molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures

Send Orders for Reprints to [email protected]

Current Pharmaceutical Design, 2013, 19, 5531-5542 5531

Molecular Farming of Pharmaceutical Proteins Using Plant Suspension Cell and Tissue Cultures

Stefan Schillberg1,2,*, Nicole Raven1, Rainer Fischer1,3, Richard M. Twyman4 and Andreas Schiermeyer1

1Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany; 2Justus-Liebig University Giessen, Institute for Phytopathology and Applied Zoology, Phytopathology Department, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany; 3RWTH Aachen University, Institute for Molecular Biotechnology, Worringerweg 1, 52074 Aachen, Ger-many; 4TRM Ltd, P. O. Box 93, York, YO43 3WE, UK

Abstract: Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sus-tainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the prod-uct and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production plat-forms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in contain-ment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower com-pared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process op-timization, helping to increase yields and scalability.

Keywords: bioreactor, downstream processing, fermentation, recombinant proteins.

1. MOLECULAR FARMING - WHOLE PLANTS AND CELL/TISSUE CULTURES Molecular farming is the use of whole plants or plant cells/tissues cultured in vitro for the production of valuable recom-binant proteins, which has been established as an economically-viable alternative to mainstream production systems such as mi-crobes and mammalian cells cultivated in large-scale bioreactors. The advantages of plant-based systems can be summarized as fol-lows. (1) Plants are less expensive to set up and maintain than cul-tured mammalian cells. (2) They lack the undesirable components found in conventional systems, e.g. endotoxins in bacteria, hyper-glycosylated proteins produced by yeast, and human/animal patho-gens in mammalian cell cultures and transgenic livestock. (3) There are no limits to the production scale and the cost of scaling up is low, in contrast to mammalian cells where media costs increase in line with the production scale and eliminate any economic benefits of large-scale manufacturing. (4) Plant-based systems are extremely versatile. There are now nearly 100 different platforms based on a wide range of species, gene transfer strategies, expression strate-gies, protein targeting and recovery strategies. A suitable platform is therefore likely to be available for any conceivable product, and species can also be chosen because they are compatible with local environments and infrastructures. (5) Plants are higher eukaryotes and can therefore fold and assemble complex/multimeric proteins and carry out most of the post-translational modifications that occur in mammals. (6) Food crops are generally recognized as safe (GRAS) which means that they are suitable vehicles for the deliv-ery of oral vaccines and topical microbicides with limited (and therefore inexpensive) processing. These topics are discussed in recent reviews and by other contributors to this issue [1-4]. Despite the well-recognized advantages of plants, the take-up of plant-based production technology has been slow, and only a few

*Address correspondence to this author at the Fraunhofer IME, Forcken-beckstrasse 6, 52074 Aachen, Germany; Tel: +49-241-608511050; E-mail: [email protected]

recombinant proteins derived from whole plants have reached the market. Currently, a handful of technical reagents for diagnostic and cosmetic purposes are available that have been produced in maize, barley and rice [5,6], and a small number of pharmaceutical products have reached clinical trials [7-10]. The only licensed pharmaceutical-related product manufactured in intact plants is an antibody affinity reagent used in Cuba for the purification of a hepatitis B vaccine. The vaccine itself is produced in yeast, but the entire production process has to be approved so the plant-derived antibody was evaluated with the same stringency as the active pharmaceutical ingredient (API) [11]. Several factors are responsible for the slow uptake of molecular farming and none of them reflect genuine problems with the tech-nology. One reason is the negative public perception of genetic modification (GM) technology (field-grown transgenic plants in particular) mainly in Europe but also in other areas of the world. This perception is based on exaggerated fears of widespread out-crossing and environmental damage that are not supported by scien-tific evidence [12]. The knock-on effect is that the regulators re-sponsible for overseeing field trials and the commercial exploitation of GM crops have been extremely cautious when considering the approval of field-grown pharmaceutical crops, and have imposed onerous restrictions and disproportionate penalties for noncompli-ance, even though the benefits of plant-derived pharmaceuticals are accepted by patient advocacy groups [13]. Many promising small and medium enterprises (SMEs) have therefore abandoned their research and some of the bigger industry players such as Monsanto have withdrawn their support for molecular farming. Another contributory factor is the uncertain regulatory land-scape surrounding the products themselves. There are two problems here. First, plant-derived pharmaceuticals fall somewhere between the jurisdictions of the agencies responsible for GM agriculture and those responsible for biotechnology-derived drugs. Second, phar-maceutical regulations have been developed around the concept of cells grown in fermenters and many of these concepts (e.g. mas-ter/working cell banks, virus inactivation steps) are difficult to ap-

1873-4286/13 $58.00+.00 © 2013 Bentham Science Publishers

5532 Current Pharmaceutical Design, 2013, Vol. 19, No. 31 Schillberg et al.

ply to plants [14]. For example, good manufacturing practice (GMP) demands the growth of cells and microorganisms under precise, documented conditions to ensure batch-to-batch consis-tency of the API, conditions that are impossible to achieve in open-field cultivation due to variations in weather, climate, soil consis-tency and the impact of pests, herbivores and microorganisms. Therefore, most of the academic and industrial groups supporting molecular farming seek to develop plant systems that are grown in containment. The use of controlled environments, e.g. growing plants in greenhouses, reduces as much as possible the variability caused by unpredictable weather, climate, soil heterogeneity and interactions with other organisms and helps to address regulatory concerns about batch-to-batch product consistency. Although the implementation of contained production systems was a major step towards GMP compliance, the clinching factor was the approval of a separated process, with plant cultivation, harvesting and initial downstream processing in the greenhouse or growth chambers, followed by the transfer of the process into a cleanroom area for final product purification and formulation. GMP processes are now available for whole transgenic plants and transient expression sys-tems, so the first products are undergoing clinical trials [15]. In contrast, plant tissues and cell suspension cultures can be controlled in a manner that is exactly analogous to the approaches currently approved by the US Food and Drugs Administration (FDA) and the European Medicines Agency (EMA) for mammalian cells and microbes, and it is therefore not surprising that the first plant-derived human pharmaceutical which received market ap-proval is produced in such a system (see section 2.3).

2. PLANT CELL/TISSUE SUSPENSION CULTURES AS PLATFORMS FOR THE PRODUCTION OF PHARMACEU-TICAL PROTEINS Until the 1980s, plant cell suspension cultures, tissues (e.g. hairy roots), moss and algae were mainly used for the production of anthocyanins, fatty acids or therapeutic secondary metabolites such as ajmalicine, scopolamine and paclitaxel [16]. For example, Phy-ton Biotech achieved the commercial production of paclitaxel (Taxol) in Taxus spp. cell suspension cultures up to a volume of 75,000 L, and this has provided a sustainable source of the drug since 1995 [17]. In 1990, Sijmons et al. reported the production of the first recombinant human pharmaceutical protein (human serum albumin) in tobacco suspension cell cultures [18]. Since then, nu-merous pharmaceutical proteins have been produced in plant cell suspension cultures and tissues, including antibodies [19], cytokines [20], enzymes [21], growth factors [22,23], hormones [24] and vaccines [25]. A comprehensive list of recombinant pharmaceutical proteins produced in plant cells and tissues has been published re-cently [26].

2.1. Plant Cell and Tissue Culture Systems Plant cell and tissue culture systems are grown in vitro under contained and defined conditions and include suspension cells and hairy roots, which have a long track record for the production of valuable compounds, and more recently established platforms such as moss, microalgae and aquatic plants. They require different me-dia and cultivation conditions to achieve maximum protein yields, but the major difference is that cells derived from roots like tobacco hairy roots, tobacco BY-2 cells, carrot and rice cells do not need light and grow on sugar-based media and therefore can grow to very high densities, whereas moss, algae and duckweed (Lemna minor) are derived from green tissue and therefore require light but need only water as a medium, which facilitates product recovery from the culture supernatant [27-31]. 2.1.1. Cell Suspension Cultures Plant cell suspension cultures grow as individual cells or small aggregates and are usually derived from callus tissue by the disag-gregation of friable callus pieces in shake bottles, and are later

scaled up for bioreactor-based production. Recombinant protein production is achieved using transgenic explants to derive the cul-tures, or by transforming the cells after disaggregation, usually by co-cultivation with Agrobacterium tumefaciens [32]. The co-cultivation of plant cell suspensions and recombinant A. tumefa-ciens has also been used for the transient expression of proteins [33]. Tobacco is the most popular source of suspension cells for recombinant protein production, since these proliferate rapidly and are easy to transform. The main representatives are cells from the cultivars Nicotina tabacum cv Bright Yellow 2 (BY-2) and N. taba-cum 1 (NT-1), which are thought to be derived from the embryonic root meristem cells [34]. However, other plant species have also been used to generate suspension cells, including rice and Arabi-dopsis thaliana [35,36] alfalfa [37], soybean [38], tomato [39] and carrot [31], the latter used by the Israeli company Protalix biothera-peutics for the production of recombinant glucocerebrosidase, the first plant-derived human pharmaceutical protein approved for the market (see section 2.3). Recombinant proteins expressed in plant cell suspension cul-tures, including large protein complexes such as IgGs, are secreted into the culture supernatant. A portion of the target protein may be retained within the cells depending on its folding and size relative to the pores in the cell wall. 2.1.2. Hairy Root Cultures The hairy root phenotype in plants is caused by the infection of wounded plant tissues with Agrobacterium rhizogenes, which has been subverted as a gene transfer vector allowing hairy roots to be grown in bioreactors for the production of secondary metabolites and recombinant proteins. This has resulted in excellent culture, scale-up and genetic engineering techniques that pave the way for the industrial exploitation of the hairy root system. Engineering hairy roots for the production of recombinant therapeutic proteins in contained bioreactors is an exciting spin-off of the technology [40]. In contrast to ordinary root cultures (nontransformed roots), which often require auxin and cytokinin to maintain their growth and phe-notype, hairy roots grow rapidly on hormone-free medium and are highly branched. They are phenotypically and genetically stable, and selected hairy root lines can be exploited indefinitely. Stably-transformed hairy root cultures have been established from numer-ous species of dicotyledonous plants, but for production of recom-binant proteins hairy roots have been derived mainly from tobacco [41]. Similar to cell suspension cultures, the recombinant proteins expressed by hairy roots are often secreted into the culture medium, from which they can be extracted [30,42]. Attempts to establish in vitro systems for the infection of plant suspension cells with viruses have been unsuccessful, but virus-based vectors for the transient expression of recombinant proteins in hairy roots have been developed [43]. Infection and virus accu-mulation do not affect hairy root growth, and successful expression requires the presence of fixed connections (plasmodesmata) be-tween the root cells, facilitating cell-to-cell movement. In contrast, cell-to-cell movement in plant cell suspension cultures relies on the formation of cell aggregates, and therefore this strategy suffers from low recombinant protein yields. 2.1.3. Moss Cultures The moss Physcomitrella patens is a haploid bryophyte that can be grown in bioreactors under controlled conditions, using synthetic growth media and artificial light [28]. The P. patens genome has been fully sequenced and a unique feature of this organism, relative to all other plants, is its ability to promote efficient homologousrecombination [44]. This means the moss can be stably transformed with new genetic information, and that endogenous genes can also be disrupted by gene targeting. The major application of gene tar-geting in molecular farming is the modification of the glycosylation pathway (by knocking out enzymes that add non-human glycans to proteins) thus allowing the production of humanized glycoproteins

