses regulatory, technical and commercial issues relating to the production of proteins in plants for use as human pharmaceuticals.
39. Turpen TH: Tobacco mosaic virus and the virescence of
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40. Crawford KM, Zambryski PC: Plasmodesmata signaling: many
roles, sophisticated statutes. Curr Opin Plant Biol 1999, 2:382-387.
41. Kusnadi AR, Evangelista RL, Hood EE, Howard JA, Nikolov ZL:
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Processing of transgenic corn seed and its effect on the recovery
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The authors investigate various options for the recovery and purification of
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stability during storage and processing, are also discussed.
42. Kermode AR: Mechanisms of intracellular protein transport and
targeting in plant cells. Crit Rev Plant Sci 1996, 15:285-423.
43. Conrad U, Fiedler U, Artsaenko O, Phillips J: High-level and stable
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44. McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK,
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mouse IgG expressed in transgenic tobacco plants. Glycobiology
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A comprehensive investigation of the glycoforms present on plant-produced IgG1 monoclonal antibodies, including comparison with the corresponding IgG1 of murine origin. The N-linked glycans on the plant protein
exhibited a high degree of structural diversity, with 60% of the oligosaccharides possessing β(1, 2)-xylose and α(1, 3)-fucose residues characteristic of plant glycans.
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Foreign protein production in plant tissue cultures
Pauline M Doran
Foreign proteins synthesised by plants are now in the
marketplace, and clinical trials for plant-derived therapeutic
proteins are underway. Economic analysis of plant production
systems has helped identify the types of protein that would be
most suitable for manufacture using tissue culture methods.
The major advantages associated with in vitro plant systems
include the ability to manipulate environmental conditions for
better control over protein levels and quality, the rapidity of
production compared with agriculture, and the use of simpler
and cheaper downstream processing schemes for product
recovery from the culture medium.
Addresses
Department of Biotechnology, University of New South Wales, Sydney,
NSW 2052, Australia; e-mail: [email protected]
Current Opinion in Biotechnology 2000, 11:199–204
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
ER
endoplasmic reticulum
GMP
Good Manufacturing Practice
Introduction
Foreign proteins can be synthesised using microbial cell
culture, animal cell culture, plant tissue culture, transgenic
animals and transgenic plants. Choosing the most appropriate method for commercial production requires
case-by-case analysis. A wide range of factors must be considered, including cost of production, market volume, the
efficacy, safety and stability of the product, and whether
the foreign protein has different biochemical or pharmacological properties compared with material from existing
sources. Although most interest during the past 15–20
years has been focused on microbial and animal cell cultures, plants and plant cells are now considered as viable
and competitive expression systems for large-scale protein
production. The development of transgenic plants and
plant viral vectors for foreign protein synthesis has been
reviewed extensively [1•,2–6].
Factors in favour of plant systems as sources of animalderived proteins include: the potential for large-scale,
low-cost biomass production using agriculture; the low risk
of product contamination by mammalian viruses, bloodborne pathogens, oncogenes and bacterial toxins; the
capacity of plant cells to correctly fold and assemble multimeric proteins; low downstream processing requirements
for proteins administered orally in plant food or feed; the
ability to introduce new or multiple transgenes by sexual
crossing of plants; and the avoidance of ethical problems
associated with transgenic animals and the use of animal
materials. There are, however, potential issues of concern
for plant protein production: containment of genetically
modified plants in the environment; allergic reactions to
plant protein glycans and other plant antigens; product
contamination by mycotoxins, pesticides, herbicides and
endogenous plant secondary metabolites; and regulatory
uncertainty, particularly for proteins requiring approval for
human drug use.
An important indicator of the cost competitiveness of
plant-based foreign protein synthesis is the increasing
involvement of industrial groups. Commercial processes
for the production of avidin, β-glucuronidase and aprotinin
from transgenic corn have already been developed
[7,8•,9•]. Plant-derived non-injectable immunotherapeutic
and vaccine proteins are also progressing through human
clinical trials; these include a secretory IgA/G antibody
against oral bacterial colonisation [10•], an edible vaccine
against an Escherichia coli enterotoxin [11•] and an edible
vaccine against hepatitis B [12•]. Details of company activity in selected areas, including plant production of
monoclonal antibodies, vaccines and industrial enzymes,
are provided in recent reviews [2,3,5,13••].
