Putting the Spotlight Back on Plant Suspension Cultures

Abstract

Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein derived from plants—is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.

Full text

REVIEW
published: 11 March 2016
doi: 10.3389/fpls.2016.00297
Frontiers in Plant Science | www.frontiersin.org 1March 2016 | Volume 7 | Article 297
Edited by:
Domenico De Martinis,
ENEA Italian National Agency for New
Technologies, Energy and Sustainable
Economic Development, Italy
Reviewed by:
Heiko Rischer,
VTT Technical Research Centre of
Finland Ltd., Finland
Karen Ann McDonald,
University of California, Davis, USA
*Correspondence:
Tanja Holland
tanja.holland@ime.fraunhofer.de
Specialty section:
This article was submitted to
Plant Biotechnology,
a section of the journal
Frontiers in Plant Science
Received: 15 December 2015
Accepted: 25 February 2016
Published: 11 March 2016
Citation:
Santos RB, Abranches R, Fischer R,
Sack M and Holland T (2016) Putting
the Spotlight Back on Plant
Suspension Cultures.
Front. Plant Sci. 7:297.
doi: 10.3389/fpls.2016.00297
Putting the Spotlight Back on Plant
Suspension Cultures
Rita B. Santos 1, Rita Abranches 1, Rainer Fischer 2, 3, Markus Sack 3and Tanja Holland 2*
1Plant Cell Biology Laboratory, Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica António Xavier,
Oeiras, Portugal, 2Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME), Integrated Production
Platforms, Aachen, Germany, 3Biology VII, Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany
Plant cell suspension cultures have several advantages that make them suitable for the
production of recombinant proteins. They can be cultivated under aseptic conditions
using classical fermentation technology, they are easy to scale-up for manufacturing,
and the regulatory requirements are similar to those established for well-characterized
production systems based on microbial and mammalian cells. It is therefore no surprise
that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein
derived from plants—is produced in plant cell suspension cultures. But despite this
breakthrough, plant cells are still largely neglected compared to transgenic plants and
the more recent plant-based transient expression systems. Here, we revisit plant cell
suspension cultures and highlight recent developments in the field that show how the
rise of plant cells parallels that of Chinese hamster ovary cells, currently the most
widespread and successful manufacturing platform for biologics. These developments
include medium optimization, process engineering, statistical experimental designs,
scale-up/scale-down models, and process analytical technologies. Significant yield
increases for diverse target proteins will encourage a gold rush to adopt plant cells as
a platform technology, and the first indications of this breakthrough are already on the
horizon.
Keywords: plant suspension cultures, biopharmaceuticals, BY-2, protein production, plant cell cultures
INTRODUCTION
Protein-based drugs are big business. The market for biopharmaceuticals is growing faster than
the pharmaceuticals market as a whole, and a recent projection suggested the value of this
segment could reach $US 278.2 billion by 2020 (PMR, 2015). There are more than 200 approved
biopharmaceuticals on the market today and many more in the clinical pipeline (Walsh, 2014).
Currently, most biologics are produced in microbes or mammalian cells growing in fermenters.
Microbes are simple and inexpensive but often fail to produce complex proteins or those requiring
specific post-translational modifications, whereas mammalian cells can achieve these folding and
modification tasks with aplomb but only at a much higher cost. Both systems also have the potential
for undesirable contaminants—endotoxins in the case of bacteria, and viruses or other pathogens
in the case of mammalian cells. The extra steps required during downstream processing to remove
these contaminants can increase production costs even further (BOX 1).
The choice of expression hosts has more recently expanded to include plants because they offer
unique features compared to the current dominant production systems (Stoger et al., 2014; Ma
et al., 2015). The production of recombinant proteins in plants, where the protein itself is the

Santos et al. Focusing on Plant Cell Suspensions
BOX 1 | COMPARISON OF MAJOR PRODUCTION PLATFORMS
Industry platforms for the production of recombinant proteins are based mainly on microbes and mammalian cells. The major microbial system is the bacterium
Escherichia coli which was the first species used to produce a recombinant human protein (somatostatin in 1977, Itakura et al., 1977) and the first to be used for the
production of a commercial therapeutic protein (recombinant human insulin, approved in 1982 and marketed by Eli Lilly & Co. under license from Genentech). Many
simple and unmodified proteins are produced commercially in E. coli but more complex proteins are difficult to fold unless targeted to the periplasm and this is not a
scalable process (Baneyx and Mujacic, 2004; Choi and Lee, 2004). E. coli is simple and inexpensive but problems include the accumulation of proteins as insoluble
inclusion bodies and the production of endotoxins that can cause septic shock. Yeasts are sometimes preferred because they share the advantages of bacteria but
they are eukaryotes and thus support protein folding and modification, although the glycan chains are often longer than in mammals. Saccharomyces cerevisiae was
the first yeast used to express recombinant proteins and it is still used commercially to produce a Hepatitis B virus vaccine, but other yeasts such as Pichia pastoris
and Hansenula polymorpha are now favored during process development because they are more suitable for in-process inducible expression (Gerngross, 2004).
Mammalian cells have dominated the biopharmaceutical industry since the 1990s because they can produce high titers (1–5 g/L) of complex proteins with mammalian
glycan structures (Chu and Robinson, 2001). They are much more expensive than microbes but most pharmaceuticals are glycoproteins and the quality of the product
is superior when mammalian cells are used. CHO cells are preferred by the industry but others that are widely used include the murine myeloma cells lines NS0 and
SP2/0, BHK and HEK-293, and the human retinal line PER-C6. The major disadvantage of mammalian cells remains the cost of production, purification, and the risk
of contamination with human pathogens.
BOX 2 | DIVERSITY OF MOLECULAR FARMING TECHNOLOGIES
The immense diversity of molecular farming systems reflects the fact that recombinant proteins have been produced in many different plant species wherein there
is a choice of whole plants or various cell/tissue culture formats (Twyman et al., 2003, 2005). Each of these may be suitable for stable expression (including nuclear
and plastid transformation is some species) and transient expression (which can be achieved using Agrobacterium tumefaciens, plant virus vectors or combinations of
both; Paul et al., 2013). Transgenic terrestrial plants are the most established platform and following a period of extensive diversification the field has now consolidated
mainly to support tobacco as the primary leafy crop and the cereals maize, rice, and barley (Nandi et al., 2005; Tremblay et al., 2010; Sabalza et al., 2013). The main
difference between these platforms is that leaves are watery tissues and the recombinant protein must be extracted quickly to avoid degradation whereas cereal seeds
are desiccated and the protein remains stable for long periods. Cereal seeds are also suitable for direct oral administration. Aquatic plants such as duckweed and moss
are also used as platforms (Reski et al., 2015). These have properties in common with terrestrial plants (differentiated whole plants) and cell suspension cultures (grown
in containment in simple medium). The technology for aquatic plants and cell suspension cultures is similar but aquatic plants require light, whereas undifferentiated cell
suspension cultures are grown in the dark but require a carbon source. After transgenic whole plants and cell suspension cultures, the third major technology platform is
transient expression, which involves the introduction of non-integrating (episomal) vectors into leaves. The two main transient expression strategies are agroinfiltration,
where leaves are infiltrated with A. tumefaciens by injection or vacuum leading to the transfection of millions of cells and the production of large amounts of recombinant
protein in a short time (Komarova et al., 2010), and the use of recombinant plant viruses that infect cells directly, replicate within them and spread by cell-to-cell
movement and systemic spreading through the vascular network to produce recombinant protein in every cell (Yusibov et al., 2006). A midway strategy that achieves
biocontainment is the use of deconstructed virus genomes delivered by A. tumefaciens, which results in the transfection of many cells with the virus genome followed
by its cell-to-cell movement but no systemic spreading (Peyret and Lomonossoff, 2015). All three major platforms have advantages and disadvantages—transgenic
plants have a slow development cycle but are the most scalable, cell suspension cultures have a quick development cycle and allow contained production but are the
least scalable, and transient expression allows the rapid production of high protein yields ideal for emergencies such as vaccines and prophylactic antibodies, as seen
in the recent outbreak of Ebola virus disease in West Africa (Arntzen, 2015), but the large number of bacteria introduced into the leaves increases the endotoxin load
(Arfi et al., 2015).
desired product, is often described as molecular farming. If
the proteins are pharmaceuticals then a bit of wordplay offers
molecular pharming as an alternative. Plants combine the
advantages of higher eukaryotic cells (efficient protein folding
and post-translational modification) with the use of simple and
inexpensive growth media. The diversity of molecular farming
technologies is much greater than other production platforms,
which can be advantageous or disadvantageous depending on the
perspective (BOX 2).
One niche of molecular farming technology that is now
coming back into the limelight is the use of plant cells,
specifically plant cell suspension cultures, rather than whole
plants (Doran, 2000; Hellwig et al., 2004). Although molecular
farming conjures up images of greenhouses bursting with dense
green leaves containing valuable pharmaceutical proteins, much
of the technical and commercial progress made in molecular
farming has been based on plant cells. These combine the
advantages of plants with those of traditional fermenter systems:
contained, controlled and sterile production environments,
chemically defined media lacking animal components, and
compatibility with the toughest regulatory guidelines in
existence—pharmaceutical good manufacturing practice (GMP).
Recent advances in process engineering have seen plant cells
leap forward toward commercial viability much faster than the
established platforms achieved during their own development
phases. The first molecular farming product approved for human
use is manufactured in plant cells—and this is only the beginning
(Zimran et al., 2011; Tekoah et al., 2015).
PLANT CELL SUSPENSION
CULTURES—PLATFORMS AND
PRODUCTS
The production of recombinant proteins in plant cell suspension
cultures was first demonstrated more than 25 years ago (Sijmons
et al., 1990) but progress over the subsequent decade was
overshadowed by whole plants, and only a small number of
studies involving cultivated plant cells as production hosts were
published before the turn of the century (Table 1). The status of
plant cells began to change after the first bubble of commercial
interest in molecular farming collapsed due to the absence of
a regulatory pathway, the opposition to GM crops (particularly
in Europe), and the lack of support from an industry already
heavily invested in fermenters. Whereas, some in the molecular
farming community worked toward establishing regulations for
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Santos et al. Focusing on Plant Cell Suspensions
pharmaceuticals derived from whole plants (Arfi et al., 2015;
Ma et al., 2015; Sack et al., 2015) others realized that plant cells
were already similar in many ways to microbial and mammalian
cells and could be handled under the existing regulations
(Ramachandra Rao and Ravishankar, 2002; Zimran et al., 2011).