Molecular Farming in Plant Suspension and Tissue Cultures Current Pharmaceutical Design, 2013, Vol. 19, No. 31 5533

[45-47]. The P. patens system is being developed by the German biotechnology company greenovation Biotech GmbH. The com-pany has developed transient expression systems that allow feasibil-ity studies [47,48], and stable production strains that can be scaled up to several thousand liters [22,49]. These strains achieve high yields and the recombinant protein can be secreted into the medium [45]. 2.1.4. Microalgae Microalgae are found in both marine and freshwater environ-ments and comprise a heterogeneous group of prokaryotic and eu-karyotic microbes, some of which are photosynthetic and others heterotrophic. Microalgae have been exploited in the past for the production of high-value pharmaceutical compounds [50], biofuels [51] and for bioremediation applications [52], but now especially photosynthetic microalgae such as Chlamydomonas spp. (which can also grow heterotrophically) can be used as a platform for the production of recombinant proteins. Algae undergo rapid vegetative growth on simple media, the nuclear and/or organelle genomes are sequenced and different nuclear and plastid transformation methods are available (e.g. electroporation, particle bombardment and A. tumefaciens-mediated transformation), although the plastid trans-formation approach produces much higher yields [53-55]. Unlike higher plant cells, which contain ~100 plastids microalgae such C. reinhardtii contain only a single plastid per cell [56]. The transfor-mation of this organelle is relatively easy and allows the regenera-tion of transgenic lines within 4-6 weeks. However, proteins ex-pressed in the plastids do not undergo certain post-translational modifications such as glycosylation and they are not secreted to the culture medium, which makes subsequent purification more chal-lenging. The US company PhycoBiologics Inc. is using Chlamydo-monas spp. plastid transformation to produce oral aquaculture vac-cines such as the Staphylococcus aureus fibronectin-binding do-main D2 fused to the CTB mucosal adjuvant to induce antigen-specific immune response in mice [57]. 2.1.5. Floating Plant Cultures More recent plant-based production systems comprise plants that are not cultivated in suspension but are free floating on water. Major representatives that are used for production of biopharma-ceuticals include Lemna minor [29,58], Spirodela oligorrhiza [59] and Wolffia spp. [60]. Stable transformation is usually accom-plished by Agrobacterium-mediated transformation or particle bombardment [61,62]. As is the case for plant cell and tissue sus-pensions, recombinant proteins can be secreted into the culture medium which in many cases is simply water. On the other hand, the intracellular accumulation of antigens in L. minor has the ad-vantage that the plant contains a pectic polysaccharide with adju-vant properties and oral administration of plant extract containing the target antigen promotes a stronger immune response than the pure protein [63]. The American company Biolex Therapeutics Inc. developed L.minor and S. oligorrhiza as platforms for the production of recom-binant human pharmaceutical proteins such as IFN-�2b (Locteron) which is ready for Phase III clinical trials. The company has also humanized glycosylation in L. minor by RNA interference, target-ing the endogenous �1,3-fucosyltransferase and �1,2-xylosyl-transferase genes. A human CD20-specific antibody produced in these engineered cells did not contain any plant-specific N-glycans and showed an improved cell-mediated cytotoxicity and effector cell reporter binding than the corresponding antibody produced in CHO cells [64]. The company has recently sold its technology and product portfolio to the Dutch company Synthon BV.

2.2. Advantages of Plant Cell/Tissue Suspensions Compared to Whole Plant Systems Like plant cell/tissue suspensions, whole plants can also be grown in containment under controlled conditions, in glasshouses

or growth chambers that even eliminate fluctuations in the intensity of sunlight. The recent development of automated cultivation sys-tems, with LED lights and advanced nutrient supply using nutrient film technology, focus on improving the reproducibility and consis-tency of plant growth and protein production even further [65-67]. However, despite this progress in whole plant cultivation, plant cell and tissue culture systems offer several advantages: (1) Rapid growth. Plant cells and tissues grow more rapidly than

whole plants, with cell doubling times as short as one day, or 8 h in the case of C. reinhardtii [68]. One batch fermentation run for tobacco BY-2 cells therefore takes 1-2 weeks, whereas the timeframe from seeding to harvesting in transgenic plants is several months [36]. The scaling up of whole plant production requires only the parallel cultivation of more plants, which does not increase the overall production period. In contrast, scaled up fermentation processes require additional pre-cultures that increase in line with higher final cultivation vol-umes. This prolongs the production cycles, but the overall process time is still shorter than the cultivation of whole plants even if volumes >10,000 L are processed. Alternatively, paral-lel cultivation capacity or continuous fermentation processes can be used to shorten the production periods for larger fer-mentation volumes. Shorter production processes are only pos-sible when transient expression systems based on whole plants [69-71] or plant cell and tissue suspensions (see above) are used. However, GMP compliance is more difficult to achieve with transient expression systems due to the complexity intro-duced by the infiltration procedure, and the regulatory burden inherent in the large-scale use of genetically modified bacteria and/or viruses [15].

(2) Simplified product recovery and purification. Recombinant proteins can be secreted into the medium of plant suspension cell and tissue cultures using N-terminal signal peptides, which are cleaved off when the recombinant proteins enter the secre-tory pathway. Accumulation in the well-defined culture me-dium facilitates protein recovery because cells contain most of the contaminating proteins, and these can easily be separated by filtration. Because extraction is not required and most of the plant-derived proteins are already removed, downstream proc-essing is simpler and less expensive compared to whole plant systems where product recovery requires the mechanical dis-ruption of plant tissue and the removal of insoluble compounds and endogenous proteins.

(3) Greater protein integrity and homogeneity. The secretion of target proteins into the culture medium also improves product integrity and homogeneity because all proteins are fully proc-essed, i.e. the signal peptide must be removed and complex glycan structures added if the product is a glycoprotein. Al-though many recombinant proteins accumulate to higher levels inside cells, especially when retained in the endoplasmic re-ticulum [72,73], the liberation of proteins by cell disruption promotes the co-purification of unprocessed, immature poly-peptides with intact signal peptides and/or heterogeneous gly-cans. For example, our unpublished data show that antibodies isolated from the spent medium of tobacco BY-2 cultures dis-play homogeneous complex glycan structures (three glyco-forms, dominant form representing 87% of the total) whereas the same antibody isolated from intact tobacco plants is repre-sented by six different glycoforms.

(4) Improved process and product consistency/GMP compliance. Plant cell and tissue suspension cultures are grown in con-tained bioreactors, under precise and documented conditions that are fully controlled so that the nutritional and physical process parameters can be monitored and adjusted. Fermenta-tion processes are therefore less prone to biotic and abiotic variations that reduce the batch-to-batch consistency of green-


house plants, and are therefore more compliant with GMP guidelines for the production of pharmaceutical proteins.

2.3. Commercial Production of Pharmaceutical Proteins Using Plant Cell/Tissue Suspension Cultures Many recombinant pharmaceutical proteins have been produced successfully in plant cell/tissue suspension cultures [26]. Antibodies are often chosen because they represent an important class of bio-pharmaceuticals [74], and they are also relatively stable and there-fore accumulate to high levels (>100 mg/L). Antibodies can also be purified easily from the medium or extracts by protein A affinity chromatography, and various assays are available to verify their functionality. Therefore, they are considered an ideal choice to demonstrate the advantages and efficiency of plant-based produc-tion systems. The yield of recombinant antibodies in mammalian cells has significantly improved during the last few decades and now routinely exceeds 5 g/L culture volume [75]. This is 10 times higher than the maximum levels achieved using plant cells, so it is unlikely that plant cells will become a competitive system for anti-body production in the near future. Instead, the intrinsic strengths of the plant cell suspension cul-tures should be exploited, e.g. by using the plant matrix to improve the immunogenicity of oral vaccines [63], by using antigens in non-toxic plant extracts to avoid intensive downstream processing or by taking advantage of plant-specific glycosylation. Indeed, the first two recombinant proteins from plant cells that have been approved for commercial production take advantage of the unique properties of the production platform: in one case a poultry vaccine produced in tobacco BY-2 cells that does not require extensive purification from the plant extract and can be injected as a crude extract, and in the other case a recombinant glucocerebrosidase produced in carrot cells that does not require processing in vitro because the undesir-able terminal glycans added by mammalian cells are missing when the product is manufactured in plants. 2.3.1. Hemagglutinin-neuraminidase Glycoprotein The Newcastle disease vaccine for poultry was developed by Dow AgroSciences LLC and was the first plant cell-derived phar-maceutical product to be approved. The vaccine is the recombinant hemagglutinin-neuraminidase glycoprotein (HN) of Newcastle dis-ease virus (NDV). HN is one of two viral surface glycoproteins and the major surface antigen that induces neutralizing antibodies. It is used as a vaccine to induce a protective immune response against NDV in domestic poultry and other avian species, therefore helping to prevent the spread of this acute and contagious disease. The vac-cine was produced in transgenic tobacco NT-1 suspension cells and accumulated within the cells. Processing comprised a basic extrac-tion followed by injection of the crude extract containing recombi-nant HN into chickens, thus reducing the production costs enough to make a plant-derived veterinary vaccine economically viable. The activity of the unpurified vaccine was demonstrated in vitrousing hemagglutination assays and the HN protein conferred full protection on chickens when challenged with the virus. Although the vaccine was approved by the USDA in February 2006 for use in poultry [76], a strategic decision was made by Dow AgroSciences not to introduce the product onto the market. 2.3.2. Glucocerebrosidase Recombinant glucocerebrosidase (prGCD, taliglucerase alpha, Elelyso™) manufactured in carrot cells by Protalix Biotherapeutics and distributed by Pfizer Inc. was approved by the FDA in May 2012 for human use (http://www.fda.gov/NewsEvents/Newsroom/ PressAnnouncements/ucm302549.htm). Recombinant glucocere-brosidase is needed to replace the nonfunctional enzyme in patients with the monogenic disorder Gaucher’s disease, who otherwise cannot degrade glucosylceramides, which therefore accumulate in the lysosomes of phagocytes. Clinical symptoms of the disease include hepatosplenomegalia, anemia, and thrombocytopenia. Pa-