An alternative but less well-developed technology for producing foreign proteins is plant tissue culture. Using this
approach, plant cells in differentiated or dedifferentiated
states are grown axenically in nutrient medium in bioreactors under controlled conditions, with foreign protein
harvested from either the biomass or culture liquid, or a
combination of both. Although plant tissue culture may
not be suitable for all applications of foreign protein synthesis, such as food-based production of edible vaccines, it
offers a number of advantages for the manufacture of proteins that are extracted and purified after synthesis. The
purpose of this review is to outline the potential advantages and limitations associated with plant tissue culture
for foreign protein production, and to summarise recent
developments in the area. The review focuses on the production of proteins of direct commercial value. Application
of foreign proteins in plants for metabolic modulation or to
improve pathogen resistance or nutritional value are discussed elsewhere [14–16].
Recent applications of plant tissue cultures for
production of foreign proteins
Suspended plant cells have been used in several recent
studies as a means of producing a variety of foreign proteins. These include recombinant antibodies and antibody
fragments [17–23], enzymes such as β-glucuronidase [24]
and invertase [25], and proteins of therapeutic value such
as human interleukin (IL)-2 and IL-4 [26], ribosome-inactivating protein [27], ricin [28], and human α1-antitrypsin
[29•,30•]. The most commonly used host species for protein synthesis in suspension cultures is tobacco [17–28],
although rice cell cultures have also been tested [29•,30•].
200
Biochemical engineering
Several stirred bioreactor studies at volumes up to 40 L
have been reported, using batch [22,23], continuous [25]
and two-stage [29•] modes of operation. Hairy root cultures
of transgenic tobacco have also been tested for production
of recombinant IgG1 antibody [18,31], including in a 2 L
sparged bioreactor [18]. In other work, bacterial xylanase
and human placental alkaline phosphatase were produced
using hydroponic culture of transgenic tobacco plants in
sterile, sucrose-containing medium in flasks [32].
Large-scale plant tissue culture versus
agriculture
For bulk manufacture of proteins such as those used in
industrial, analytical and diagnostic applications, most production systems will find it hard to compete with the low
cost of agriculture. A detailed economic evaluation of
β-glucuronidase extraction and purification from transgenic corn yielded a production cost of only US$43 g–1 for
an initial seed protein concentration of 0.015% dry weight,
a product purity of 83%, and an annual production volume
of 137 kg [33••]. Costs of research and development, royalties and sales were not included in the analysis. In
comparison, the cost of producing 100 kg of protein from
transgenic goats’ milk has been estimated at US$105 g–1,
whereas manufacture using mammalian (Chinese hamster
ovary) cell culture costs US$300–3000 g–1 depending on
the product yield and other operating factors [34].
Agricultural production of protein has been reported to be
10–50 times cheaper than E. coli fermentation, even
though product levels in bacteria are higher than those in
plants [35]. Foreign protein production using greenhousecultivated plants is considerably more expensive than with
field-grown crops; recent estimates are US$500–600 g–1
based on operations in a 250 m2 greenhouse and including
heating, labour and consumables for protein extraction and
purification [36].
accumulate protein levels at least an order of magnitude
higher than those achievable in agricultural systems. This
would reduce the costs associated with product recovery to
offset the substantially higher cost of biomass production.
Plant cell cultures provide greater opportunity for manipulation of foreign protein levels, as culture conditions can be
altered much more readily in reactors than in the field. For
example, supplementation of suspended plant cells with
amino acids prior to harvest resulted in a transient threefold
increase in recombinant antibody levels [21], while accumulation of recombinant ricin in tobacco culture medium
was sensitive to the concentration of auxin provided [28]. In
other work, exposure of suspended plant cells to dark/light
cycles was used to control foreign gene expression and protein titre [24]. Whereas these examples demonstrate how
protein production might be enhanced in plant cell cultures, some studies have shown that recombinant protein
levels are significantly lower in plant cell suspensions than
in whole plants or organ cultures such as hairy roots [19,20].