The production of recombinant proteins in plant cell
suspension cultures can be achieved by transforming wild-
type cells already in suspension and selecting those carrying
a co-introduced marker gene, or by initiating cultures from
transgenic plants. As with other fermenter-based systems, the
scalability of plant cell cultures is limited by the bioreactor
capacity but the product can be recovered from the medium
allowing continuous production, or it can be directed to a specific
internal compartment if this is more appropriate (Schillberg et al.,
2013). Although inducible promoters in plants allow production
to be divided into a growth phase and a production phase
analogous to the inducible production systems used in bacteria
and yeast, there is currently no counterpart of the amplification
technologies used with mammalian cells so the product yields in
plant cells are much lower—however, the assembly of an artificial
system in plants is conceivable (BOX 3).
Several platforms have emerged as contenders for a
standardized production technology including cell suspension
cultures derived from tobacco (Nicotiana tabacum), rice (Oryza
sativa), and carrot (Daucus carota), which are the front runners
today. The most widely used tobacco cell line is derived from
the cultivar Bright Yellow 2 (BY-2). Tobacco BY-2 suspension
cell cultures can multiply up to 100-fold within 7 days with a
doubling time of 16–24 h under ideal conditions. The BY-2 cell
line was developed in 1968 at the Hatano Tobacco Experimental
Station, Japan Tobacco Company (Kato et al., 1972). The
transformation of BY-2 cells using A. tumefaciens is highly
efficient (Nagata et al., 1992) and therefore many different
products have been successfully produced using this system
(Table 1). One of the drawbacks of molecular farming in whole
tobacco plants is that the leaves contain nicotine, but BY-2 cells
BOX 3 | THE CHO AMPLIFICATION SYSTEM AND CAN WE REPLICATE IT IN PLANTS?
The CHO system is the most widely used mammalian cell line platform in the industry because it was the first to market and is therefore backed by years of cumulative
experience and process optimization, and it is compatible with serum-free medium which reduces the potential bioburden (Wurm, 2004). Most of all it has a highly
effective gene amplification system, paired with an unstable genome that facilitates amplification and other genetic changes (Cacciatore et al., 2010). This was discovered
accidentally when rare individual CHO cells were shown to survive toxic concentrations of the drug methotrexate, which inhibits the enzyme dihydrofolate reductase
(DHFR). The analysis of surviving cells showed that some carried point mutations conferring resistance but others contained multiple copies of the dhfr locus and
produced enough of the enzyme to outcompete the inhibitor.
Stepwise selection at higher concentrations isolated cells with massively amplified dhfr gene arrays allowing survival at 10,000 times the normal toxic dose of
methotrexate. The amplified genes were present as homogeneously staining regions within chromosomes or as small extra chromosomes called double minutes.
Importantly, these arrays contain flanking regions as well as the dhfr gene itself so adjacent genes can also be amplified even if though they do not contribute to
methotrexate-resistant phenotype (Cacciatore et al., 2010). The current industry CHO platform is based on the mutant cell line DG44 which lacks an endogenous
dhfr gene. This cell line is transfected with a tandem dhfr-X construct, where X encodes the desired recombinant protein. Both genes are amplified under selection
and the yield of the recombinant protein is boosted substantially. Many different amplifiable markers have been identified but only dhfr-methotrexate and glutamine
synthase-methionine sulfoxamine are used for commercial pharmaceutical production.
There is no equivalent amplifiable marker system in plants although many of the markers which work in mammalian cells as amplifiable markers can be used for
simple one-step selection in plants, including dhfr (Eichholtz et al., 1987). The failure of amplifiable selection therefore suggests that plants lack an intrinsic ability to
generate massive arrays of small regions of the genome under selection, which indicates a difference in the capacity for homologous recombination. Such differences
have been observed before, and explain the difference in gene targeting efficiency between mammals and plants (Puchta and Fauser, 2013). One potential solution to
this issue is the use of extra chromosomal replicating vectors for amplification in plants, as reported by Regnard et al. (2010). Even without amplification, plant cells are
moving toward parity with mammalian cells. For example, cell-specific production rates of 8 pg/cell/day have been reported for the monoclonal antibody M12 produced
in tobacco BY-2 cells (Havenith et al., 2014) compared to typical production rates of 20–40 pg/cell/day for CHO cells carrying thousands of gene copies, showing that
the difference between these systems is less than an order of magnitude.
do not produce significant amounts of this metabolite even
when induced by jasmonates, and instead produce the related
compound anatabine as well as low levels of other alkaloids
(Shoji and Hashimoto, 2008).
Rice cell suspension cultures are used almost as widely as
tobacco BY-2 cells due to the availability of the carbohydrate-
sensitive α-amylase promoter system (RAmy3D) that works in
synchronization with the fermentation cycle. This promoter is
induced by sugar starvation, and gene expression can therefore
be optimized by timing media exchanges so that cells are exposed
to consecutive growth and production phases (Lee et al., 2007).
Most rice varieties can be dedifferentiated but Japonica varieties
appear more amenable than Indica varieties, such that callus can
easily be produced from almost every part of the plant. Rice
cell suspension cultures have a doubling time of 1.5–1.7 days
(Trexler et al., 2005). Many pharmaceutical products have been
expressed in rice cells (Table 1) and at least one major company
has adopted rice cells as an industrial production platform,
albeit for non-pharmaceutical-grade cosmetics ingredients and
research reagents (Natural Bio-Materials, Jeollabuk-do, Korea;
http://www.nbms.co.kr/).
Carrot cell lines can be derived from hypocotyl, epicotyl,
or cotyledon tissues. The transformation of carrot cells can
be achieved by co-cultivation with A. tumefaciens, particle
bombardment or the electroporation of protoplasts (Rosales-
Mendoza and Tello-Olea, 2015). The first plant-derived
biopharmaceutical protein approved by the FDA for human use
was taliglucerase alfa, produced in carrot cell suspension cultures
by the Israeli company Protalix Biotherapeutics (http://www.
protalix.com) and licensed by Pfizer (Table 1).
In addition to these three commercially-relevant platforms,
several other plant species have been used to produce cell
suspension cultures for molecular farming. The model legume
Medicago truncatula (Abranches et al., 2005) is typically used for
the analysis of secondary metabolism (Cook, 1999; Broeckling
et al., 2005) but this species has been developed more recently for
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TABLE 1 | Biopharmaceuticals produced in different plant cell suspension cultures.
Host cell Variety Protein Indication Yield References
Tobacco cells BY2 Hepatitis B Surface Antigen (HBsAg) Hepatitis B vaccine 6.5 µg/g FW Smith et al., 2002
PRX-102 (α-Galactosidase-A) Fabry disease Kizhner et al., 2015
EPO Tissue protective function Low Matsumoto et al., 1995;
Pires et al., 2012
Granulocyte-Macrophage Colony-
Stimulating Factor (GM-CSF)
Production of white cells Up to 250 µg/L James et al., 2000; Lee
et al., 2002
IL-4 Immunoregulation 0.18 µg/L Magnuson et al., 1998
α-HBsAg Mab Hepatitis B antibody Up to 15 mg/ L Yano et al., 2004; Sunil
Kumar et al., 2007
2G12 monoclonal α-HIV Ab Anti-HIV antibody 12 mg/L SN Holland et al., 2010
Human Growth Hormone Growth hormone Up to 35 mg/L Xu et al., 2010
Human Interferon α2b Anti-viral and immunomodulator 0.2–3% TSP Xu et al., 2007
IL-10 Immunoregulation Up to 3% TSP Kaldis et al., 2013
Norwalk virus capsid protein Acute gastroenteritis vaccine Up to 1.2% TSP Zhang and Mason, 2006
IL-12 Immunoregulation Up to 160 µg/L Kwon et al., 2003b
Rice cells Oryza sativa Human α1-antitrypsin Emphysema 4.5–7.7 mg/L
Up to 150 mg/L
McDonald et al., 2005;
Trexler et al., 2005
hCTLA4Ig Immunosuppressive agent Up to 31.4 mg/L Lee et al., 2007
Der p 2-FIP-fve fusion protein Immunomodulator and
immunotherapeutic for allergies
10.5% TSP Su et al., 2012
hGM-CSF Production of white cells 2% TSP Kim et al., 2008b
Human Serum Albumin Treatment of hypoalbuminemia up to 25 mg/L Huang et al., 2005
Human CTLS4Ig Immunosupressive agent Up to 31.4 mg/L Lee et al., 2007; Kang
et al., 2015
Human Growth Hormone Growth Hormone Up to 120 mg/L Kim et al., 2008a
Granulocyte-Macrophage Colony-
Stimulating Factor (GM-CSF)
Production of white cells Up to 200 mg/L Lee et al., 2007; Shin
et al., 2011
Medicago
cells
Medicago
truncatula cv.
Jemalong
EPO Tissue protective Pires et al., 2012
Prostaglandin D2Synthase Clinical marker Pires et al., 2014
Carrot cells Daucus
carota
Taliglucerase alfa Gaucher disease Shaaltiel et al., 2007
PEGylated recombinant human
acetylcholinesterase (PRX-105)
Biodefense program Protalix Biotherapeutics
(www.protalix.com)
α1-antitrypsin (PRX-107) Emphysema Protalix Biotherapeutics
(www.protalix.com)
Tomato cells Lycopersicon
esculentum
hGM-CSF Immunosuppressive and
immunomodulator
Up to 45 µg/L Kwon et al., 2003a
Soybean cells Glycine max HBsAg Vaccine against Hepatitis B 65 µg/g FW Smith et al., 2002
Siberian
Ginseng cells
Acanthopanax
senticosus
Human lactoferrin Immunosupressive and
immunomodulator
0.2–2.3% TSP Jo et al., 2006
Korean
ginseng cells
Panax
ginseng
Human lactoferrin Immunosupressive and
immunomodulator
3% TSP Kwon S. Y. et al., 2003
Sweet Potato
cells
Ipomoea
batatas
Human lactoferrin Immunosupressive and
immunomodulator
3.2 µg/mg TSP Min et al., 2006
SN, supernatant; TSP, total soluble protein, FW, fresh weight.