tients have been treated with a recombinant version of the enzyme (imiglucerase, Cerezyme) produced in CHO cells [7], which is one of the most expensive biopharmaceuticals on the market, with an annual treatment cost of USD 200,000 per patient [77]. The recom-binant imiglucerase is purified from the CHO cell culture medium and must then be processed enzymatically to expose terminal man-nose residues that are required for the efficient uptake of the en-zyme into macrophages. Taliglucerase alpha from carrot cells does not require these additional processing steps because it is targeted to the cell vacuole where the complex type N-glycans are trimmed to the paucimannose form, exposing terminal mannose residues [78]. The marketing of glucocerebrosidase has taken advantage of an abbreviated approval process that allows direct progression from phase I to phase III trials because the drug falls within the scope of the Orphan Drug Act.

3. ENHANCING PLANT CELL/TISSUE SUSPENSION CUL-TURES FOR THE PRODUCTION OF PHARMACEUTICAL PROTEINS A major objective in the field of molecular farming is to im-prove the yield of recombinant proteins, which is typically <1% of total soluble proteins. Many strategies have focused on improving upstream production, and improvements have come about by simul-taneous developments in several areas - through enhanced gene transcription using new promoters, efficient mRNA processing and translation, newer gene transfer strategies such as plastid transfor-mation and transient expression using plant viruses [79-82] and perhaps most importantly, the realization that protein targeting within the plant cell has a profound impact on product stability and recovery [83-86]. The problems that remain are those related to the quality of the protein, influenced by post-translational modifications such as gly-cosylation and protein degradation. Cultivation parameters and downstream processing may also affect product quantity, quality and homogeneity, so pharmaceutical products of consistent purity and quality must be achieved by regulating both the cultivation and downstream processing components of the process between very tight parameters. The cultivation process is more difficult to control because it is based mainly on biological principles (e.g. cell growth and productivity) whereas downstream processing is based on physical and chemical principles (e.g. filtration and chromatogra-phy) allowing control to be exerted by maintaining a constant envi-ronment. This is not possible with field-grown plants or plants grown conventionally in a greenhouse, so it is unreasonable to ex-pect either the plants themselves or the product to be consistent in terms of yield and quality.

3.1. Humanization of Glycan Structures Approximately 30% of all approved biopharmaceuticals contain N-linked glycans, so N-glycosylation is the most important post-translational modification that needs to be taken into account when manufacturing recombinant proteins in plants. The glycosylation machinery in plants is similar but not identical to its mammalian counterpart. In both cases, the N-glycosylation of a polypeptide begins with the co-translational transfer of an oligosaccharide pre-cursor to asparagine residues within the consensus sequence N-X-S/T (where X is any amino acid except proline) in the endoplasmic reticulum. As the protein matures, the oligosaccharide precursor is trimmed to eventually yield a glycoform known as the high-mannose type, which is identical in plants and mammals. When the glycoprotein travels further down the secretory pathway, the gly-cans are modified stepwise by enzymes located in the Golgi appara-tus. The final complex-type N-glycans differ between plants and mammals. Plant glycoproteins contain core �1,2-xylose and �1,3-fucose residues that are not present in endogenous mammalian gly-coproteins, whereas mammalian glycoproteins contain �1,4 galac-tose and terminal neuraminic acid residues that are not synthesized


in plants [87,88]. Plant glycans are more of a problem in theory than in practice because there is little evidence that plant-type gly-cosylation negatively affects the activity of recombinant human proteins, but nevertheless there have been efforts to humanize plant-derived glycoproteins in case the presence of plant glycans affects the half-life, stability and immunogenicity of the product in mammals [89]. The simplest way to avoid plant-like glycan residues is to add the tetrapeptide H/KDEL motif to the C-terminus of pharmaceutical proteins, causing them to be retrieved to the endoplasmic reticulum before the plant-type �1,2-xylose and �1,3-fucose glycans are added in the Golgi apparatus. This strategy has been used to pro-duce many high-mannose recombinant glycoproteins in whole plants and plant cell suspension cultures [90-92]. However, the addition of this small tag changes the structure of the recombinant protein and may affect its pharmacokinetic properties or biological activity, certainly inviting additional regulatory scrutiny. Further-more, retention in the endoplasmic reticulum means that cells must be disrupted to access the protein, adding complexity and expense to the downstream processing step. Alternative strategies have therefore focused on inhibiting the endogenous plant glycosyltransferases that transfer �1,2-xylose and �1,3-fucose residues onto nascent proteins. In P. patens, the genes for �1,2-xylosyltransferase and �1,3-fucosyltransferase have been disrupted by gene targeting [46]. This double knockout mutant has been used to produce secreted human erythropoietin that lacks core �1,2-xylose and �1,3-fucose glycans [49]. Higher plants are less amenable to gene targeting, but RNA interference (RNAi) can be used to silence the endogenous �1,2-xylosyltransferase and �1,3-fucosyltransferase genes. For example, L. minor was transformed with a vector encoding the human anti-CD20 antibody MDX-060 and an inverted repeat construct homologous to �1,2-xylosyl-transferase and �1,3-fucosyltransferase, resulting in the production of a recombinant antibody lacking plant-type N-glycans [64]. Com-pared to a counterpart produced in Chinese hamster ovary (CHO) cells, the MDX-060 antibody had a >10-fold higher affinity for the human Fc receptor and more than 20-fold higher antibody-dependent cell-mediated cytotoxicity (ADCC) against a tumor cell line in vitro. The prevalence of N-linked core �1,2-xylose and �1,3-fucose glycans can be also reduced in tobacco BY-2 cells by expressing rat �1,4-N-acetylglucosaminyltransferase (GntIII) along with the target protein [93]. This enzyme is not found in plants, but in humans and certain other mammals it adds a bisecting GlcNAc residue to the �-mannose of N-linked carbohydrates, creating a bisected complex or hybrid oligosaccharide. Transgenic tobacco BY-2 cells accumulat-ing rat GntIII in the Golgi apparatus also produced up to 59% hu-man-compatible N-glycans on endogenous secreted proteins [93]. This strategy has yet to be applied to recombinant proteins pro-duced in cell suspension cultures, although feasibility has been demonstrated in intact tobacco plants using a human GntIII [94]. N-glycosylation in plant cell cultures can also be affected by medium components [95,96]. Feeding tobacco BY-2 cells with N-acetylglucosamine causes a dose-dependent shift from pauciman-nosidic or hybrid to complex N-glycans in endogenous proteins [97]. Sugar starvation in rice suspension cell cultures can also alter the N-glycan profiles of secreted glycoproteins [98]. Medium sup-plementation and design may therefore provide a new tool to con-trol the glycosylation profiles of recombinant proteins and is par-ticularly suitable for plant cell/tissue suspension cultures where defined and controlled media are used. Whereas N-glycosylation has significant structural and func-tional implications, much less is known about O-glycosylation and its impact on the production of plant-derived biopharmaceuticals. The O-linked glycosylation of hydroxyproline (hyp) is unique to higher plants and green algae, but the O-glycosylation of serine and

threonine residues has also been observed [99]. Proteins with hyp-O-linked glycans are abundant in plant cells and may act as immu-nogens, although there is no evidence that the hyp-O-glycans pro-duced in tobacco BY-2 cells induce a more potent immune response [100,101]. When a synthetic O-glycosylation motif comprising a tandem repeat of the dipeptide serine/proline (SP) was fused to the C-terminus of human interferon �2b, the chimeric protein had a higher molecular mass due to the presence of O-linked glycans and a serum half-life 13 times longer than standard IFN�2 when in-jected intravenously into mice, reflecting the slower renal clearance and greater resistance towards proteolytic degradation [101]. It is now possible to produce almost any recombinant protein in plant cells with a glycan structure compatible with humans. How-ever, for the formulation of more potent vaccines and other bio-pharmaceuticals for cancer immunotherapy, plant-specific glycans might be advantageous by increasing the immune visibility of the antigen, and targeting antigen-presenting cells particularly through lectins or mannose-fucose receptors on the surface of dendritic cells. In addition, therapeutic proteins with plant-derived glycans could function in a superior manner to their native counterparts (such products are known as ‘biobetters’ as an extension of ‘biosimilars’). The recombinant glucocerebrosidase produced in carrot cells with vacuole-specific glycans to improve uptake by human macrophages is one such example (see above).