The reasons for this are unknown at present, but could
relate more to post-synthesis product stability rather than to
expression levels per se. Technology to improve foreign protein accumulation in culture systems must be developed if
product levels significantly greater than those achievable in
whole plants are required for economic feasibility.
In vitro cell culture offers intrinsic advantages, or could
even be a necessity, for foreign protein synthesis in certain
situations. Plant tissue culture may be the most suitable
production system when small-to-medium quantities of
specialty, high-price, high-purity proteins are needed, such
as in some therapeutic applications. The time involved for
production using plant cell culture is significantly shorter
than the growth cycle of whole plants; therefore, compared
with the months needed for agriculture, proteins could be
manufactured in days or weeks on a time-scale compatible
with clinical demands. Because bioreactors provide a much
more controlled and reproducible environment than the
open field, regulatory requirements for therapeutic proteins can be met more easily using reactor-grown cells. In
addition, when the protein of interest is secreted into the
medium of plant cell cultures, product recovery and purification could be greatly simplified and much cheaper than
from plant biomass, while eliminating the need to destroy
the cells. However, if the most probable niche for plant cell
cultures is the production of therapeutic proteins, an
important issue is post-translational processing of proteins
and the effects of plant glycans on the human immune system. These factors, together with recent developments
influencing the choice of plant cell culture as a production
vehicle for foreign proteins, are discussed below.
The cost of transgenic corn seed for extraction of foreign
protein has been estimated as only US$0.20 kg–1 [33••].
This includes a premium of US$0.09 kg–1 to provide
incentive to farmers and grain handlers, and to cover any
additional costs associated with growing the transgenic
plants and keeping them and their seeds isolated from
other plants and grain products. Although specific cost
analyses for protein production using plant tissue culture
have not been provided in the literature, it would be difficult to produce a kilogram of plant biomass in a reactor at
such a low price. Despite plant culture medium being substantially cheaper than animal culture medium, the cost of
the medium alone for generation of 1 kg of plant cells is
substantially greater than US$0.20, without factoring in
other costs associated with bioreactor operation, such as
labour, utilities, waste treatment, equipment depreciation,
and so on.
Good Manufacturing Practice, process
reproducibility and product quality
Plant cell cultures are likely to be uncompetitive for the
majority of proteins required in the large quantities deliverable by agricultural production, unless they are able to
The regulatory issues relevant to Good Manufacturing
Practice (GMP) for production of therapeutic proteins in
plants have been outlined in recent papers [37,38••].
Present indications are that there is no a priori barrier to
Foreign protein production in plant tissue cultures Doran
the production of therapeutic proteins using plants grown
in open fields, even though this production system is subject to the vagaries of weather, insect activity, soil
variability and other factors that influence product yield
and quality (Z Nikolov, personal communication). If, as
seems likely, regulatory acceptance of the product will
depend mainly on its compliance with purity, efficacy and
biocomparability standards at the end of the production
chain, field-grown protein will be able to satisfy requirements if sufficient safeguards are designed into the process
so that undesirable components are either prevented from
forming or removed during purification. Reactor culture
could make regulatory compliance easier by reducing variation in production conditions and increasing average
batch quality; whether or not this is a crucial advantage will
depend on the properties of the particular system and their
susceptibility to environmental influences. Although variation in the level of protein accumulation has been
reported for transgenic plants in different growing locations [7], lot-to-lot consistency of purified plant vaccine
proteins and conformity with regulatory specifications
have also been demonstrated [39].
Developments in the area of inducible promoters may lead
to future alleviation of problems with environment-related
variability in field-grown plants by separating the biomass
growth and protein synthesis steps. For example, using a
recently patented post-harvest expression system [P1], plant
tissues harvested from the field can be induced to produce
foreign protein during storage in laboratory or GMP facilities. Because conditions during actual product synthesis are
controlled to a greater extent than is possible with constitutive promoters and outdoor plants, better product yield and
less variation in product quality can be expected. Such
improvements in agriculture-based production systems
could reduce the potential benefits associated with plant tissue culture with respect to regulatory compliance.