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molecular farming because suspension cells can be derived from
the mature leaf, root and seedling cotyledon, and transformation
is highly efficient (Araujo et al., 2004). A cell line derived from
this species was shown to achieve high recombinant protein
yields (Pires et al., 2008) although only two biopharmaceutical
products have been reported thus far (Pires et al., 2012, 2014).
Other proteins have been produced in cell lines derived from
tomato (Solanum lycopersicum), soybean (Glycine max), potato
(Solanum tuberosum), sunflower (Helianthus annus), sweet
potato (Ipomoea batatas), and medicinal plants such as Siberian
ginseng (Eleutherococcus senticosus) and Korean ginseng (Panax
ginseng). Biopharmaceuticals produced in these species are listed
in Table 1.
PROGRESS AND CHALLENGES
Although plant cells are relative newcomers in the commercial
environment and the yields they achieve still lag behind
those of microbes and mammalian cells, it is important to
remember that the yields produced by microbes and mammalian
cells have increased substantially during their 30 years as
industrial platform leaders. These increases have been achieved
incrementally by several routes, including strain optimization,
genetic modification to improve production characteristics,
medium optimization, and process engineering (e.g., bioreactor
design and fermentation conditions). In contrast, plant cells
have been used commercially for less than 10 years and
already the improvements have been striking, mainly because
the lessons learned during the development of microbial and
mammalian cell platforms have been applied to plant cells
comparatively much earlier in their history as a platform
technology, and novel approaches adopted by the industry
more recently have been used with plant cells immediately, and
implemented during early process development. These include
strategies such as high-throughput clone selection, medium
and process optimization using statistical experimental designs
(typically design-of-experiments approaches) and the application
of in-process monitoring systems, known as process analytical
technology (PAT). Given the lead time, it is clear that current
industry-standard mammalian cell lines such as Chinese hamster
ovary (CHO) cells will remain superior in terms of overall yields
for some time to come, but plant cells are gaining ground due
to the many advantageous properties they offer (Table 2). The
main challenges that plant cells still face are the absence of a gene
amplification system comparable to the systems used with CHO
cells (BOX 3) and the convenience of handling issues that are
important for GMP compliance, such as cryopreservation and
cell banking (Eck and Keen, 2009; Mustafa et al., 2011).
SPECIFIC CHALLENGES—CELL
CLUSTERS, GROWTH CHARACTERISTICS,
AND CULTURE HETEROGENEITY
Almost all plant cell suspension cultures share one key property
that sets them aside from microbial and mammalian cells—they
do not grow as single cells but instead form clusters (Mavituna
and Park, 1987; Nagata et al., 2013). Moreover, plant cells can
grow significantly by elongation, increasing the volume and wet
biomass without increasing the cell number. Both issues must
be addressed by adopting specific methodologies. Although cell
clusters can be advantageous, e.g., aggregation can be used as
the basis for self-immobilization methods (Kieran et al., 1997;
Kolewe et al., 2011), large cell clusters are generally undesirable
because cells in the center may have limited oxygen and nutrient
availability.
Cell clusters are also challenging during the generation of
transgenic cell lines because monoclonal cultures cannot be
generated by plating or limiting dilution. Cell suspension cultures
generated de novo by the transformation of wild-type cells are
always polyclonal because transformation is not 100% efficient
and different cells can be transformed at different loci (Muller
et al., 1996; Nocarova and Fischer, 2009). Even cell lines derived
from transgenic callus are rarely monoclonal because the callus
tissue may be chimeric. In both cases, the resulting transgenic
cell lines can also undergo somaclonal variation, generating cell
populations with heterogeneous expression levels (James and Lee,
2006). Therefore, even if an advanced technology such as the
CRISPR/Cas9 system is used to specify a targeted integration site,
one or more rounds of screening and selection is still necessary to
identify and isolate the most productive cells to seed monoclonal
production lines. Screening can be carried out at the callus
stage and the use of fluorescent marker proteins facilitates the
identification of chimeric callus tissue, allowing the selection of
cell material for sequential rounds of sub culturing (Figure 1).
An alternative is the preparation of protoplasts with subsequent
selection by flow cytometry, although single protoplasts are
TABLE 2 | Comparison among the available systems for biopharmaceutical production.
System N-glycosylation ability Contamination
risk
Time to
production
Scalability Overall cost
Plant cell suspensions Yes Very low Medium High Medium
no terminal galactose or sialic acid;
Core-xylose; different fucose linkage
Whole plant systems Low High Very high Low
Plant transient expression Low High High Low
Mammalian Cells Yes but different potential sialic acid
(NGNA) and alpha-Gal epitope both
potential immunogenic
High Medium Medium High
Overall cost: Low, $20–100/g; Medium, $50–1000/g; High, $1000–10,000/g.
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FIGURE 1 | Sectored callus cultures. (A,C) Images were taken under
normal white light, (B,D) Images were taken under green light with a red filter
for the macroscopic visualization of DsRed fluorescence.
fragile and plating on feeder cells is often required. Recently,
flow sorting has been used to separate the most productive cells
from a heterogeneous tobacco BY-2 cell culture producing a full-
length human antibody, by selecting the co-expressed fluorescent
marker protein DsRed located on the same T-DNA (Kirchhoff
et al., 2012). Using a feeder strategy, single protoplasts selected by
flow cytometry were regenerated into stable monoclonal cell lines
with homogeneous DsRed fluorescence and antibody yields up to
13-fold higher than the parent culture.
The productivity and growth characteristics of cell lines at the
callus stage and in suspension are often unrelated thus raising
additional challenges. Although fluorescent marker proteins can
be used to screen callus tissue, it is good practice to continue
screening the suspension cells under realistic production
conditions to ensure a compromise between protein production
and cell growth rates. CHO cell lines also display idiosyncratic
behavior with respect to stability, media requirements and other
process performance parameters. The current industry solution
is to screen a sufficiently large number of clones under rigorous
selection criteria to ensure that high-performance clones are
identified, and this strategy is equally applicable to plant cell
suspension cultures.
Because plant cells are large and tend to grow in clusters,
it can be difficult to determine accurate cell densities. Plant
cells are 50–200 µM in length and range in morphology from
spherical to cylindrical depending on the growth phase. Cells
in the exponential growth phase undergoing rapid division are
spherical or elliptical, with a length of 50–100 µm, whereas those
at the end of the exponential growth phase grow mainly by
elongation and tend to be more cylindrical, with a length of
up to 200 µm (Mavituna and Park, 1987; Holland et al., 2013).
Aggregation occurs when daughter cells fail to separate after
cell division, and is promoted by extracellular polysaccharides.
The tendency for form clumps varies between cell lines and
depends on the age of the cells and the growth conditions. Cell
counting is the most precise method to establish cell density
but it becomes more difficult when the cells clump together.
Alternative methods such as the measurement of turbidity
or light scattering are also unsuitable due to the size of the
clumps. Therefore, the density of plant cell suspension cultures
is often determined by measuring the packed cell volume or
wet cell weight after gentle centrifugation and aspiration of the
supernatant. Alternatively, the pellet can be dried and cell density
can be extrapolated from the dry weight. However, these are
invasive and destructive off-line procedures. The use of non-
invasive radio frequency impedance spectroscopy (RFIS) offers
significant benefits because it can achieve continuous in-line real-
time measurement suitable for PAT. Although RFIS measures
the volume of viable cells, this parameter correlates well with
the packed cell volume, wet cell weight, and dry biomass weight.
Continuous measurement can also pinpoint the transition from
cell division to cell elongation (Holland et al., 2013).
SPECIFIC CHALLENGES—MEDIUM
OPTIMIZATION
The productivity of cell suspension cultures can be improved
by optimizing the expression construct and by selecting highly-
productive monoclonal cultures, but it is also necessary to
optimize the culture conditions starting with the growth medium
(Schillberg et al., 2013). Unlike whole plants, cell suspension
cultures are not phototrophic so they require a carbon source.
Plant cell media therefore usually contain sucrose, inorganic
salts, vitamins, plant hormones and water, and a wide range
of different media are commercially available depending on the
species, growth characteristics, and purpose of the cultivation
(Fawcett, 1954; Murashige and Skoog, 1962; Gamborg et al.,
1968). In many cases, the growth medium is a variant of the
MS recipe developed by Murashige and Skoog (1962), which
provides nitrogen as a mixture of nitrate and ammonium salts.
However, the addition of more nitrogen to MS medium can
improve the productivity of BY-2 cells by up to 150-fold in the
stationary phase, ultimately improving yields of recombinant
proteins by up to 20-fold (Holland et al., 2010; Ullisch et al.,
2012). In contrast to CHO cells, where medium optimization
needs to be done for each product and cell line on a case-by-case
basis (Wurm, 2004), for plant cell cultures it appears to similarly
benefit all products (Holland et al., 2010; Ullisch et al., 2012). This
was one catalyst for the introduction of statistical experimental
designs that can simultaneously test the impact of varying
several different medium components simultaneously, as well
as other conditions such as pH, temperature and aeration rate.
Accordingly, product-specific medium optimization achieved
a five-fold increase in the yield of a recombinant antibody
produced by tobacco BY-2 cells following the application of
a statistical experimental design (Vasilev et al., 2013). The
impact of changes in medium composition during fermentation,
and the introduction of compensatory in-line adjustments,
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Santos et al. Focusing on Plant Cell Suspensions
has also boosted product yields substantially. For example,
a respiration activity monitoring system (RAMOS) revealed
metabolic changes in cultivated BY-2 cells caused by ammonia
depletion, and the replacement of this missing ammonia resulted
in a 100% increase in product yields (Ullisch et al., 2012).