3.2. Cell Culture Approaches 3.2.1. Cell Line Development and Improvement The transformation of plant cells and tissues typically produces primary transformants with different transgene copy numbers and insertion sites, which can have a significant impact on productivity [102,103]. Therefore, several transformants must be screened to identify elite producers and many must be screened to increase the probability of finding extreme producers (jackpot clones). Usually this is done at the callus stage on agar plates, or individual events can be screened after cultivation in microtiter plates or small biore-actors. Screening transformants in 50-mL disposable bioreactors has many advantages (Fig. 1) because it mimics the conditions of the subsequent scaled-up production process, space requirements are low and no sophisticated equipment is needed. Parallel cultiva-tion is also useful for high-throughput process and media optimiza-tion.

Fig. (1). Parallel cultivation of plant suspension cells in 50-mL disposable bioreactors for medium optimization and process development. Up to 120 tubes can be incubated in parallel using an orbital shaker (left). Each biore-actor is equipped with a vented screw cap with five holes of different sizes for optimal gas exchange (right). A membrane in the cap serves as a sterile barrier and minimizes liquid loss by evaporation.

Variation in recombinant protein accumulation can also arise during prolonged cultivation and periodic subculturing, with trans-gene silencing and recombination events diminishing or even abol-ishing desirable characteristics such as the high-level production of recombinant proteins [104]. Cultures with heterogeneous and in-consistent expression include only a small number of cells produc-ing high yields of the target protein (Fig. 2). Often this is because


the most productive cells grow more slowly than, and tend to be outcompeted by, their less productive peers [104,105]. It can there-fore be useful to isolate elite individual cells and use them to seed highly-productive monoclonal cell suspension cultures. 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 [106]. Using a feeder cell strategy, single cells selected by flow cytometry were regenerated into monoclonal cells lines with homogeneous DsRed fluorescence and antibody yields up to 13-fold higher than the pa-rental culture (182 ± 10 �g⁄g fresh weight) that remained stable for 10-12 months. Selection by flow cytometry has also been used to improve the productivity of a transgenic BY-2 cell line secreting the same antibody into the culture medium. However, the yield increased only 1.2-fold suggesting that other factors in the culture medium (e.g. the presence of extracellular proteases) limit the fur-ther accumulation of the protein (unpublished data).

Fig. (2). Isolation of high-performance cells by flow cytometry improves the homogeneity and productivity of DsRed expression in tobacco BY-2 callus cultures. Left: Example of primary callus following transformation with a construct encoding the fluorescent protein DsRed. Right: Callus regenerated from cells showing intense DsRed fluorescence isolated by flow cytometry shows uniform DsRed expression and improved productivity (not shown).

3.2.2. Medium Optimization Whereas microalgae and duckweed are cultivated in water, plant suspension cells require a more complex medium, generally based on Murashige-Skoog (MS) salts which provide two nitrogen sources (nitrate and ammonium) in a defined ratio [107]. The ratio of nitrate to ammonium ions plays an important role in the metabo-lism of different plant cells [108,109]. Nitrogen is a constituent of both nucleic acids and proteins and is thus essential to plant life. Nitrogen plays a pivotal role in plant cell metabolism and is directly connected to amino acid and protein synthesis [110,111]. Species-dependent nutrient requirements have been described for different plant cell cultures including rice [112], soybean [113] and tobacco [107]. Media compositions are optimized empirically, using a trial and error approach, but in most cases the objective has been to in-crease biomass accumulation and the improvement of recombinant protein accumulation has been neglected. Rational analysis has recently shown that additional nitrogen in the MS medium can improve recombinant protein accumulation in the culture supernatant of tobacco BY-2 cells [114]. In the nitrogen-enriched MS medium, the cells produced 10-20 times more anti-body than cells in standard medium, resulting in 150-fold greater yields in the stationary phase when recombinant protein levels usu-ally decrease. Importantly, the optimized medium did not affect antibody quality or activity, and the increased productivity in shake flasks could be replicated in stirred tank bioreactors. The higher antibody levels in the culture medium are thought to reflect the enhanced secretion and higher stability of the recombinant protein. It is unclear whether the addition of nitrogen also affects protease activity in the culture medium, but it seems that the additional ni-trogen compensates for the reduced osmolality caused by the deple-tion of nutrients at the end of the cultivation phase, as shown by the significant loss of conductivity in the culture medium [115].

The first rational approach to increase recombinant protein yields in cell suspension cultures has demonstrated that there is room for further improvement and that modern methods (including non-invasive online monitoring, design of experiments and factorial designs [116,117]) are required for the targeted high-throughput optimization of novel culture media. Ullisch and colleagues [115] have recently shown that the on-line analysis of oxygen consump-tion using a respiration activity monitoring system (RAMOS) is a useful tool for process and medium optimization. This allowed the detection of metabolic changes in transgenic BY-2 cells cultivated in shake flasks, and further analysis of the nutrients in the spent MS medium revealed that the metabolic shift was caused by ammonia depletion. Supplementing the medium with additional ammonium doubled the amount of recombinant GFP produced by these cells, and the same medium also doubled the productivity of transgenic NT-1 cells producing the influenza hemagglutinin protein [115]. 3.2.3. Minimizing Protein Degradation Despite progress in vector design, cell line development and medium optimization, the degradation of recombinant proteins by intracellular or extracellular plant proteases is a major obstacle limiting the yield of recombinant proteins in plant cell/tissue sus-pension cultures, and the resulting protein fragments must be re-moved during downstream processing (Fig. 3). Proteolytic degrada-tion of recombinant proteins has been observed irrespective of sub-cellular localization, but the extracellular compartments (apoplast and culture medium) appear to be particularly rich in proteolytic enzymes [118-123]. Degradation may occur in vivo, during extrac-tion and/or during subsequent downstream processing [124]. One strategy to avoid degradation in the culture medium is retention in the endoplasmic reticulum by adding a C-terminal KDEL tag. This compartment contains molecular chaperones that facilitate protein folding/assembly and enhance their stability, and many recombi-nant proteins accumulate to higher levels in the endoplasmic reticu-lum (10-100-fold) than secreted counterparts [125,126]. As men-tioned above however, this strategy affects the glycan structures of recombinant proteins, increases the complexity of downstream processing and changes the primary structure of the recombinant protein. Therefore, alternative approaches have been investigated to reduce protease activity and hence recombinant protein degradation in the culture medium.

Fig. (3). Recombinant human serum albumin is degraded in spent tobacco BY-2 medium. Culture medium from transgenic tobacco BY-2 cells secret-ing human serum albumin for 5 days was analyzed by SDS-PAGE and immunoblot using a specific antibody (+ indicates cultures were supple-mented with 5x CompleteTM proteinase inhibitor, added on day 4 for 24 h, and - indicates the control culture without additives. Arrows indicate the intact full-length protein and a major degradation product.

48

33

62

25

17

83 HSA

M - +

degradation product

kDa


The characterization of protease activity is complex because several hundred proteases contribute to intracellular and extracellu-lar proteolytic activity, and the relative contribution of different proteases differs throughout the production cycle [118,127]. Initial analysis has revealed the characteristics of certain proteases se-creted into the culture medium but a broader range of substrates is required so that other protease activities are not overlooked (Fig. 4).The involvement of specific protease classes in the degradation of recombinant proteins secreted into the plant cell culture medium has been reported only occasionally. There is evidence for the in-volvement of cysteine and serine proteases in the degradation of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in rice cell suspension cultures [128,129], and met-alloproteinases in the degradation of recombinant DSPA�1 pro-duced by tobacco BY-2 cells [122,123].

Fig. (4). Protease activity in the culture medium of wild type tobacco BY-2 cells during cultivation. Protease activity was measured in 100 �L spent medium from three independent 50-mL cultures cultivated in Erlenmeyer flasks on an orbital shaker using fluorescence-labeled casein as a substrate (EnzCheck assay, Invitrogen). Fresh cell weight was determined after re-moval of the medium by vacuum filtration.

It has been possible to inhibit certain protease activities in plant cell cultures by co-expressing plant-derived protease-specific in-hibitors together with the recombinant proteins, thus protecting the proteins from proteolytic degradation to some extent [130,131]. For example, the co-secretion of the serine protease inhibitor PI-II from Nicotiana alata inhibits proteolytic degradation and doubles the accumulation of recombinant hGM-CSF in rice cell suspension cultures [132]. Equivalent protease inhibitors remain to be discov-ered in other plants, and there is thus far no evidence for the exis-tence of plant-derived matrix metalloproteinase-specific protease inhibitors similar to the tissue inhibitor of metalloproteinase (TIMP) found in animals. Alternative strategies include the removal of the product during cultivation [133] or the co-secretion of unre-lated proteins that might act as bait for the proteases. For example, the co-expression of human serum albumin in P. patens enhanced the recovery of secreted recombinant human growth factor [22]. Unrelated bait proteins or compounds such as gelatin or polyethyl-ene glycol can also be added directly to the culture medium, al-though animal proteins must be avoided during the production of pharmaceuticals to reduce the risk of contamination with human or animal pathogens [134]. When the specific proteases responsible for the degradation of recombinant proteins are known, knockout and knockdown strate-gies can be used to reduce their abundance. Proteases play a signifi-cant role in many aspects of plant development, stress responses and pathogen defense, so their elimination may be detrimental in intact plants [127,135]. However, the same limitations may not

apply in cell suspension cultures that are grown under controlled conditions that minimize stress and exclude pathogens. Indeed, we have found that the simultaneous expression of antisense RNAs against endogenous aspartate, cysteine, serine and metalloproteases in tobacco BY-2 cells reduced protease activity at the late stages of the cultivation cycle and increased the accumulation of a recombi-nant antibody four-fold compared to wild type cells expressing the same protein, by reducing proteolytic degradation of the heavy chain (unpublished results).