The ability of plant cells and organs to propagate indefinitely in tissue culture without the need for sexual reproduction
offers a solution to other problems relating to gene segregation and long-term transgene stability in agricultural crops.
Variations in foreign protein expression within and between
successive generations of corn have been reported [7,9•], as
well as loss of fertility and/or pollen transmission of the transgene [7] and loss of the original selection marker [7,9•].
Greater stability may develop with increasing number of
generations [9•]; however, it is probable that breeding programs with continuous selection of elite lines will be
necessary for consistent high-level protein production using
whole plants. This situation could be improved by the use of
perennial host species [36]. In contrast, there are several
reports of stable, long-term foreign protein synthesis in plant
tissue cultures, such as carrot invertase in tobacco suspensions over four years [25] and IgG1 antibody in tobacco hairy
root cultures over 19 months [18]. Gene silencing occurs in
plant tissue cultures as well as in whole plants (EJ Finnegan,
personal communication); however, if transmission of signals
201
for systemic post-transcriptional silencing occurs via plasmodesmata and the vascular system [40], the conditions of
minimal cell–cell contact existing in plant suspensions could
be advantageous for stable foreign protein production. On
the other hand, dedifferentiated plant cells, such as those in
suspension cultures, are subject to a range of genetic modifications through the mechanisms of somaclonal variation.
Further studies are needed to verify whether better longterm stability of protein production can be achieved in vitro
compared with whole-plant systems.
Downstream processing, protein secretion
and stability
Recovery and purification of proteins from plant biomass is
an expensive and technically challenging business, requiring multiple separation steps such as precipitation,
adsorption, chromatography and diafiltration. In a recent
cost analysis of β-glucuronidase production from transgenic
corn seed, milling, protein extraction and purification operations accounted for ~94% of the production cost [33••]. As
the corn itself contributed only 6%, the importance of downstream processing in determining the economic feasibility of
plant protein production is very clear. Costs of protein recovery from seed could be reduced if most of the protein is
localised in a separable component such as the embryo
[8•,41•], or by targeting protein expression to seed oil bodies
for easier extraction and purification [13••].
Because plant nutrient media are relatively simple solutions with no added proteins, if a foreign protein is
produced in tissue culture and secreted into the medium
rather than stored inside the cells, product recovery and
purification could be carried out in the absence of large
quantities of contaminating proteins. Economic analysis of
protein recovery from spent plant culture medium has not
been reported in the literature. Yet, significant cost advantages may be derived from this type of production system,
despite the protein being diluted to low concentrations in
the relatively large liquid volume. Extracellular protein
secretion is effected in plant cells using signal peptides
that direct transport of peptides into the lumen of the
endoplasmic reticulum (ER). After folding and assembly
in the ER, unless the protein is carrying a signal that targets it for retention in the ER or transport to another
organelle such as the vacuole, proteins leaving the ER
enter the default secretory pathway to the Golgi apparatus
and finally to the extracellular space [42].
Whether or not secretion is the best strategy for protein accumulation depends on the relative stability of the protein in
the extracellular environment. For some recombinant proteins, highest accumulation is achieved by retention in the
ER. For example, carboxy-terminal fusion of the KDEL signal peptide to single-chain antibody variable-region
fragments (scFvs) and subsequent ER retention has been
found to increase antibody levels by a factor of up to 10–100
compared with either extracellular secretion or expression in
the cytosol [1•,20,43]. There is also evidence that secreted
202
Biochemical engineering
0.30
3.0
0.25
2.5
0.20
2.0
0.15
1.5
0.10
1.0
0.05
0.5
0.00
0
10
20
30
40
50
Antibody in the medium (µg mL-1)
Antibody in the biomass (mg)
Figure 1
Expression and secretion of assembled IgG1
antibody during culture of transgenic tobacco
hairy roots. (䊉) Antibody in the biomass; (䊊)
measured antibody concentration in the
medium. The error bars indicate standard
errors from triplicate cultures; the initial
medium volume was 50 mL. The broken line
indicates the medium antibody concentration
calculated by mass balance, assuming that all
antibody secreted from the biomass
accumulates in the medium. The difference
between the calculated and measured
antibody levels in the medium indicates the
extent of antibody degradation. Data adapted
from [19].