SPECIFIC CHALLENGES—PROTEIN
DEGRADATION
Degradation caused by intracellular and extracellular proteases
reduces the yield and quality of biopharmaceuticals produced in
plant cells, and extra purification steps are required to remove
degradation products. Extracellular degradation can be avoided
by targeting the protein to accumulate within an intracellular
compartment, and the endoplasmic reticulum (ER) is often used
for this purpose because complex proteins fold efficiently and
accumulate to higher levels than those secreted to the apoplast,
i.e., the space under the cell wall (Twyman et al., 2013). However,
the benefits of intracellular accumulation must be balanced
against two drawbacks—the need to extract the protein by
breaking the cell, which releases more contaminants (including
proteases) during downstream processing (Buyel et al., 2015),
and the impact on glycosylation, which is discussed in the next
section. A better approach is to allow secretion but to counter the
effect of proteases directly. Plants produce hundreds of proteases
and it is not always possible to identify which is responsible for
degrading a recombinant protein, particularly because different
products are susceptible to different protease classes (Mandal
et al., 2010, 2014; Navarre et al., 2012; Niemer et al., 2014). If
a particular protease can be identified then it may be possible
to knock out the corresponding gene or co-express a protease
inhibitor to prevent product degradation (Kim et al., 2008b;
Benchabane et al., 2009). Decoy proteins such as gelatin or
bovine serum albumin can also be added to the medium but
proteinaceous additives from animal sources must be evaluated
carefully because they pose a risk of contamination with prions,
thus compromising the economic and regulatory advantages of
plant cells (James et al., 2000; Baur et al., 2005). Non-protein
additives such as polyvinylpyrrolidone (Magnuson et al., 1996;
LaCount et al., 1997), Pluronic F-68 and polyethylene glycol (Lee
and Kim, 2002) can also reduce the damage caused by proteases
but may be difficult to remove in subsequent processing steps
(Baur et al., 2005). Osmotic stress has also been proposed to
increase product accumulation, although this may inhibit cell
growth so the timing of application must be optimized carefully
(Tsoi and Doran, 2002; Soderquist and Lee, 2005). Medium
optimization and process control are promising tools to avoid
protein degradation during production—for example, a balanced
supply of nitrogen not only dramatically increases the amount
of a secreted antibody but also stabilizes the secreted product
toward the end of the cultivation process (Holland et al., 2010).
SPECIFIC CHALLENGES—PLANT
GLYCANS
The early steps of protein glycosylation in plants and mammals
are identical, but once a nascent protein moves from the ER
to the Golgi apparatus subtle differences in the oligosaccharide
structures begin to appear. Plant glycoproteins tend to contain
core α1,3-fucose (rather than core α1,6-fucose which is
present in mammals) and core β1,2-xylose, whereas mammalian
glycoproteins contain β1,4-galactose and terminal sialic acid
residues that are not present in plants (Gomord et al.,
2010). Initially there was concern that plant glycans could be
immunogenic in humans and much effort was expended to
ensure that plant glycans were avoided. This involved either
targeting the proteins to be retained in the ER resulting in generic
high-mannose glycans, or engineering plant lines in which the
glycosylation pathway was modified to abolish the enzymes
responsible for plant glycans and, in some cases, introduce
enzymes that produced human-like glycans instead (Castilho and
Steinkellner, 2012; Bosch et al., 2013). The glycan panic has since
abated given the lack of evidence that plant glycans are harmful
in humans (Gomord et al., 2010). The first-in-class Protalix drug
taliglucerase alfa (Table 1) contains the aforementioned core
α1,3-fucose and β1,2-xylose residues but no adverse effects have
been reported in clinical trials or post-market use (Tekoah et al.,
2015).
Although plant glycans can affect the properties of
recombinant proteins, including stability and functionality, in
some cases the plant-derived version is superior—not biosimilar
but bio-better. In the case of taliglucerase alfa, targeting the
protein to the vacuole of carrot cells exposes terminal mannose
residues that are required for the efficient uptake of the enzyme
into macrophages by mannose receptors. The equivalent protein
produced in CHO cells (imiglucerase, marketed as Cerezyme R
)
has terminal sialic acid residues that prevent uptake, and these
must be enzymatically removed in vitro during processing, which
increases the production costs dramatically. The comparison
between taliglucerase alfa and imiglucerase also highlights the
safety advantages of plant cells. The production of imiglucerase
in CHO cells by Genzyme was shut down for a significant time
due to viral contamination in the production plant (European
Medicines Agency, 2009). This resulted in an acute shortage of
the product because the plant was responsible for ∼20% of the
global supply at that time (Hollak et al., 2010).
SPECIFIC CHALLENGES—UPSTREAM
PROCESSING STRATEGIES
Plant cell cultures are often successful in the laboratory because
they can be grown in well-aerated shake flasks and the products
can be extracted in small volumes of buffer, allowing the use of
protease inhibitors and other expensive additives that cannot be
used at the process scale. Cell line selection and optimization
also tends to be carried out using small flasks or even microtiter
plates, so scaling up production is a significant challenge (Fischer
et al., 2015). Plant cell cultures have been cultivated in many
different bioreactors, like stirred tanks reactors (STR’s; Hooker
et al., 1990; Doran, 1999; Trexler et al., 2002), wave reactors
(Eibl and Eibl, 2006), wave and undertow (Terrier et al., 2007),
bubble column (Terrier et al., 2007), single use bubble column
reactor (Shaaltiel et al., 2007), air-life reactors (Wen Su et al.,
1996), membrane reactors (McDonald et al., 2005), and rotation
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Santos et al. Focusing on Plant Cell Suspensions
drum reactors (Tanaka et al., 1983). The homogeneous nature
of plant cell suspension cultures requires a fermentation broth
similar to that used for microbes and mammalian cells, which
is fed with nutrients and oxygen and mixed to achieve an even
distribution (Hellwig et al., 2004). In many reports, stirred-tank
bioreactors with large impellers and a ring sparger have been used
to reduce shear stress, delivering maximum biomass values of
60–70% packed cell volume. In the context of cell suspension
cultures the bioreactor must be matched to the production
line to synergize with its biological characteristics, e.g., growth
rate, morphology, aggregation tendency, shear sensitivity, oxygen
demand, and rheological properties. The relative merits of the
different reactor designs have been intensively discussed and
reviewed elsewhere (Xu et al., 2010; Huang and McDonald,
2012).
The cultivations in the different reactor designs covering
a range of volumes and offering three main fermentation
strategies: batch, fed-batch and continuous processes. The batch
fermentation is the simplest and most commonly used process.
A batch fermentation is filled with medium, inoculated and
after inoculation the reactor is a closed system except for a
few additives like oxygen and base/acid for controlling the
pH-value. The cell culture undergoes a lag, exponential and
stationary growth phase and cell growth generally occurs under
varying and sometimes unfavorable conditions. A more advance
fermentation modes is the fed-batch fermentation, which starts
with a classical batch phase and once certain conditions are
reached the feed is started, i.e., additional nutrients are provided.
In fed-batch also several culture parameters are changing and
the cells undergo classical growth phases. Furthermore, batch
and fed-batch fermentations suffer from low running-to-set-up-
times ratio (preparation, sterilization before the cultivation, and
cleaning afterwards), which can be compensated by investments
of men-power and infrastructure. To overcome low running-
to-set-up-times different continuous fermentation strategies for
plant cell cultures has been developed. Classical continuous
fermentations strategies are perfusion and chemostat processes.
In perfusion processes the cells in the reactor are supported
with continuous feed of fresh media and cell free fermentation
broth is constantly being removed in the same volume. Perfusion
fermentation have successfully realized with plant cell cultures
(Su and Arias, 2003; De Dobbeleer et al., 2006). In a chemostat
fermentation the culture is also being supported by a continuous
feed of fresh medium but in this case the same volume of
fermentation broth which is being removed also contains cells,
thus in a perfusion the cell density is increasing while in a
chemostat the cell density stays constant. A chemostat cultivation
can run over a long time period and the cells are maintained
at the exponential growth phase, which make this strategy
attractive for large scale production (Miller et al., 1968; van Gulik
et al., 2001). Despite the ideal characteristics of the continuous
bioreactor, the process itself is sensitive and subjected to influence
from various factors such as risks of contaminations, genetic
instability, and changes in the biotic phase of the bioreactor.
To avoid these semi-continuous fermentation strategies have
been developed, where a fraction of the fermentation broth is
removed once and replaced by fresh medium (Hogue et al.,
1990; Huang et al., 2010). The merits of the different cultivation
strategies is summarized and discussed intensively from Xu
et al. (2011). Some recent examples for large scale cultivations
are the manufacture of antibodies in tobacco BY-2 cells, which
has been scaled up from shake flasks to 200-L disposable
bioreactors without loss of yield (Raven et al., 2015). Scaling
up from 50-ml shake flasks to 600-L bioreactors (a factor
of 12,000) has also been achieved without any impact on
growth characteristics (Reuter et al., 2014). Taliglucerase alfa and
other products in the Protalix pipeline are produced in carrot
cells cultivated in bubble column-type bioreactors fitted with
disposable polyethylene bags (Shaaltiel et al., 2007; Tekoah et al.,
2015).
SPECIFC CHALLENGES—CELL BANKING
To meet the regulatory requirements it will be necessary to
ensure cell line stability over the entire production process time.
Many cell cultures are maintained by a weekly sub culturing
routine and are stable over long time period, nevertheless there
are only few early studies on long term production stability
done (Gao et al., 1991; Sierra et al., 1992; Kirchhoff et al.,
2012). Cell banking for the supply of well-defined starting
material and a routine procedure for the cryopreservation
of plant cells is one key feature to enable plant suspension
culture as a biopharmaceutical production platform. The first
successful cryo preservations of plant cell cultures have been
reported in the late 1960s and 1970s (Quatrano, 1968; Nag and
Street, 1973). Since then several protocols have been developed,
e.g., for particular cell lines like BY-2 cells and tobacco cell
lines (Menges and Murray, 2004; Schmale et al., 2006) or
arabidopsis cells (Menges and Murray, 2004; Ogawa et al., 2008).