3.3. Cryopreservation Long-term subculturing of cell suspension cultures is expen-sive, labor-intensive and increases the risk of contamination, but most importantly the performance of cell lines can change over time (in terms of growth rate and recombinant protein productivity) as mutations and epigenetic modifications increase the heterogene-ity of the population. The cryopreservation of plant cells and tissues by storing cultures at -196°C in liquid nitrogen is therefore neces-sary for long-term genetic conservation, and this is also a prerequi-site for the establishment of master and working cell banks under GMP guidelines. Plant cells and tissues have different properties according to origin, so individual cryopreservation protocols must be established and optimized for cells and tissues from different species [136-139]. The essence of cryopreservation is controlled dehydration to avoid the formation of intracellular ice crystals dur-ing freezing and thawing, which is achieved by the addition of cryoprotectants such as dimethylsulfoxide (DMSO), glycerol or sucrose. Currently there are three main techniques available for the cryopreservation of plant cell cultures. (1) Controlled rate cooling, also called two-step/equilibrium freezing, involves a slow cooling gradient (1°C per minute) to an intermediate low temperature (usu-ally between �30 and �40°C) followed by rapid cooling in liquid nitrogen to vitrify the cell contents, i.e. it forms a glass without ice crystals. (2) Vitrification, which involves the exposure of cells to high (5-8 M) concentrations of cryoprotectants at non-freezing temperatures to promote dehydration, followed by ultra-rapid cool-ing in liquid nitrogen. (3) Desiccation with or without encapsulation in alginate beads. If beads are used, these are incubated for several days in a concentrated sucrose medium and then transferred to liq-uid nitrogen after drying. Cells are recovered by slowly warming the beads. Successful cryopreservation has been achieved in many species as shown by the recovery of viable biomass, but the protocols are usually optimized using non-transgenic cell lines and evaluated using criteria such as cell growth and morphology. There are lim-ited data for transgenic lines, which also need to be evaluated in terms of productivity after recovery. Schmale et al. [140] have de-veloped a cryopreservation protocol for a transgenic BY-2 cells producing human serum albumin. Growth and recombinant protein productivity remained stable after cryopreservation but the cells were stored in liquid nitrogen only for one week and further optimi-zation is required to establish reproducible procedures for the long-term cryopreservation of plant cells and tissues.

3.4. Bioreactor Systems for Plant Suspension Cultures Plant cell cultures must be scaled up during process develop-ment to achieve commercial productivity. Cell line development and optimization are usually carried out using small-scale vessels such as microtiter plates, shake flasks or disposable 50-mL bioreac-tors (Fig. 1). However, there are many different bioreactor designs for larger-scale cultivation. Floating plants and hairy root cultures require specialized bioreactors, but most plant cell cultures are grown as homogeneous suspensions similar to microbial and mammalian cell fermentations, and these can be maintained in stirred-tank bioreactors, bubble column bioreactors, air-lift bioreac-tors, single-use stirred-tank bioreactors, wave mixed bioreactors and membrane bioreactors. The choice depends on the species and

2

4

6

8

10

12

14

fluo

resc

ence

[uni

ts/m

in]

0 3 4 5 6 7 8 9 10

cultivation [days]

0.4

0.2

0.3

0.1 0.05

0.15

0.25

0.35

fresh cell weight [m

g/mL]


culture type, based on characteristics such as cell or tissue growth, morphology and aggregation, shear sensitivity, oxygen demand and culture rheological properties. For example, moss cultures need light and cannot be grown in stainless steel fermenters, whereas tobacco BY-2 cells are heterotrophic and are well suited to such vessels. The status and characteristics of different types of bioreac-tors for recombinant protein production in plant cell/tissue suspen-sion have been recently discussed [141]. Plant cell/tissue suspensions are often maintained in reusable bioreactor systems made of glass or stainless steel. These are often stirred-tank bioreactors with large, slow-moving axial and radial flow impellers, delivering maximum biomass values of 60-70% packed cell volume [142]. The typical design incorporates marine or pitched blade impellers and a ring sparger for aeration, which ensures cell growth without cell damage even though many cultures become non-Newtonian fluids with increasing culture broth viscos-ity [143]. Foam can form on the culture surface reflecting the pres-ence of sugars, polysaccharides, fatty acids, secreted proteins and air bubbles in the culture medium, and can block tubing and filters if not addressed using appropriate anti-foaming agents [144,145]. Reusable bioreactors require regular cleaning to avoid contami-nation, which involves the use of harsh chemicals for cleaning-in-place (CIP) and steam venting for sterilization-in-place (SIP). These expensive and time-consuming operations can be avoided by using disposable bioreactors, where the fermentation takes place within a disposable plastic bag [146]. Although the cost of disposable sys-tems is high, this is offset at higher production scales by the time and cost savings by avoiding CIP/SIP procedures. The Protalix recombinant glucocerebrosidase is produced in carrot cells culti-vated in a bubble column-type bioreactor fitted with a disposable polyethylene bag (440 L working volume) and this is currently the largest disposable bioreactor used with plant cells [147]. All the other single-use systems currently available are surface-aerated bioreactors like Nalgene vessels [148], wave-mixed bioreactors or orbitally-shaken bioreactors such as the OrbShake SB200-X (Adolf Kuhner AG, Birsfelden, Switzerland). We have developed a large-scale process for tobacco BY-2 suspension cultures using orbitally-shaken bioreactors (Fig. 5).Scaling up from shake flasks to the final production volume was straightforward because the same cultivation and shaking condi-tions were applied at all three stages, i.e. small-scale (500-mL shake flasks), medium-scale (20-L Nalgene vessels), and large-scale (200-L OrbShake SB200-X bioreactor, 100 L nominal working volume). The cell suspension culture performed similarly in terms of growth and recombinant protein productivity at all three scales and the process time was significantly shorter than the cultivation of whole

plants in the greenhouse even though pre-cultures were included in the process chain and several fermentation runs were completed.

3.5. Downstream Processing Most biopharmaceuticals are formulated as a purified product so the majority of pharmaceutical proteins produced in plants must be extracted from plant tissue or recovered from culture medium and then purified and formulated in the same way as conventional biopharmaceutical products. Regardless of the upstream production platform, downstream processing can account for up to 80% of the total manufacturing costs for a pharmaceutical protein [149], but the first downstream processing steps are largely determined by the properties of the production host [150]. If the protein is produced in an intact plant and/or if it accumulates inside the plant cell, addi-tional steps are required to release it by mechanical disruption in the presence of an appropriate extraction buffer and the extract must be clarified by filtration and/or centrifugation to remove debris, fibers and other particulates, which increase the overall downstream proc-essing costs. Therefore, the secretion of recombinant proteins into the plant cell culture medium is advantageous, since the purification process can begin directly after the cells have been removed from the culture broth by filtration or centrifugation. After clarification, the product may be captured from the feed by chromatography if a suitable affinity resin is available, allowing a high level of purifica-tion in a single step. A wide range of natural and synthetic affinity ligands are available, particularly for the capture of antibodies, e.g. the natural ligand protein A and the synthetic ligand mercaptoeth-ylpyridine (MEP HyperCel™) [151]. After capture, polishing is usually achieved by the application of two or more orthogonal sepa-ration methods to achieve purity by efficient contaminant removal, e.g. ion exchange, hydrophobic interaction, hydroxyapatite and size exclusion chromatography [152]. If a capture step is not possible, these chromatography methods may be used for intermediate puri-fication prior to polishing. For the purification of a human full-length antibody from BY-2 cell culture medium, we have estab-lished a robust downstream process which involves the removal of cells by filtration followed by ion exchange chromatography, pro-tein A affinity chromatography and desalting by size exclusion chromatography. Using this procedure, 85% of the antibody present in the culture broth was recovered at ~90% a purity (Fig. 5) (unpub-lished results). Biopharmaceuticals produced for human clinical trials must achieve certain quality criteria that are defined by GMP guidelines. The production of pharmaceuticals using plant suspension cells and tissues is similar in concept to conventional systems based on mammalian cells, and the approval of glucocerebrosidase produced

Fig. (5). Scaled-up production of plant suspension cells in disposable orbitally-shaken bioreactors. Four liters of a transgenic tobacco BY-2 suspension pre-culture secreting a human full-length antibody were cultivated in an orbitally-shaken 20-L disposable plastic vessel (Nalgene) and used to inoculate 100 L of fresh medium. Cells were cultivated in a 200-L disposable plastic bag (Sartorius-Stedim, Goettingen, Germany) fitted into a 200-L orbital shaker (OrbShake SB200-X, Adolf Kuhner AG, Birsfelden, Switzerland). After 7 days, the culture broth was harvested, the cells removed and the antibody purified from the culture medium. Analysis by SDS-PAGE verified the integrity and high purity of the plant-derived antibody.

4 L pre-culture in 20 L vessel

104 L culture in 200 L bag

Harvest DSP + analytic


in carrot cells using a disposable bioreactor has demonstrated that the regulatory agencies now accept the use of plant cells for the manufacture of pharmaceutical proteins.

4. OUTLOOK AND FUTURE CHALLENGES Plant cells are now established as a platform for the production of recombinant pharmaceutical proteins. We have achieved a spe-cific productivity of 5 pg per cell per day using transgenic tobacco BY-2 cells, which equates to a recombinant protein yield of 70 mg per L culture medium (unpublished data). This is only an order of magnitude lower than elite CHO cell lines, bearing in mind that these cells must undergo gene amplification and multiple selection rounds to achieve such high yields [153,154]. High protein titers in CHO cells are also achieved by increasing the cell number during the fermentation process. This is difficult to achieve in plant cell suspension and tissue cultures because packed cell volumes of al-most 70% are achieved at the end of the cultivation process al-though the total cell number is very similar to comparable processes using mammalian cells where the packed cell volume is only 0.5%. This reflects the large cell size of plant cells, in which most of the cell volume is taken up by the vacuole, suggesting that one way to increase productivity would be to reduce the cell/vacuole size. In-deed, the most productive tobacco BY-2 cell lines have smaller cells with a lower packed cell volume at the end of the cultivation process, thus increasing the total cell number. Production levels could also be boosted by establishing gene amplification systems in plants that boost production levels to the same extent seen in mam-malian cells. One of the key advantages of plant cells is the simple and inex-pensive cultivation medium compared to mammalian cells, but the optimization of plant cell media to promote recombinant protein production is an emerging field, and it remains to be seen whether medium optimization requires novel ingredients that increase costs. The reduction of protease activity in the medium and the establish-ment of robust cryopreservation protocols will also help to improve productivity and ensure production consistency and homogeneity. The FDA approval of recombinant glucocerebrosidase pro-duced in carrot cells has demonstrated the competitiveness of plant cell-based production platforms in niche markets, but further candi-dates must now be produced using GMP-compliant, large-scale processes and the products entered into clinical trials to demonstrate the overall sustainability of contained and controlled plant cell and tissue culture systems. The benefits of the plant-type glycans and the use of the plant matrix to enhance the immunogenicity of ani-mal vaccines and avoid cost-intensive purification processes are unique selling points which can be exploited in the next generation of pharmaceutical products from plant cells.