0.0
60
Time (days)
Current Opinion in Biotechnology
full-length antibodies and antibody heavy chains are degraded in suspended plant cell and hairy root cultures [17–19]. As
indicated in Figure 1, a mass balance of assembled antibody
during culture of transgenic tobacco hairy roots shows the
extent to which product is lost from the system. Use of protein stabilising agents such as polyvinylpyrrolidone (PVP)
improved antibody titres in the culture fluid by up to 35-fold
[17,18], suggesting that the medium is the site of antibody
degradation. Application of bacitracin, a broad-spectrum protease inhibitor, did not prevent antibody loss [19]. Further
work is required to identify the mechanisms of extracellular
antibody instability in plant culture medium. In other studies, degradation of human α1-antitrypsin in the medium of
transgenic rice cultures was attributed to protease release
from disrupted cells; adjusting the medium osmotic pressure
inhibited cell disruption and improved the active protein
titre [30•]. Although secreted product levels of up to 3% of
total secreted protein have been reported in plant cultures
[32], in some systems, concentrations of foreign protein are
very low relative to other secreted proteins, for example,
0.05% of total medium protein [28].
For foreign proteins secreted into the apoplastic spaces of
organs such as leaves and roots, methods are available for
extraction of the protein-rich intercellular fluid without
homogenisation of the biomass. Based on vacuum infiltration of the tissue with buffer followed by mild
centrifugation [32,44], these methods have been demonstrated mainly at the laboratory scale. Whether such
techniques can be scaled up readily to handle high throughputs of biomass is unclear, although large-scale application
for purification of human α-galactosidase A from plant
leaves has been reported [39]. Protein harvesting from the
apoplast might also be feasible for in vitro systems such as
hairy root cultures. Significant enrichment of recombinant
protein can be achieved using this approach: intercellular
fluids in which the foreign protein accounted for 30% of the
total protein present have been recovered [45].
Glycosylation
There are several differences in the biosynthesis and structure of protein glycans between plants and mammals
[46,47••]. Although differences in glycosylation may not
alter the activity of a protein, other properties, such as folding, stability, solubility, susceptibility to proteases, blood
clearance rate and antigenicity, can be affected profoundly
[48,49]. Addition in the ER of high-mannose glycans at
specific asparagine (N) sites on proteins is identical in
mammalian and plant cells; however, subsequent trimming of the sugar residues in the ER and Golgi generate
complex N-glycans with very different structures and properties. Plant glycoproteins exhibit a high degree of
heterogeneity of N-glycosylation, suggesting that many
variables can affect the processes involved [46,47••].
The xylosyl and fucosyl residues on plant N-linked complex
glycans have been demonstrated to be the key epitopes
responsible for the allergenicity of plant glycoproteins in
humans [50]. The possibility that plant-derived therapeutic
proteins could elicit an immune or sensitisation response in
patients is of concern, although clinical application might still
be possible depending on the dose requirements and blood
clearance rate [37]. Glycosylation differences are not an issue
for non-glycosylated proteins and peptides, such as those
used as antigens in some vaccine applications, and may only
be a problem for therapeutic glycoproteins administered to
patients by injection. Plant-derived antibodies given orally
have been shown in human trials not to elicit abnormally
high titres of serum IgG, IgA or IgM antibodies capable of
binding to the foreign protein [10•]. This result is consistent
Foreign protein production in plant tissue cultures Doran
203
with our repeated exposure to plant oligosaccharides in
foods, and the lack of novelty of plant glycans to the mucosal immune system.
2.
Larrick JW, Yu L, Chen J, Jaiswal S, Wycoff K: Production of
antibodies in transgenic plants. Res Immunol 1998, 149:603-608.