Although different techniques have been published, including
desiccation (Nitzsche, 1980), vitrification (Uragami et al., 1989)
or encapsulation-dehydration cryopreservation (Bonnart and
Volk, 2010), there is no protocol that can generally be applied
to cell suspension of all plant species and all protocols need
to be optimized on a case-by-case basis. Only few protocols
have been verified for different cell species (Ogawa et al.,
2012).
CONCLUSIONS
In the near future, plant cell suspension cultures will most
certainly become the preferred choice among plant-based
systems for the production of high-value recombinant proteins,
because they combine the advantages of all other systems.
Although plant cells have been overshadowed by whole-plant
platforms, this trend has been inverted following the approval of
taliglucerase alfa for use in human adults in 2012 and then for
pediatric use in 2014 (Tekoah et al., 2015). This has opened the
way to the full acceptance of this technology, and several other
products are now undergoing clinical trials and are expected
to reach the market in the near future. The significant number
of drugs that are now coming off patent will contribute to this
market expansion.
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Santos et al. Focusing on Plant Cell Suspensions
Plant-based systems still face one major bottleneck that needs
to be overcome—their lower yields compared to mammalian
cell cultures. This partly reflects the much more recent
emergence of plant cells as a competitive platform, so there
has been less investment thus far in strain, medium and
process optimization compared to mammalian cells. However,
there are many researchers currently working to address
this challenge, and several recent reports discussed herein
have made breakthroughs in the development of robust
upstream production and downstream processing strategies.
These developments include medium optimization, process
engineering, statistical experimental designs, scale-up/scale-
down models, and process analytical technologies. Overall,
these optimization procedures will lead to higher yields and
will put plant cell cultures back into the spotlight. Other
factors that will also contribute to the success of plant cells
include the straightforward compliance with GMP compared
with whole plants, and the better public acceptance of
biopharmaceuticals produced in cultivated cells than GM
plants.
AUTHOR CONTRIBUTIONS
All authors listed, have made substantial, direct and intellectual
contribution to the work, and approved it for publication.
All authors contributed equally in the Introduction, Progress
and challenges and conclusion parts. RS and RA contributed
in the specific Challenges: Cell clusters, growth characteristics,
and culture heterogeneity, protein degradation and in making
and designing the tables. TH, MS, and RF contributed in
the specific challenges: Medium optimization, plant glycans,
upstream processing, and cell banking.
FUNDING
This work was funded by Fundação para a Ciência e Tecnologia
(FCT, Portugal) through grants ERA-IB/0001/2012, PTDC/BIA-
PLA/2411/2012, and UID/Multi/04551/2013 and by the
Fraunhofer Future Foundation project “Innovative technologies
to manufacture ground-breaking biopharmaceutical products in
microbes and plants” (125-300004).
REFERENCES
Abranches, R., Marcel, S., Arcalis, E., Altmann, F., Fevereiro, P., and Stoger,
E. (2005). Plants as bioreactors: a comparative study suggests that Medicago
truncatula is a promising production system. J. Biotechnol. 120, 121–134. doi:
10.1016/j.jbiotec.2005.04.026
Araujo, S. D., Duque, A. S. R. L. A., Dos Santos, D. M. M. F., and Fevereiro,
M. P. S. (2004). An efficient transformation method to regenerate a high
number of transgenic plants using a new embryogenic line of Medicago
truncatula cv. Jemalong. Plant Cell Tissue Organ Cult. 78, 123–131. doi:
10.1023/B:TICU.0000022540.98231.f8
Arfi, Z. A., Hellwig, S., Drossard, J., Fischer, R., and Buyel, J. F. (2015). Polyclonal
antibodies for specific detection of tobacco host cell proteins can be efficiently
generated following RuBisCO depletion and the removal of endotoxins.
Biotechnol. J. doi: 10.1002/biot.201500271. [Epub ahead of print].
Arntzen, C. (2015). Plant-made pharmaceuticals: from ‘Edible Vaccines’ to Ebola
therapeutics. Plant Biotechnol. J. 13, 1013–1016. doi: 10.1111/pbi.12460
Baneyx, F., and Mujacic, M. (2004). Recombinant protein folding and misfolding
in Escherichia coli.Nat. Biotechnol. 22, 1399–1408. doi: 10.1038/nbt1029
Baur, A., Reski, R., and Gorr, G. (2005). Enhanced recovery of a secreted
recombinant human growth factor using stabilizing additives and by
co-expression of human serum albumin in the moss Physcomitrella
patens. Plant Biotechnol. J. 3, 331–340. doi: 10.1111/j.1467-7652.2005.
00127.x
Benchabane, M., Rivard, D., Girard, C., and Michaud, D. (2009). Companion
protease inhibitors to protect recombinant proteins in transgenic plant extracts.
Methods Mol. Biol. 483, 265–273. doi: 10.1007/978-1-59745-407-0_15
Bonnart, R., and Volk, G. M. (2010). Increased efficiency using the encapsulation-
dehydration cryopreservation technique for Arabidopsis thaliana.Cryo Letters
31, 200–205.
Bosch, D., Castilho, A., Loos, A., Schots, A., and Steinkellner, H. (2013).
N-Glycosylation of plant-produced recombinant proteins. Curr. Pharm. Des.
19, 5503–5512. doi: 10.2174/1381612811319310006
Broeckling, C. D., Huhman, D. V., Farag, M. A., Smith, J. T., May, G. D., Mendes,
P., et al. (2005). Metabolic profiling of Medicago truncatula cell cultures reveals
the effects of biotic and abiotic elicitors on metabolism. J. Exp. Bot. 56, 323–336.
doi: 10.1093/jxb/eri058
Buyel, J. F., Twyman, R. M., and Fischer, R. (2015). Extraction and downstream
processing of plant-derived recombinant proteins. Biotechnol. Adv. 33,
902–913. doi: 10.1016/j.biotechadv.2015.04.010
Cacciatore, J. J., Chasin, L. A., and Leonard, E. F. (2010). Gene amplification
and vector engineering to achieve rapid and high-level therapeutic protein
production using the Dhfr-based CHO cell selection system. Biotechnol. Adv.
28, 673–681. doi: 10.1016/j.biotechadv.2010.04.003
Castilho, A., and Steinkellner, H. (2012). Glyco-engineering in plants to
produce human-like N-glycan structures. Biotechnol. J. 7, 1088–1098. doi:
10.1002/biot.201200032
Choi, J. H., and Lee, S. Y. (2004). Secretory and extracellular production of
recombinant proteins using Escherichia coli.Appl. Microbiol. Biotechnol. 64,
625–635. doi: 10.1007/s00253-004-1559-9
Chu, L., and Robinson, D. K. (2001). Industrial choices for protein production by
large-scale cell culture. Curr. Opin. Biotechnol. 12, 180–187. doi: 10.1016/S0958-
1669(00)00197-X
Cook, D. R. (1999). Medicago truncatula–a model in the making! Curr. Opin. Plant
Biol. 2, 301–304. doi: 10.1016/S1369-5266(99)80053-3
De Dobbeleer, C., Cloutier, M., Fouilland, M., Legros, R., and Jolicoeur, M.
(2006). A high-rate perfusion bioreactor for plant cells. Biotechnol. Bioeng. 95,
1126–1137. doi: 10.1002/bit.21077
Doran, P. M. (1999). Design of mixing systems for plant cell suspensions in stirred
reactors. Biotechnol. Prog. 15, 319–335. doi: 10.1021/bp990042v
Doran, P. M. (2000). Foreign protein production in plant tissue cultures. Curr.
Opin. Biotechnol. 11, 199–204. doi: 10.1016/S0958-1669(00)00086-0
Eck, J., and Keen, P. (2009). Continued expression of plant-made vaccines
following long-term cryopreservation of antigen-expressing tobacco cell
cultures. In Vitro Cell. Dev. Biol. Plant 45, 750–757. doi: 10.1007/s11627-009-
9231-9
Eibl, R., and Eibl, D. (2006). “Design and use of the wave bioreactor for plant cell
culture,” in Plan Tissue Culture Engineering, eds S. D. Gupta and Y. Ibaraki
(Dordrecht: Springer), 203–227.
Eichholtz, D. A., Rogers, S. G., Horsch, R. B., Klee, H. J., Hayford, M., Hoffmann,
N. L., et al. (1987). Expression of mouse dihydrofolate reductase gene confers
methotrexate resistance in transgenic petunia plants. Somat. Cell Mol. Genet.
13, 67–76. doi: 10.1007/BF02422300
European Medicines Agency (2009). Supply shortages of Cerezyme and Fabrazyme
- priority access for patient most in need of treatment recommended
[Press release]. Retrieved from: http://www.ema.europa.eu/ema/index.jsp?
curl=pages/news_and_events/news/2009/11/news_detail_000075.jsp&mid=
WC0b01ac058004d5c1
Fawcett, D. W. (1954). The Cultivation of Animal and Plant Cells, Vol. 120. New
York, NY: Ronald Press.
Frontiers in Plant Science | www.frontiersin.org 9March 2016 | Volume 7 | Article 297

Santos et al. Focusing on Plant Cell Suspensions
Fischer, R., Vasilev, N., Twyman, R. M., and Schillberg, S. (2015). High-value
products from plants: the challenges of process optimization. Curr. Opin.
Biotechnol. 32, 156–162. doi: 10.1016/j.copbio.2014.12.018
Gamborg, O. L., Miller, R. A., and Ojima, K. (1968). Nutrient requirements of
suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158. doi:
10.1016/0014-4827(68)90403-5
Gao, J., Lee, J. M., and An, G. (1991). The stability of foreign protein
production in genetically modified plant cells. Plant Cell Rep. 10, 533–536. doi:
10.1007/BF00234589
Gerngross, T. U. (2004). Advances in the production of human therapeutic
proteins in yeasts and filamentous fungi. Nat. Biotechnol. 22, 1409–1414. doi:
10.1038/nbt1028
Gomord, V., Fitchette, A. C., Menu-Bouaouiche, L., Saint-Jore-Dupas, C., Plasson,
C., Michaud, D., et al. (2010). Plant-specific glycosylation patterns in the
context of therapeutic protein production. Plant Biotechnol. J. 8, 564–587. doi:
10.1111/j.1467-7652.2009.00497.x
Havenith, H., Raven, N., Di Fiore, S., Fischer, R., and Schillberg, S. (2014). Image-
based analysis of cell-specific productivity for plant cell suspension cultures.