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS Some of the unpublished results presented here have been gen-erated within the EU-funded projects CoMoFarm (227420) and the COST Action Molecular Farming (FA0804). The authors would like to thank the members of both consortia for the stimulating dis-cussions on the current and future directions and challenges on the exploitation plant cell suspension cultures for the production of biopharmaceuticals.

REFERENCES[1] Paul M, Ma JK. Plant-made pharmaceuticals: leading products and

production platforms. Biotechnol Appl Biochem 2011; 58: 58-67. [2] Nagels B, Weterings K, Callewaert N, van Damme EJM. Produc-

tion of Plant Made Pharmaceuticals: From Plant Host to Functional Protein. Crit Rev Plant Sci 2012; 31: 148-80.

[3] Rybicki EP. Plant-made vaccines for humans and animals. Plant Biotechnol J 2010; 8: 620-37.

[4] Schiermeyer A, Schillberg S. Plant molecular pharming - pharma-ceuticals for human health, in: R.A. Meyers (Ed.), Encyclopedia of sustainability science and technology, Springer, New York 2012, pp. 8126-8141.

[5] Woodard SL, Mayor JM, Bailey MR, et al. Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol Appl Biochem 2003; 38: 123-30.

[6] Howard JA. Commercialization of biopharmaceutical and bioin-dustrial proteins from plants. Crop Sci 2005; 45: 468-72.

[7] Hollak CE, Vom Dahl S, Aerts JM, et al. Force Majeure: therapeu-tic measures in response to restricted supply of imiglucerase (Cerezyme) for patients with Gaucher disease. Blood Cells Mol Dis 2010; 44: 41-7.

[8] Leede LG de, Humphries JE, et al. Novel controlled-release Lemna-derived IFN-alpha2b (Locteron): pharmacokinetics, phar-macodynamics, and tolerability in a phase I clinical trial. J Inter-feron Cytokine Res 2008; 28: 113-22.

[9] McCormick AA, Reddy S, Reinl SJ, et al. Plant-produced idiotype vaccines for the treatment of non-Hodgkin's lymphoma: safety and immunogenicity in a phase I clinical study. Proc Natl Acad Sci U S A 2008; 105: 10131-6.

[10] Boothe J, Nykiforuk C, Shen Y, et al. Seed-based expression sys-tems for plant molecular farming. Plant Biotechnol J 2010; 8: 588-606.

[11] Valdés R, Reyes B, Alvarez T, et al. Hepatitis B surface antigen immunopurification using a plant-derived specific antibody pro-duced in large scale. Biochem Biophys Res Commun 2003; 310: 742-7.

[12] Ramessar K, Peremarti A, Gómez-Galera S, et al. Biosafety and risk assessment framework for selectable marker genes in trans-genic crop plants: a case of the science not supporting the politics. Transgenic Res 2007; 16: 261-80.

[13] IAPO (International Alliance of Patients´ Organizations). Briefing paper on plant-made pharmaceuticals. The use of genetically modi-fied plants to produce human therapeutic proteins: a summary of existing and potential benefits and risks. 2005

[14] Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK. Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res 2007; 16: 147-61.

[15] Fischer R, Schillberg S, Hellwig S, Twyman RM, Drossard J. GMP issues for recombinant plant-derived pharmaceutical proteins. Bio-technol Adv 2012; 30: 434-9.

[16] Kolewe ME, Gaurav V, Roberts SC. Pharmaceutically active natu-ral product synthesis and supply via plant cell culture technology. Mol Pharm 2008; 5: 243-56.

[17] Roberts Shuler. Large-scale plant cell culture. Curr Opin Biotech-nol 1997; 8: 154-9.

[18] Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A. Production of correctly processed human se-rum albumin in transgenic plants. Bio-Technol 1990; 8: 217-21.

[19] Sack M, Paetz A, Kunert R, et al. Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures. FASEB J 2007; 21: 1655-64.

[20] Sirko A, Vanek T, Gora-Sochacka A, Redkiewicz P. Recombinant cytokines from plants. Int J Mol Sci 2011; 12: 3536-52.

[21] Sorrentino A, Schillberg S, Fischer R, Rao R, Porta R, Mariniello L. Recombinant human tissue transglutaminase produced into to-bacco suspension cell cultures is active and recognizes autoantibod-ies in the serum of coeliac patients. Int J Biochem Cell Biol 2005; 37: 842-51.

[22] Baur A, Reski R, Gorr G. Enhanced recovery of a secreted recom-binant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. Plant Biotechnol J 2005; 3: 331-40.

[23] Kim TG, Baek MY, Lee EK, Kwon TH, Yang MS. Expression of human growth hormone in transgenic rice cell suspension culture. Plant Cell Rep 2008; 27: 885-91.

[24] Matsumoto S, Ikura K, Ueda M, Sasaki R. Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol 1995; 27: 1163-1172.

[25] Smith ML, Mason HS, Shuler ML. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen ac-


cumulation in batch culture and its intracellular form. Biotechnol Bioeng 2002; 80: 812-22.

[26] Xu J, Ge X, Dolan MC. Towards high-yield production of pharma-ceutical proteins with plant cell suspension culture. Biotechnol Adv 2011; 29: 278-99.

[27] Nagata T, Nemoto Y, Hasezawa S. Tobacco BY-2 cell line as the "HeLa" cell in the cell biology of higher plants. Int Rev Cytol 1992; 132: 1-30.

[28] Decker EL, Reski R. Moss bioreactors producing improved bio-pharmaceuticals. Curr Opin Biotechnol 2007; 18: 393-8.

[29] Gasdaska JR, Spencer D, Dickey L. Advantages of therapeutic protein production in the aquatic plant Lemna. Bioprocessing 2003; 2: 49-56.

[30] Medina-Bolívar F, Cramer C. Production of recombinant proteins by hairy roots cultured in plastic sleeve bioreactors. Methods Mol Biol 2004; 267: 351-63.

[31] Mikschofsky H, Hammer M, Schmidtke J, et al. Optimization of growth performance of freshly induced carrot suspensions concern-ing PMP production. In vitro Cell Dev-Pl 2009; 45: 740-9.

[32] An G. High efficiency transformation of cultured tobacco cells. Plant Physiol 1985; 79: 568-70.

[33] O'Neill KM, Larsen JS, Curtis WR. Scale-Up of Agrobacterium-Mediated Transient Protein Expression in Bioreactor-Grown Nico-tiana glutinosa Plant Cell Suspension Culture. Biotechnol Prog 2008; 24: 372-6.

[34] Winicur ZM, Zhang GF, Staehelin LA. Auxin deprivation induces synchronous Golgi differentiation in suspension-cultured tobacco BY-2 cells. Plant Physiol 1998; 117: 501-13.

[35] Torres E, Vaquero C, Nicholson L, et al. Rice cell culture as an alternative production system for functional diagnostic and thera-peutic antibodies. Transgenic Res 1999; 8: 441-9.

[36] Plasson C, Michel R, Lienard D, et al. Production of recombinant proteins in suspension-cultured plant cells. Methods Mol Biol 2009; 483: 145-61.

[37] Pires AS, Rosa S, Castanheira S, Fevereiro P, Abranches R. Ex-pression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago. Plant Cell Tiss Or-gan Cult 2012; 110: 171-81.

[38] Ganapathi TR, Sunil Kumar GB, Srinivas L, Revathi CJ, Bapat VA. Analysis of the limitations of hepatitis B surface antigen ex-pression in soybean cell suspension cultures. Plant Cell Rep 2007; 26: 1575-84.

[39] Kwon T, Kim Y, Lee J, Yang M. Production and secretion of bio-logically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures. Biotechnol Lett 2003; 25: 1571-4.

[40] Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M, Gantet P. Harnessing the potential of hairy roots: dawn of a new era. Trends Biotechnol 2006; 24: 403-9.

[41] Wongsamuth R, Doran PM. Production of monoclonal antibodies by tobacco hairy roots. Biotechnol Bioeng 1997; 54: 401-15.

[42] Gaume A, Komarnytsky S, Borisjuk N, Raskin I. Rhizosecretion of recombinant proteins from plant hairy roots. Plant Cell Rep 2003; 21: 1188-93.

[43] Skarjinskaia M, Karl J, Araujo A, et al. Production of recombinant proteins in clonal root cultures using episomal expression vectors. Biotechnol Bioeng 2008; 100: 814-9.

[44] Schaefer, DG. Gene targeting in Physcomitrella patens. Curr Opin Plant Biol 2001; 4: 143-50.

[45] Gorr G, Jost W. Glycosylation design in transgenic moss for better product efficacy. Bioprocessing 2005; 4: 26-30.

[46] Koprivova A, Stemmer C, Altmann F, et al. Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotechnol J 2004; 2: 517-23.

[47] Schuster M, Jost W, Mudde GC, et al. In vivo glyco-engineered antibody with improved lytic potential produced by an innovative non-mammalian expression system. Biotechnol J 2007; 2: 700-8.

[48] Baur A, Kaufmann F, Rolli H, et al. A fast and flexible PEG-mediated transient expression system in plants for high level ex-pression of secreted recombinant proteins. J Biotechnol 2005; 119: 332-42.

[49] Weise A, Altmann F, Rodriguez-Franco M, et al. High-level ex-pression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella �-fuc-t �-xyl-t mutant. Plant Biotechnol J 2007; 5: 389-401.