3.
Fischer R, Liao Y-C, Hoffmann K, Schillberg S, Emans N: Molecular
farming of recombinant antibodies in plants. Biol Chem 1999,
380:825-839.
Strategies to avoid the formation of immunogenic plant glycans may be necessary for plant proteins administered
systemically. Two possible approaches include the retention of recombinant glycoproteins in the ER to prevent
plant-specific modifications in the Golgi, and the modification of Golgi activity by removing or adding specific
enzymes [46]. For application in plant cell cultures, the first
strategy prevents extracellular protein secretion and puts
the associated cost benefits for downstream processing out
of reach; the second may be possible in the future as our
understanding of plant glycosylation improves. Gross modifications, such as complete inhibition of glycosylation or
the removal of glycosylation sites from peptide chains, can
result in protein folding defects, higher levels of aggregation, and increased protease susceptibility [49,51]. Just as
culture conditions such as pH, hormone levels, ammonium
ion concentration and reactor configuration have been
shown to affect N-linked glycosylation in mammalian cell
systems [48,51], it is also possible that some control over
plant glycan composition and structure may be achieved in
plant tissue culture by manipulation of environmental variables. As yet, however, this has not been demonstrated.
4.
Hammond J, McGarvey P, Yusibov V (Eds): Plant Biotechnology: New
Products and Applications. Berlin: Springer-Verlag; 1999.
5.
Hood EE, Jilka JM: Plant-based production of xenogenic proteins.
Curr Opin Biotechnol 1999, 10:382-386.
6.
Ma JK-C, Vine ND: Plant expression systems for the production of
vaccines. In Defense of Mucosal Surfaces: Pathogenesis, Immunity
and Vaccines. Edited by Kraehenbuhl J-P, Neutra MR. Berlin: SpringerVerlag; 1999:275-292.
7.
Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M,
Flynn P, Register J, Marshall L, Bond D et al.: Commercial production
of avidin from transgenic maize: characterization of transformant,
production, processing, extraction and purification. Mol Breed
1997, 3:291-306.
Conclusions
Plant cell and root cultures have been demonstrated to produce a wide range of recombinant proteins. The role of plant
tissue culture as a means for commercial protein production
is not clear at present, but is likely to depend on the ability
to express high levels of proteins in vitro by manipulating
culture conditions. Extracellular localisation and the stability
of secreted proteins in the medium of plant cultures may be
important keys to economic feasibility, allowing easier product recovery without destroying the biomass. Because the
controlled environment in plant cultures offers advantages
for GMP and regulatory compliance, a potential niche for
reactor-scale plant tissue culture is the rapid production of
low-to-medium volume therapeutic proteins. Technology for
modifying the glycans of plant glycoproteins may be
required before plant systems can be fully exploited for the
manufacture of injectable drugs and vaccines.
Acknowledgements
The author’s research was supported by the Australian Research Council (ARC).
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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•
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•
Witcher DR, Hood EE, Peterson D, Bailey M, Bond D, Kusnadi A,
Evangelista R, Nikolov Z, Wooge C, Mehigh R et al.: Commercial
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Outlines important practical considerations for commercial production of
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the procedures used for downstream processing, and the steps required for
physical and functional characterisation of the protein.
9.
•
Zhong G-Y, Peterson D, Delaney DE, Bailey M, Witcher DR, Register JC,
Bond D, Li C-P, Marshall L, Kulisek E et al.: Commercial production
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The authors discuss the use of a crop species to manufacture a foreign protein with potential therapeutic value. They provide information about the variability of protein expression in successive generations of corn.
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•
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immunological response.
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•
Arntzen CJ: Immunogenicity in humans of a recombinant bacterial
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An antigen derived from a bacterial enterotoxin was administered in human
trials as an edible vaccine in uncooked potato. The results showed that
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12. Kapusta J, Modelska A, Figlerowicz M, Pniewski T, Letellier M, Lisowa O,
•
Yusibov V, Koprowski H, Plucienniczak A, Legocki AB: A plantderived edible vaccine against hepatitis B virus. FASEB J 1999,
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