Plant Cell Tissue Organ Cult. 117, 393–399. doi: 10.1007/s11240-014-0448-x
Hellwig, S., Drossard, J., Twyman, R. M., and Fischer, R. (2004). Plant cell cultures
for the production of recombinant proteins. Nat. Biotechnol. 22, 1415–1422.
doi: 10.1038/nbt1027
Hogue, R. S., Lee, J. M., and An, G. (1990). Production of a foreign protein product
with genetically modified plant cells. Enzyme Microb. Technol. 12, 533–538. doi:
10.1016/0141-0229(90)90071-W
Hollak, C. E. M., vom Dahl, S., Aerts, J. M. F. G., Belmatoug, N., Bembi, B., Cohen,
Y., et al. (2010). Force majeure: therapeutic measures in response to restricted
supply of imiglucerase (Cerezyme) for patients with Gaucher disease. Blood
Cells Mol. Dis. 44, 41–47. doi: 10.1016/j.bcmd.2009.09.006
Holland, T., Blessing, D., Hellwig, S., and Sack, M. (2013). The in-line
measurement of plant cell biomass using radio frequency impedance
spectroscopy as a component of process analytical technology. Biotechnol. J.
8, 1231–1240. doi: 10.1002/biot.201300125
Holland, T., Sack, M., Rademacher, T., Schmale, K., Altmann, F., Stadlmann, J.,
et al. (2010). Optimal nitrogen supply as a key to increased and sustained
production of a monoclonal full-size antibody in BY-2 suspension culture.
Biotechnol. Bioeng. 107, 278–289. doi: 10.1002/bit.22800
Hooker, B. S., Lee, J. M., and An, G. (1990). Cultivation of plant cells in a
stirred vessel: effect of impeller design. Biotechnol. Bioeng. 35, 296–304. doi:
10.1002/bit.260350311
Huang, L.-F., Liu, Y.-K., Lu, C.-A., Hsieh, S.-L., and Yu, S.-M. (2005). Production
of human serum albumin by sugar starvation induced promoter and rice cell
culture. Transgenic Res. 14, 569–581. doi: 10.1007/s11248-004-6481-5
Huang, T. K., and McDonald, K. A. (2012). Bioreactor systems for in vitro
production of foreign proteins using plant cell cultures. Biotechnol. Adv. 30,
398–409. doi: 10.1016/j.biotechadv.2011.07.016
Huang, T. K., Plesha, M. A., and McDonald, K. A. (2010). Semicontinuous
bioreactor production of a recombinant human therapeutic protein using a
chemically inducible viral amplicon expression system in transgenic plant
cell suspension cultures. Biotechnol. Bioeng. 106, 408–421. doi: 10.1002/bit.
22713
Itakura, K., Hirose, T., Crea, R., Riggs, A. D., Heyneker, H. L., Bolivar, F., et al.
(1977). Expression in Escherichia coli of a chemically synthesized gene for the
hormone somatostatin. Science 198, 1056–1063. doi: 10.1126/science.412251
James, E., and Lee, J. M. (2006). Loss and recovery of protein productivity
in genetically modified plant cell lines. Plant Cell Rep. 25, 723–727. doi:
10.1007/s00299-005-0096-z
James, E. A., Wang, C., Wang, Z., Reeves, R., Shin, J. H., Magnuson, N. S., et al.
(2000). Production and characterization of biologically active human GM-CSF
secreted by genetically modified plant cells. Protein Expr. Purif. 19, 131–138.
doi: 10.1006/prep.2000.1232
Jo, S.-H., Kwon, S.-Y., Park, D.-S., Yang, K.-S., Kim, J.-W., Lee, K.-T., et al. (2006).
High-yield production of functional human lactoferrin in transgenic cell
cultures of siberian ginseng (Acanthopanax senticosus). Biotechnol. Bioprocess
Eng. 11, 442–448. doi: 10.1007/BF02932312
Kaldis, A., Ahmad, A., Reid, A., McGarvey, B., Brandle, J., Ma, S., et al.
(2013). High-level production of human interleukin-10 fusions in tobacco cell
suspension cultures. Plant Biotechnol. J. 11, 535–545. doi: 10.1111/pbi.12041
Kang, S. H., Jung, H. S., Lee, S. J., Park, C. I., Lim, S. M., Park, H., et al.
(2015). Glycan structure and serum half-life of recombinant CTLA4Ig, an
immunosuppressive agent, expressed in suspension-cultured rice cells with
coexpression of human beta1,4-galactosyltransferase and human CTLA4Ig.
Glycoconj. J. 32, 161–172. doi: 10.1007/s10719-015-9590-x
Kato, K., Matsumoto, T., Koiwai, S., Mizusaki, S., Nishida, K., Nogushi, M., et al.
(1972). “Liquid suspension culture of tobaco cells,” in Ferment Technology
Today, ed G. Terui (Osaka: Society of Fermentation Technology), 689–695.
Kieran, P. M., MacLoughlin, P. F., and Malone, D. M. (1997). Plant cell suspension
cultures: some engineering considerations. J. Biotechnol. 59, 39–52. doi:
10.1016/S0168-1656(97)00163-6
Kim, T.-G., Baek, M.-Y., Lee, E.-K., Kwon, T.-H., and Yang, M.-S. (2008a).
Expression of human growth hormone in transgenic rice cell suspension
culture. Plant Cell Rep. 27, 885–891. doi: 10.1007/s00299-008-0514-0
Kim, T. G., Lee, H. J., Jang, Y. S., Shin, Y. J., Kwon, T. H., and Yang,
M. S. (2008b). Co-expression of proteinase inhibitor enhances recombinant
human granulocyte-macrophage colony stimulating factor production in
transgenic rice cell suspension culture. Protein Expr. Purif. 61, 117–121. doi:
10.1016/j.pep.2008.06.005
Kirchhoff, J., Raven, N., Boes, A., Roberts, J. L., Russell, S., Treffenfeldt, W., et al.
(2012). Monoclonal tobacco cell lines with enhanced recombinant protein
yields can be generated from heterogeneous cell suspension cultures by flow
sorting. Plant Biotechnol. J. 10, 936–944. doi: 10.1111/j.1467-7652.2012.00722.x
Kizhner, T., Azulay, Y., Hainrichson, M., Tekoah, Y., Arvatz, G., Shulman, A., et al.
(2015). Characterization of a chemically modified plant cell culture expressed
human alpha-Galactosidase-A enzyme for treatment of Fabry disease. Mol.
Genet. Metab. 114, 259–267. doi: 10.1016/j.ymgme.2014.08.002
Kolewe, M. E., Henson, M. A., and Roberts, S. C. (2011). Analysis of aggregate
size as a process variable affecting paclitaxel accumulation in Taxus suspension
cultures. Biotechnol. Prog. 27, 1365–1372. doi: 10.1002/btpr.655
Komarova, T. V., Baschieri, S., Donini, M., Marusic, C., Benvenuto, E., and
Dorokhov, Y. L. (2010). Transient expression systems for plant-derived
biopharmaceuticals. Expert Rev. Vaccines 9, 859–876. doi: 10.1586/erv.10.85
Kwon, S. Y., Jo, S. H., Lee, O. S., Choi, S. M., Kwak, S. S., and Lee, H. S. (2003).
Transgenic ginseng cell lines that produce high levels of a human lactoferrin.
Planta Med. 69, 1005–1008. doi: 10.1055/s-2003-45146
Kwon, T. H., Kim, Y. S., Lee, J. H., and Yang, M. S. (2003a). Production
and secretion of biologically active human granulocyte-macrophage colony
stimulating factor in transgenic tomato suspension cultures. Biotechnol. Lett.
25, 1571–1574. doi: 10.1023/A:1025409927790
Kwon, T. H., Seo, J. E., Kim, J., Lee, J. H., Jang, Y. S., and Yang, M. S. (2003b).
Expression and secretion of the heterodimeric protein interleukin-12 in plant
cell suspension culture. Biotechnol. Bioeng. 81, 870–875. doi: 10.1002/bit.10528
LaCount, W., An, G., and Lee, J. M. (1997). The effect of polyvinylpyrrolidone
(PVP) on the heavy chain monoclonal antibody production from plant
suspension cultures. Biotechnol. Lett. 19, 93–96. doi: 10.1023/A:1018383524389
Lee, J. H., Kim, N. S., Kwon, T. H., Jang, Y. S., and Yang, M. S. (2002).
Increased production of human granulocyte-macrophage colony stimulating
factor (hGM-CSF) by the addition of stabilizing polymer in plant suspension
cultures. J. Biotechnol. 96, 205–211. doi: 10.1016/S0168-1656(02)00044-5
Lee, S. J., Park, C. I., Park, M. Y., Jung, H. S., Ryu, W. S., Lim, S. M., et al.
(2007). Production and characterization of human CTLA4Ig expressed in
transgenic rice cell suspension cultures. Protein Expr. Purif. 51, 293–302. doi:
10.1016/j.pep.2006.08.019
Lee, S.-Y., and Kim, D.-I. (2002). Stimulation of murine granulocyte macrophage-
colony stimulating factor production by Pluronic F-68 and polyethylene glycol
in transgenic Nicotiana tabacum cell culture. Biotechnol. Lett. 24, 1779–1783.
doi: 10.1023/A:1020609221148
Ma, J. K., Drossard, J., Lewis, D., Altmann, F., Boyle, J., Christou, P., et al.
(2015). Regulatory approval and a first-in-human phase I clinical trial of a
monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnol.
J. 13, 1106–1120. doi: 10.1111/pbi.12416
Magnuson, N. S., Linzmaier, P. M., Gao, J. W., Reeves, R., An, G., and Lee, J. M.