[50] Guedes AC, Amaro HM, Malcata FX. Microalgae as sources of high added-value compounds--a brief review of recent work. Bio-technol Prog 2011; 27: 597-613.

[51] Malcata FX. Microalgae and biofuels: a promising partnership? Trends Biotechnol 2011; 29: 542-9.

[52] Shimoda K, Hamada H. Bioremediation of Bisphenol A and Ben-zophenone by Glycosylation with Immobilized Marine Microalga Pavlova sp. Environ Health Insights 2009; 3: 89-94.

[53] Rasala BA, Muto M, Lee PA, et al. Production of therapeutic pro-teins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 2010; 8: 719-33.

[54] Michelet L, Lefebvre-Legendre L, Burr SE, Rochaix J, Gold-schmidt-Clermont M. Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas. Plant Biotechnol J 2011; 9: 565-74.

[55] Rasala BA, Muto M, Sullivan J, Mayfield SP. Improved heterolo-gous protein expression in the chloroplast of Chlamydomonas rein-hardtii through promoter and 5' untranslated region optimization. Plant Biotechnol J 2011; 9: 674-83.

[56] Rasala BA, Mayfield SP. The microalga Chlamydomonas reinhard-tii as a platform for the production of human protein therapeutics. Bioeng Bugs 2011; 2: 50-4.

[57] Dreesen IAJ, Charpin-El Hamri G, Fussenegger M. Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. J Biotechnol 2010; 145: 273-80.

[58] Woodard SL, Wilken LR, Barros GO, White SG, Nikolov ZL. Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development. Biotechnol Bioeng 2009; 104: 562-71.

[59] Rival S, Wisniewski JP, Langlais A, et al. Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin. Transgenic Res 2008; 17: 503-13.

[60] L. Dickey J. Gasdaska K. Cox. Expression of biologically active polypeptides in duckweed 2011; US 8,022,270 B2.

[61] Yamamoto YT, Rajbhandari N, Lin XH, Bergmann BA, Nishimura Y, Am Stomp. Genetic transformation of duckweed Lemna gibba and Lemna minor. In vitro Cell Dev-Pl 2001; 37: 349-53.

[62] Kruse C, Boehm R, Voeste D, Barth S, Schnabl H. Transient trans-formation of Wolffia columbiana by particle bombardment. Aquat Bot 2002; 72: 175-81.

[63] Popov SV, Golovchenko VV, Ovodova RG, et al. Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L. Vaccine 2006; 24: 5413-9.

[64] Cox KM, Sterling JD, Regan JT, et al. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 2006; 24: 1591-7.

[65] Kim H, Wheeler RM, Sager JC, Yorio NC, Goins GD. Light-emitting diodes as an illumination source for plants: a review of re-search at Kennedy Space Center. Habitation (Elmsford) 2005; 10: 71-8.

[66] Buemi F, Massa M, Sandini G, Costi G. The AGROBOT project. Adv Space Res 1996; 18: 185-9.

[67] Graves CJ. The nutrient film technique. Horticultural Reviews 1983; 5: 1-44.

[68] Harris EH. Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 2001; 52: 363-406.

[69] Gleba Y, Klimyuk V, Marillonnet S. Magnifection--a new platform for expressing recombinant vaccines in plants. Vaccine 2005; 23: 2042-8.

[70] Pogue GP, Vojdani F, Palmer KE, et al. Production of pharmaceu-tical-grade recombinant aprotinin and a monoclonal antibody prod-uct using plant-based transient expression systems. Plant Biotech-nol J 2010; 8: 638-54.

[71] Sainsbury F, Sack M, Stadlmann J, Quendler H, Fischer R, Lo-monossoff GP. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody. PLoS ONE 2010; 5: e13976.

[72] Conrad U, Fiedler U. Compartment-specific accumulation of re-combinant immunoglobulins in plant cells: an essential tool for an-tibody production and immunomodulation of physiological func-tions and pathogen activity. Plant Mol Biol 1998; 38: 101-9.

[73] Fischer R, Liao YC, Hoffmann K, Schillberg S, Emans N. Molecu-lar farming of recombinant antibodies in plants. Biol Chem 1999; 380: 825-39.


[74] Muynck B de, Navarre C, Boutry M. Production of antibodies in

plants: status after twenty years. Plant Biotechnol J 2010; 8: 529-63.

[75] Hacker DL, Jesus M de, Wurm FM. 25 years of recombinant pro-teins from reactor-grown cells - where do we go from here? Bio-

technol Adv 2009; 27: 1023-7. [76] Fox JL. Turning plants into protein factories. Nat Biotechnol 2006;

24: 1191-3. [77] Kaiser J. Is the drought over for pharming? Science 2008; 320:

473-5. [78] Shaaltiel Y, Bartfeld D, Hashmueli S, et al. Production of glu-

cocerebrosidase with terminal mannose glycans for enzyme re-placement therapy of Gaucher's disease using a plant cell system.

Plant Biotechnol J 2007; 5: 579-90. [79] Desai PN, Shrivastava N, Padh H. Production of heterologous

proteins in plants: strategies for optimal expression. Biotechnol Adv 2010; 28: 427-35.

[80] Boivin EB, Lepage E, Matton DP, Crescenzo G de, Jolicoeur M. Transient expression of antibodies in suspension plant cell suspen-

sion cultures is enhanced when co-transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing. Biotechnol

Prog 2010; 26: 1534-43. [81] Collens JI, Mason HS, Curtis WR. Agrobacterium-mediated viral

vector-amplified transient gene expression in Nicotiana glutinosa plant tissue culture. Biotechnol Prog 2007; 23: 570-576.

[82] Dietz-Pfeilstetter A. Stability of transgene expression as a chal-lenge for genetic engineering. Plant Sci 2010; 179: 164-7.

[83] Schillberg S, Fischer R, Emans N. Molecular farming of recombi-nant antibodies in plants. Cell Mol Life Sci 2003; 60: 433-45.

[84] Hood EE, Love R, Lane J, et al. Subcellular targeting is a key condition for high-level accumulation of cellulase protein in trans-

genic maize seed. Plant Biotechnol J 2007; 5: 709-19. [85] Gruber V, Berna PP, Arnaud T, et al. Large-scale production of a

therapeutic protein in transgenic tobacco plants: effect of subcellu-lar targeting on quality of a recombinant dog gastric lipase. Mol

Breeding 2001; 7: 329-40. [86] Yang J, Barr LA, Fahnestock SR, Liu ZB. High yield recombinant

silk-like protein production in transgenic plants through protein targeting. Transgenic Res 2005; 14: 313-24.

[87] Saint-Jore-Dupas C, Faye L, Gomord V. From planta to pharma with glycosylation in the toolbox. Trends Biotechnol 2007; 25:

317-23. [88] Gomord V, Fitchette AC, Menu-Bouaouiche L, et al. Plant-specific

glycosylation patterns in the context of therapeutic protein produc-tion. Plant Biotechnol J 2010; 8: 564-87.

[89] Bardor M, Faveeuw C, Fitchette AC, et al. Immunoreactivity in mammals of two typical plant glyco- epitopes, core alpha(1,3)-

fucose and core xylose. Glycobiology 2003; 13: 427-34. [90] Triguero A, Cabrera G, Cremata JA, Yuen C, Wheeler J, Ramirez

NI. Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homoge-

neously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnol J 2005; 3: 449-57.

[91] Petruccelli S, Otegui MS, Lareu F, et al. A KDEL-tagged mono-clonal antibody is efficiently retained in the endoplasmic reticulum

in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J 2006; 4: 511-27.

[92] Sriraman R, Bardor M, Sack M, et al. Recombinant anti-hCG anti-bodies retained in the endoplasmic reticulum of transformed plants

lack core-xylose and core- (1,3)-fucose residues. Plant Biotechnol J 2004; 2: 279-87.

[93] Karg SR, Frey AD, Kallio PT. Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltrans-

ferase III in tobacco BY-2 cells depends on Golgi enzyme localiza-tion domain and genetic elements used for expression. J Biotechnol

2010; 146: 54-65. [94] Rouwendal GJ, Wuhrer M, Florack DE, et al. Efficient introduction

of a bisecting GlcNAc residue in tobacco N-glycans by expression of the gene encoding human N-acetylglucosaminyltransferase III.

Glycobiology 2007; 17: 334-44. [95] Baker KN, Rendall MH, Hills AE, Hoare M, Freedman RB, James

DC. Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol Bioeng 2001; 73: 188-202.

[96] Chen P, Harcum SW. Effects of elevated ammonium on glycosyla-tion gene expression in CHO cells. Metab Eng 2006; 8: 123-32.

[97] Becerra-Arteaga A, Shuler ML. Influence of culture medium sup-

plementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase. Biotechnol

Bioeng 2007; 97: 1585-93. [98] Shin Y, Chong Y, Yang M, Kwon T. Production of recombinant

human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure. Plant

Biotechnol J 2011; 9: 1109-19. [99] Kieliszewski MJ. The latest hype on Hyp-O-glycosylation codes.

Phytochemistry 2001; 57: 319-23. [100] Xu J, Okada S, Tan L, Goodrum KJ, Kopchick JJ, Kieliszewski

MJ. Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum

life. Transgenic Res 2010; 19: 849-67. [101] Xu J, Tan L, Goodrum KJ, Kieliszewski MJ. High-yields and ex-

tended serum half-life of human interferon alpha2b expressed in tobacco cells as arabinogalactan-protein fusions. Biotechnol Bio-

eng 2007; 97: 997-1008. [102] Nocarova E, Fischer L. Cloning of transgenic tobacco BY-2 cells;

an efficient method to analyse and reduce high natural heterogene-ity of transgene expression. BMC Plant Biol 2009; 9: 44.

[103] Muller E, Lorz H, Lutticke S. Variability of transgene expression in clonal cell lines of wheat. Plant Sci 1996; 114: 71-82.