(1996). Enhanced recovery of a secreted mammalian protein from suspension
culture of genetically modified tobacco cells. Protein Expr. Purif. 7, 220–228.
doi: 10.1006/prep.1996.0030
Magnuson, N. S., Linzmaier, P. M., Reeves, R., An, G., HayGlass, K., and Lee, J. M.
(1998). Secretion of biologically active human interleukin-2 and interleukin-4
Frontiers in Plant Science | www.frontiersin.org 10 March 2016 | Volume 7 | Article 297

Santos et al. Focusing on Plant Cell Suspensions
from genetically modified tobacco cells in suspension culture. Protein Expr.
Purif. 13, 45–52. doi: 10.1006/prep.1998.0872
Mandal, M. K., Fischer, R., Schillberg, S., and Schiermeyer, A. (2010). Biochemical
properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum
cv. BY-2 suspension cells. Planta 232, 899–910. doi: 10.1007/s00425-010-1221-y
Mandal, M. K., Fischer, R., Schillberg, S., and Schiermeyer, A. (2014). Inhibition of
protease activity by antisense RNA improves recombinant protein production
in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells. Biotechnol. J.
9, 1065–1073. doi: 10.1002/biot.201300424
Matsumoto, S., Ikura, K., Ueda, M., and Sasaki, R. (1995). Characterization of a
human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant
Mol. Biol. 27, 1163–1172. doi: 10.1007/BF00020889
Mavituna, F., and Park, J. M. (1987). Size distribution of plant cell aggregates in
batch culture. Chem. Eng. J. 35, B9–B14. doi: 10.1016/0300-9467(87)80045-x
McDonald, K. A., Hong, L. M., Trombly, D. M., Xie, Q., and Jackman, A. P.
(2005). Production of human alpha-1-antitrypsin from transgenic rice cell
culture in a membrane bioreactor. Biotechnol. Prog. 21, 728–734. doi: 10.1021/
bp0496676
Menges, M., and Murray, J. A. (2004). Cryopreservation of transformed and wild-
type Arabidopsis and tobacco cell suspension cultures. Plant J. 37, 635–644. doi:
10.1046/j.1365-313X.2003.01980.x
Miller, R. A., Shyluk, J. P., Gamborg, O. L., and Kirkpatrick, J. W. (1968). Phytostat
for continuous culture and automatic sampling of plant-cell suspensions.
Science 159, 540–542. doi: 10.1126/science.159.3814.540
Min, S. R., Woo, J. W., Jeong, W. J., Han, S. K., Lee, Y. B., and Liu, J. R. (2006).
Production of human lactoferrin in transgenic cell suspension cultures of sweet
potato. Biol. Plant 50, 131–134. doi: 10.1007/s10535-005-0087-5
Muller, E., Lorz, H., and Lutticke, S. (1996). Variability of transgene expression
in clonal cell lines of wheat. Plant Sci. 114, 71–82. doi: 10.1016/0168-
9452(95)04312-8
Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and
bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. doi:
10.1111/j.1399-3054.1962.tb08052.x
Mustafa, N. R., de Winter, W., van Iren, F., and Verpoorte, R. (2011). Initiation,
growth and cryopreservation of plant cell suspension cultures. Nat. Protoc. 6,
715–742. doi: 10.1038/nprot.2010.144
Nag, K. K., and Street, H. E. (1973). Carrot embryogenesis from frozen cultured
cells. Nature 245, 270–272. doi: 10.1038/245270a0
Nagata, T., Hasezawa, S., and Depicker, A. (2013). Tobacco BY-2 Cells. Berlin;
Heidelberg: Springer.
Nagata, T., Nemoto, Y., and Hasezawa, S. (1992). Tobacco BY-2 cell line as the
“HeLa” cell in the cell biology of higher plants. Int. Rev. Cytol. 132, 1–30. doi:
10.1016/S0074-7696(08)62452-3
Nandi, S., Yalda, D., Lu, S., Nikolov, Z., Misaki, R., Fujiyama, K., et al.
(2005). Process development and economic evaluation of recombinant
human lactoferrin expressed in rice grain. Transgenic Res. 14, 237–249. doi:
10.1007/s11248-004-8120-6
Navarre, C., De Muynck, B., Alves, G., Vertommen, D., Magy, B., and Boutry, M.
(2012). Identification, gene cloning and expression of serine proteases in the
extracellular medium of Nicotiana tabacum cells. Plant Cell Rep. 31, 1959–1968.
doi: 10.1007/s00299-012-1308-y
Niemer, M., Mehofer, U., Torres Acosta, J. A., Verdianz, M., Henkel, T., Loos,
A., et al. (2014). The human anti-HIV antibodies 2F5, 2G12, and PG9
differ in their susceptibility to proteolytic degradation: down-regulation of
endogenous serine and cysteine proteinase activities could improve antibody
production in plant-based expression platforms. Biotechnol. J. 9, 493–500. doi:
10.1002/biot.201300207
Nitzsche, W. (1980). One year storage of dried carrot callus. Zeitschrift
Pflanzenphysiologie 100, 269–271. doi: 10.1016/S0044-328X(80)80255-8
Nocarova, E., and Fischer, L. (2009). Cloning of transgenic tobacco BY-2 cells; an
efficient method to analyse and reduce high natural heterogeneity of transgene
expression. BMC Plant Biol. 9:44. doi: 10.1186/1471-2229-9-44
Ogawa, Y., Sakurai, N., Oikawa, A., Kai, K., Morishita, Y., Mori, K., et al.
(2012). High-throughput cryopreservation of plant cell cultures for functional
genomics. Plant Cell Physiol. 53, 943–952. doi: 10.1093/pcp/pcs038
Ogawa, Y., Suzuki, H., Sakurai, N., Aoki, K., Saito, K., and Shibata, D.
(2008). Cryopreservation and metabolic profiling analysis of Arabidopsis T87
suspension-cultured cells. Cryo Letters 29, 427–436.
Paul, M. J., Teh, A. Y. H., Twyman, R. M., and Ma, J. K. C. (2013). Target product
selection - where can molecular pharming make the difference? Curr. Pharm.
Des. 19, 5478–5485. doi: 10.2174/1381612811319310003
Peyret, H., and Lomonossoff, G. P. (2015). When plant virology met
Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol. J. 13,
1121–1135. doi: 10.1111/pbi.12412
Pires, A. S., Rosa, S., Castanheira, S., Fevereiro, P., and Abranches, R. (2012).
Expression of a recombinant human erythropoietin in suspension cell cultures
of Arabidopsis, tobacco and Medicago. Plant Cell Tissue Organ Cult. 110,
171–181. doi: 10.1007/s11240-012-0141-x
Pires, A. S., Cabral, M. G., Fevereiro, P., Stoger, E., and Abranches, R. (2008).
High levels of stable phytase accumulate in the culture medium of transgenic
Medicago truncatula cell suspension cultures. Biotechnol. J. 3, 916–923. doi:
10.1002/biot.200800044
Pires, A. S., Santos, R. B., Nogueira, A. C., and Abranches, R. (2014). Production of
human lipocalin-type prostaglandin D synthase in the model plant Medicago
truncatula.In Vitro Cell. Dev. Biol.–Plant 50, 276–281. doi: 10.1007/s11627-
013-9584-y
PMR (2015). Global Market Study on Biopharmaceuticals: Asia to Witness Highest
Growth by 2020. New York, NY: Persistence Market Research.
Puchta, H., and Fauser, F. (2013). Gene targeting in plants: 25 years later. Int. J.
Dev. Biol. 57, 629–637. doi: 10.1387/ijdb.130194hp
Quatrano, R. S. (1968). Freeze-preservation of cultured flax cells utilizing dimethyl
sulfoxide. Plant Physiol. 43, 2057–2061.
Ramachandra Rao, S., and Ravishankar, G. A. (2002). Plant cell cultures:
chemical factories of secondary metabolites. Biotechnol. Adv. 20, 101–153. doi:
10.1016/S0734-9750(02)00007-1
Raven, N., Rasche, S., Kuehn, C., Anderlei, T., Klöckner, W., Schuster, F., et al.
(2015). Scaled-up manufacturing of recombinant antibodies produced by plant
cells in a 200-L orbitally-shaken disposable bioreactor. Biotechnol. Bioeng. 112,
308–321. doi: 10.1002/bit.25352
Regnard, G. L., Halley-Stott, R. P., Tanzer, F. L., Hitzeroth, I. I., and Rybicki, E.
P. (2010). High level protein expression in plants through the use of a novel
autonomously replicating geminivirus shuttle vector. Plant Biotechnol. J. 8,
38–46. doi: 10.1111/j.1467-7652.2009.00462.x
Reski, R., Parsons, J., and Decker, E. L. (2015). Moss-made pharmaceuticals: from
bench to bedside. Plant Biotechnol. J. 13, 1191–1198. doi: 10.1111/pbi.12401
Reuter, L. J., Bailey, M. J., Joensuu, J. J., and Ritala, A. (2014). Scale-up
of hydrophobin-assisted recombinant protein production in tobacco BY-2
suspension cells. Plant Biotechnol. J. 12, 402–410. doi: 10.1111/pbi.12147
Rosales-Mendoza, S., and Tello-Olea, M. A. (2015). Carrot cells: a pioneering
platform for biopharmaceuticals production. Mol. Biotechnol. 57, 219–232. doi:
10.1007/s12033-014-9837-y
Sabalza, M., Vamvaka, E., Christou, P., and Capell, T. (2013). Seeds as a production
system for molecular pharming applications: status and prospects. Curr.
Pharm. Des. 19, 5543–5552. doi: 10.2174/1381612811319310009
Sack, M., Rademacher, T., Spiegel, H., Boes, A., Hellwig, S., Drossard, J., et al.
(2015). From gene to harvest: insights into upstream process development for
the GMP production of a monoclonal antibody in transgenic tobacco plants.
Plant Biotechnol. J. 13, 1094–1105. doi: 10.1111/pbi.12438
Schillberg, S., Raven, N., Fischer, R., Twyman, R. M., and Schiermeyer,
A. (2013). Molecular farming of pharmaceutical proteins using plant
suspension cell and tissue cultures. Curr. Pharm. Des. 19, 5531–5542. doi:
10.2174/1381612811319310008
Schmale, K., Rademacher, T., Fischer, R., and Hellwig, S. (2006). Towards
industrial usefulness - cryo-cell-banking of transgenic BY-2 cell cultures.
J. Biotechnol. 124, 302–311. doi: 10.1016/j.jbiotec.2006.01.012
Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill-Almon, E., Galili, G., et al.
(2007). Production of glucocerebrosidase with terminal mannose glycans for
enzyme replacement therapy of Gaucher’s disease using a plant cell system.
Plant Biotechnol. J. 5, 579–590. doi: 10.1111/j.1467-7652.2007.00263.x
Shin, Y. J., Chong, Y. J., Yang, M. S., and Kwon, T. H. (2011). Production of
recombinant human granulocyte macrophage-colony stimulating factor in rice
cell suspension culture with a human-like N-glycan structure. Plant Biotechnol.
J. 9, 1109–1119. doi: 10.1111/j.1467-7652.2011.00636.x
Shoji, T., and Hashimoto, T. (2008). Why does anatabine, but not nicotine,
accumulate in jasmonate-elicited cultured tobacco BY-2 cells? Plant Cell
Physiol. 49, 1209–1216. doi: 10.1093/pcp/pcn096
Frontiers in Plant Science | www.frontiersin.org 11 March 2016 | Volume 7 | Article 297

Santos et al. Focusing on Plant Cell Suspensions
Sierra, M. I., Heijden, R., Leer, T., and Verpoorte, R. (1992). Stability of
alkaloid production in cell suspension cultures of Tabernaemontana divaricata
during long-term subculture. Plant Cell Tissue Organ Cult. 28, 59–68. doi:
10.1007/BF00039916
Sijmons, P. C., Dekker, B. M. M., Schrammeijer, B., Verwoerd, T. C., van den
Elzen, P. J. M., and Hoekema, A. (1990). Production of correctly processed
human serum-albumin in transgenic plants. Nat.Biotechnol. 8, 217–221. doi:
10.1038/nbt0390-217
Smith, M. L., Mason, H. S., and Shuler, M. L. (2002). Hepatitis B surface antigen
(HBsAg) expression in plant cell culture: kinetics of antigen accumulation in
batch culture and its intracellular form. Biotechnol. Bioeng. 80, 812–822. doi:
10.1002/bit.10444
Soderquist, R. G., and Lee, J. M. (2005). Enhanced production of recombinant
proteins from plant cells by the application of osmotic stress and protein
stabilization. Plant Cell Rep. 24, 127–132. doi: 10.1007/s00299-005-0918-z
Stoger, E., Fischer, R., Moloney, M., and Ma, J. K. C. (2014). Plant molecular
pharming for the treatment of chronic and infectious diseases. Annu. Rev. Plant
Biol. 65, 743–768. doi: 10.1146/annurev-arplant-050213-035850
Su, C. F., Kuo, I. C., Chen, P. W., Huang, C. H., Seow, S. V., Chua, K. Y., et al.
(2012). Characterization of an immunomodulatory Der p 2-FIP-fve fusion
protein produced in transformed rice suspension cell culture. Transgenic Res.
21, 177–192. doi: 10.1007/s11248-011-9518-6
Su, W. W., and Arias, R. (2003). Continuous plant cell perfusion culture: bioreactor
characterization and secreted enzyme production. J. Biosci. Bioeng. 95, 13–20.
doi: 10.1016/S1389-1723(03)80142-1
Sunil Kumar, G. B., Ganapathi, T. R., Srinivas, L., Revathi, C. J., and Bapat, V. A.
(2007). Hepatitis B surface antigen expression in NT-1 cells of tobacco using
different expression cassettes. Biol. Plant. 51, 467–471. doi: 10.1007/s10535-
007-0098-5
Tanaka, H., Nishijima, F., Suwa, M., and Iwamoto, T. (1983). Rotating drum
fermentor for plant cell suspension cultures. Biotechnol. Bioeng. 25, 2359–2370.
doi: 10.1002/bit.260251007
Tekoah, Y., Shulman, A., Kizhner, T., Ruderfer, I., Fux, L., Nataf, Y., et al. (2015).
Large-scale production of pharmaceutical proteins in plant cell culture-the
protalix experience. Plant Biotechnol. J. 13, 1199–1208. doi: 10.1111/pbi.12428
Terrier, B., Courtois, D., Hénault, N., Cuvier, A., Bastin, M., Aknin, A., et al. (2007).
Two new disposable bioreactors for plant cell culture: the wave and undertow
bioreactor and the slug bubble bioreactor. Biotechnol. Bioeng. 96, 914–923. doi:
10.1002/bit.21187
Tremblay, R., Wang, D., Jevnikar, A. M., and Ma, S. W. (2010). Tobacco, a highly
efficient green bioreactor for production of therapeutic proteins. Biotechnol.
Adv. 28, 214–221. doi: 10.1016/j.biotechadv.2009.11.008
Trexler, M. M., McDonald,K. A., and Jackman, A. P. (2002). Bioreactor production
of human alpha(1)-antitrypsin using metabolically regulated plant cell cultures.
Biotechnol. Prog. 18, 501–508. doi: 10.1021/bp020299k
Trexler, M. M., McDonald, K. A., and Jackman, A. P. (2005). A cyclical
semicontinuous process for production of human alpha 1-antitrypsin using
metabolically induced plant cell suspension cultures. Biotechnol. Prog. 21,
321–328. doi: 10.1021/bp0498692
Tsoi, B. M., and Doran, P. M. (2002). Effect of medium properties and additives
on antibody stability and accumulation in suspended plant cell cultures.
Biotechnol. Appl. Biochem. 35, 171–180. doi: 10.1042/BA20010105
Twyman, R. M., Schillberg, S., and Fischer, R. (2005). Transgenic plants in
the biopharmaceutical market. Expert Opin. Emerg. Drugs 10, 185–218. doi:
10.1517/14728214.10.1.185
Twyman, R. M., Schillberg, S., and Fischer, R. (2013). Optimizing the yield
of recombinant pharmaceutical proteins in plants. Curr. Pharm. Des. 19,
5486–5494. doi: 10.2174/1381612811319310004
Twyman, R. M., Stoger, E., Schillberg, S., Christou, P., and Fischer, R. (2003).
Molecular farming in plants: host systems and expression technology. Trends
Biotechnol. 21, 570–578. doi: 10.1016/j.tibtech.2003.10.002
Ullisch, D. A., Müller, C. A., Maibaum, S., Kirchhoff, J., Schiermeyer, A., Schillberg,
S., et al. (2012). Comprehensive characterization of two different Nicotiana
tabacum cell lines leads to doubled GFP and HA protein production by
media optimization. J. Biosci. Bioeng. 113, 242–248. doi: 10.1016/j.jbiosc.2011.
09.022
Uragami, A., Sakai, A., Nagai, M., and Takahashi, T. (1989). Survival of
cultured cells and somatic embryos of Asparagus officinalis cryopreserved by
vitrification. Plant Cell Rep. 8, 418–421. doi: 10.1007/BF00270083
van Gulik, W. M., ten Hoopen, H. J., and Heijnen, J. J. (2001). The application
of continuous culture for plant cell suspensions. Enzyme Microb. Technol. 28,
796–805. doi: 10.1016/S0141-0229(01)00331-3
Vasilev, N., Grömping, U., Lipperts, A., Raven, N., Fischer, R., and Schillberg,
S. (2013). Optimization of BY-2 cell suspension culture medium for the
production of a human antibody using a combination of fractional factorial
designs and the response surface method. Plant Biotechnol. J. 11, 867–874. doi:
10.1111/pbi.12079
Walsh, G. (2014). Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32,
992–1000. doi: 10.1038/nbt.3040
Wen Su, W., Jun He, B., Liang, H., and Sun, S. (1996). A perfusion
air-lift bioreactor for high density plant cell cultivation and secreted
protein production. J. Biotechnol. 50, 225–233. doi: 10.1016/0168-1656(96)
01568-4
Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated
mammalian cells. Nat. Biotechnol. 22, 1393–1398. doi: 10.1038/nbt1026
Xu, J., Ge, X., and Dolan, M. C. (2011). Towards high-yield production of
pharmaceutical proteins with plant cell suspension cultures. Biotechnol. Adv.
29, 278–299. doi: 10.1016/j.biotechadv.2011.01.002
Xu, J., Okada, S., Tan, L., Goodrum, K. J., Kopchick, J. J., and Kieliszewski,
M. J. (2010). Human growth hormone expressed in tobacco cells as an
arabinogalactan-protein fusion glycoprotein has a prolonged serum life.
Transgenic Res. 19, 849–867. doi: 10.1007/s11248-010-9367-8
Xu, J., Tan, L., Goodrum, K. J., and Kieliszewski, M. J. (2007). High-yields and
extended serum half-life of human interferon alpha2b expressed in tobacco
cells as arabinogalactan-protein fusions. Biotechnol. Bioeng. 97, 997–1008. doi:
10.1002/bit.21407
Yano, A., Maeda, F., and Takekoshi, M. (2004). Transgenic tobacco cells producing
the human monoclonal antibody to hepatitis B virus surface antigen. J. Med.
Virol. 73, 208–215. doi: 10.1002/jmv.20077
Yusibov, V., Rabindran, S., Commandeur, U., Twyman, R. M., and Fischer, R.
(2006). The potential of plant virus vectors for vaccine production. Drugs R
D7, 203–217. doi: 10.2165/00126839-200607040-00001
Zhang, X., and Mason, H. (2006). Bean Yellow Dwarf Virus replicons for high-level
transgene expression in transgenic plants and cell cultures. Biotechnol. Bioeng.
93, 271–279. doi: 10.1002/bit.20695
Zimran, A., Brill-Almon, E., Chertkoff, R., Petakov, M., Blanco-Favela, F., Muñoz,
E. T., et al. (2011). Pivotal trial with plant cell-expressed recombinant
glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy
for Gaucher disease. Blood 118, 5767–5773. doi: 10.1182/blood-2011-07-
366955
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