[104] James E, Lee JM. Loss and recovery of protein productivity in genetically modified plant cell lines. Plant Cell Rep 2006; 25: 723-

7. [105] Ikeda T, Matsumoto T, Obi Y, Kisaki T, Noguchi M. Characteris-

tics of Cultured Tobacco Cell Strains Producing High-Levels of Ubiquinone-10 Selected by a Cell Cloning Technique. Agr Biol

Chem Tokyo 1981; 45: 2259-63. [106] Kirchhoff J, Raven N, Boes A, et al. Monoclonal tobacco cell lines

with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting. Plant Bio-

technol J 2012; 10: 936-44. [107] Murashige T, Skoog F. A revised medium for rapid growth and

bioassay with tobacco tissue cultures. Physiol Plantarum 1962; 15: 473-97.

[108] Grimes HD, Hodges TK. The Inorganic NO3--NH4

+ Ratio Influ-ences Plant-Regeneration and Auxin Sensitivity in Primary Callus

Derived from Immature Embryos of Indica Rice (Oryza-sativa L). J Plant Physiol 1990; 136: 362-7.

[109] Niedz RP. Growth of embryogenic sweet orange callus on media varying in the ration of nitrate to ammonium nitrogen. Plant Cell

Tiss Organ Cult 1994; 39: 1-5. [110] Krouk G, Ruffel S, Gutiérrez RA, et al. A framework integrating

plant growth with hormones and nutrients. Trends Plant Sci. 2011; 16: 178-82.

[111] Crawford NM. Nitrate: nutrient and signal for plant growth. Plant Cell 1995; 7: 859-68.

[112] Ohira K, Ojima K, Fujiwara A. Studies on nutrition of rice cell-culture. I. Simple, defined medium for rapid growth in suspension

culture. Plant Cell Physiol 1973; 14: 1113-21. [113] Gamborg OL, Miller RA, Ojima K. Nutrient requirements of sus-

pension cultures of soybean root cells. Exp Cell Res 1968; 50: 151-8.

[114] Holland T, Sack M, Rademacher T, et al. Optimal nitrogen supply as a key to increased and sustained production of a monoclonal

full-size antibody in BY-2 suspension culture. Biotechnol Bioeng 2010; 107: 278-89.

[115] Ullisch DA, Müller CA, Maibaum S, et al. Comprehensive charac-terization of Nicotiana tabacum BY-2 cell growth leads to doubled

GFP concentration by media optimization. J Biosci Bioeng 2012; 113: 242-8.

[116] Gorret N, bin Rosli AK, Oppenheim SF, et al. Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inocu-

lum size, and conditioned medium on biomass production. J Bio-technol 2004; 108: 253-63.

[117] Nas MN, Eskridge KM, Read PE. Experimental designs suitable for testing many factors with limited number of explants in tissue

culture. Plant Cell Tiss Organ Cult 2005; 81: 213-20. [118] Navarre C, Muynck B de, Alves G, Vertommen D, Magy B, Boutry

M. Identification, gene cloning and expression of serine proteases in the extracellular medium of Nicotiana tabacum cells. Plant Cell

Rep 2012; 31: 1959-68.


[119] Delannoy M, Alves G, Vertommen D, Ma J, Boutry M, Navarre C. Identification of peptidases in Nicotiana tabacum leaf intercellular fluid. Proteomics 2008; 8: 2285-98.

[120] Muynck B de, Navarre C, Nizet Y, Stadlmann J, Boutry M. Differ-ent subcellular localization and glycosylation for a functional anti-body expressed in Nicotiana tabacum plants and suspension cells. Transgenic Res 2009; 18: 467-82.

[121] Doran PM. Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 2006; 24: 426-32.

[122] Schiermeyer A, Schinkel H, Apel S, Fischer R, Schillberg S. Pro-duction of Desmodus rotundus salivary plasminogen activator a1 (DSPA�1) in tobacco is hampered by proteolysis. Biotechnol Bio-eng 2005; 89: 848-58.

[123] Mandal MK, Fischer R, Schillberg S, Schiermeyer A. Biochemical properties of the matrix metalloproteinase NtMMP1 from Nico-tiana tabacum cv. BY-2 suspension cells. Planta 2010; 232: 899-910.

[124] Rivard D, Anguenot R, Brunelle F, et al. An in-built proteinase inhibitor system for the protection of recombinant proteins recov-ered from transgenic plants. Plant Biotechnol J 2006; 4: 359-68.

[125] Spiegel H, Schillberg S, Sack M, et al. Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells. Plant Sci 1999; 149: 63-71.

[126] Fiedler U, Phillips J, Artsaenko O, Conrad U. Optimization of scFv antibody production in transgenic plants. Immunotechnology 1997; 3: 205-216.

[127] van der Hoorn RA. Plant proteases: from phenotypes to molecular mechanisms. Annu Rev Plant Biol 2008; 59: 191-223.

[128] Kim NS, Kim TG, Kim OH, et al. Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture. Plant Mol Biol 2008; 68: 263-75.

[129] Kim TG, Lee HJ, Jang YS, et al. Co-expression of proteinase in-hibitor enhances recombinant human granulocyte-macrophage col-ony stimulating factor production in transgenic rice cell suspension culture. Protein Expr Purif 2008; 61: 117-21.

[130] Komarnytsky S, Borisjuk N, Yakoby N, Garvey A, Raskin I. Cose-cretion of protease inhibitor stabilizes antibodies produced by plant roots. Plant Physiol 2006; 141: 1185-93.

[131] Benchabane M, Rivard D, Girard C, Michaud D. Companion pro-tease inhibitors to protect recombinant proteins in transgenic plant extracts. Methods Mol Biol 2009; 483: 265-73.

[132] Kim TG, Kim HM, Lee HJ, et al. Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor. Protein Expr Purif 2007; 53: 270-4.

[133] James E, Mills DR, Lee JM. Increased production and recovery of secreted foreign proteins from plant cell cultures using an affinity chromatography bioreactor. Biochem Eng J 2002; 12: 205-13.

[134] Lee SY, Cho JM, Kim DI. Stability enhancement of hGM-CSF in transgenic Nicotiana tabacum suspension cell cultures. Biotechnol Bioprocess Eng 2003; 8: 187-91.

[135] van der Hoorn RAL, Jones JDG. The plant proteolytic machinery and its role in defence. Curr Opin Plant Biol 2004; 7: 400-7.

[136] Grout BWW. Cryopreservation of plant cell suspensions. Methods Mol Biol 2007; 368: 153-61.

[137] Mustafa NR, Winter W de, van Iren F, Verpoorte R. Initiation, growth and cryopreservation of plant cell suspension cultures. Nat Protoc 2011; 6: 715-42.

[138] Ogawa Y, Sakurai N, Oikawa A, et al. High-throughput Cryopre-servation of Plant Cell Cultures for Functional Genomics. Plant Cell Physiol 2012; 53: 943-52.

[139] Yin Z, Chen L, Zhao B, Zhu Y, Wang Q. Cryopreservation of embryogenic cell suspensions by encapsulation-vitrification and encapsulation-dehydration. Methods Mol Biol 2012; 877: 81-93.

[140] Schmale K, Rademacher T, Fischer R, Hellwig S. Towards indus-trial usefulness - cryo-cell-banking of transgenic BY-2 cell cul-tures. J Biotechnol 2006; 124: 302-11.

[141] Huang T, McDonald KA. Bioreactor systems for in vitro produc-tion of foreign proteins using plant cell cultures. Biotechnol Adv 2012; 30: 398-409.

[142] Eibl R, Eibl D. Design of bioreactors suitable for plant cell and tissue culture. Phytochem Rev 2008; 7: 593-8.

[143] Kieran PM, MacLoughlin PF, Malone DM. Plant cell suspension cultures: some engineering considerations. J Biotechnol 1997; 59: 39-52.

[144] Abdullah MA, Ariff AB, Marziah M, Am Ali Lajis NH. Strategies to overcome foaming and wall-growth during the cultivation of Morinda elliptica cell suspension culture in a stirred-tank bioreac-tor. Plant Cell Tiss Organ Cult 2000; 60: 205-12.

[145] Wongsamuth R, Doran PM. Foaming and Cell Flotation in Sus-pended Plant-Cell Cultures and the Effect of Chemical Antifoams. Biotechnol Bioeng 1994; 44: 481-8.

[146] Raven N, Schillberg S, Kirchhoff J, Brändli J, Imseng N, Eibl R. Growth of BY-2 suspension cells and plantibody production in sin-gle-use bioreactors, in: R. Eibl, D. Eibl (Eds.), Single-use technol-ogy in biopharmaceutical manufacture, John Wiley & Sons, Hoboken, NJ 2011, pp. 251-261.

[147] Shaaltiel Y Kirshner Y, Shtainiz AY, Naos Y, Shneor Y. Large scale disposable bioreactor. WO 2008135991.

[148] Raval K, Kato Y, Buechs J. Comparison of torque method and temperature method for determination of power consumption in disposable shaken bioreactors. Biochem Eng J 2007; 34: 224-7.

[149] Roque AC, Lowe CR, Taipa MA. Antibodies and genetically engi-neered related molecules: production and purification. Biotechnol Prog 2004; 20: 639-54.

[150] Menkhaus TJ, Bai Y, Zhang C, Nikolov ZL, Glatz CE. Considera-tions for the recovery of recombinant proteins from plants. Bio-technol Prog 2004; 20: 1001-14.

[151] Low D, O'Leary R, Pujar NS. Future of antibody purification. Journal of chromatography 2007; 848: 48-63.

[152] Drossard J. Downstream processing of plant-derived recombinant therapeutic proteins, in: R. Fischer, S. Schillberg (Eds.), Molecular farming: plant-made pharmaceuticals and technical proteins, Wiley-VCH, Weinheim 2004, pp. 217-231.

[153] Chusainow J, Yang YS, Yeo JH, et al. A study of monoclonal antibody-producing CHO cell lines: What makes a stable high pro-ducer? Biotechnol Bioeng 2009; 102: 1182-96.

[154] Kim JY, Kim Y, Lee GM. CHO cells in biotechnology for produc-tion of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 2012; 93: 917-30.

Received: November 30, 2012 Accepted: January 31, 2013

molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures

Documents