Biotechnology Advances 30 (2012) 398409

Contents lists available at ScienceDirect

Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures
Ting-Kuo Huang, Karen A. McDonald
Department of Chemical Engineering and Materials Science, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States

a r t i c l e

i n f o

a b s t r a c t
Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20 years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems. 2011 Published by Elsevier Inc.

Available online 6 August 2011 Keywords: Heterologous protein Plant cell culture Bioreactor Bioprocess Biomanufacturing

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Foreign protein production using in vitro plant cell culture . . . . . . . . . . 2.1. Foreign protein production in suspended dedifferentiated plant cells . . . 2.2. Foreign protein production in differentiated plant organ and tissue culture 2.2.1. Moss and duckweed in vitro culture . . . . . . . . . . . . . 2.2.2. Hairy root in vitro cell culture . . . . . . . . . . . . . . . . 2.3. Characteristics of in vitro plant cell cultures . . . . . . . . . . . . . . Bioreactor systems for in vitro plant cell cultures . . . . . . . . . . . . . . . 3.1. Reusable bioreactor systems . . . . . . . . . . . . . . . . . . . . . 3.1.1. Stirred-tank bioreactors . . . . . . . . . . . . . . . . . . . 3.1.2. Pneumatic bioreactors . . . . . . . . . . . . . . . . . . . . 3.1.3. Fixed-bed bioreactors . . . . . . . . . . . . . . . . . . . . 3.1.4. Rotary drum bioreactors . . . . . . . . . . . . . . . . . . . 3.2. Disposable bioreactor systems (single-use technology) . . . . . . . . . 3.2.1. Disposable standard bioreactors . . . . . . . . . . . . . . . 3.2.2. Wave-mixed bioreactors . . . . . . . . . . . . . . . . . . . 3.2.3. Immersion bioreactors . . . . . . . . . . . . . . . . . . . . 3.2.4. Membrane bioreactors . . . . . . . . . . . . . . . . . . . . 3.2.5. Microbioreactors . . . . . . . . . . . . . . . . . . . . . . . 3.3. Special considerations for moss bioreactor systems . . . . . . . . . . . 3.4. Special considerations for hairy root bioreactor systems . . . . . . . . Bioreactor operation mode optimization . . . . . . . . . . . . . . . . . . . 4.1. Constitutive expression of foreign proteins in plant cell culture bioreactors 4.2. Inducible expression of foreign proteins in plant cell culture bioreactors . . . 4.3. Strategies to enhance foreign protein stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 399 399 401 401 401 401 401 401 401 403 404 404 404 404 404 405 405 405 405 406 406 406 406 407

3.

4.

Corresponding author. Tel.: + 1 530 752 8314; fax: + 1 530 752 1031. E-mail address: kamcdonald@ucdavis.edu (K.A. McDonald). 0734-9750/$ see front matter 2011 Published by Elsevier Inc. doi:10.1016/j.biotechadv.2011.07.016

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

399

5. Opportunities for improving foreign protein production in plant cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

407 407 407

1. Introduction Biomanufacturing of heterologous protein products has gained increasing importance due to the expanded market demand for therapeutic and industrial applications leading to the development of alternative safe and cost-efcient expression platforms and emerging technologies (Hacker et al., 2009; Karg and Kallio, 2009). At present, several gene expression systems have been developed for foreign protein production. Each system offers distinct features consisting of protein expression enhancement, ease of genetic manipulation, and protein quality (Durocher and Butler, 2009). In the past decade, the plant cell-based expression platform as a bioreactor system (whole plants and in vitro plant cell, organ and tissue cultures) have been investigated as a potential alternative for the large scale production of foreign proteins including plant-made pharmaceuticals (PMP) and plant-made industrial products (PMIP) (Davies, 2010; Sharma and Sharma, 2009). Plant cell bioreactors provide attractive features over traditional expression systems that utilize microbial and mammalian host cells, including their intrinsic safety (plant cells do not propagate mammalian viruses and pathogens and can be easily grown without any animal-derived components which are important considerations for therapeutic applications), cost-effective biomanufacturing that leads to lower production costs (Shadwick and Doran, 2005), and the capability for post-translation modications (ability to produce glycoproteins and complex multimeric proteins with similarity to their native counterparts in terms of N-glycan structure compared to mammalian cells) (Gomord et al., 2010). The features of various host cellbased protein expression systems have been discussed extensively (Demain and Vaishnav, 2009; Yin et al., 2007). Compared to the use of whole plants as a production platform, in vitro plant cell cultures (suspended plant cells, tissue and organ cultures, etc.) grown in controllable bioreactor systems offer additional features for economical, sustained foreign protein production (Hellwig et al., 2004) such as 1) shorter production cycles in that the timescale for foreign protein production in plant cell culture requires days or weeks compared to months in transgenic plants; 2) more consistency in batch-to-batch product yield, quality and homogeneity of the target protein N-glycan pattern due to the homogeneous culture of plant cells under controlled bioreactor conditions (De Muynck et al., 2010; Lienard et al., 2007); 3) cheaper and simpler downstream recovery and purication particularly for products secreted into the extracellular medium and lower secondary metabolite and host cell protein concentration (Rawel et al., 2007); 4) elimination of the need for intensive labor for cultivation of greenhouse or eld-grown plants; 5) reduced contamination from endotoxin and mycotoxin; 6) safer production platform in a closed bioreactor system, avoiding gene ow in the environment and contamination of the food chains (Franconi et al., 2010), and 7) ease of compliance with cGMP requirements and product registration process, etc. Remarkable progress for improving foreign protein production yield and quality through bioreactor engineering (Huang and McDonald, 2009) and genetic engineering approaches (Desai et al., 2010; Streateld, 2007), such as enhanced transgene transcription, post-transcription stability, and translation efciency, has demonstrated that in vitro plant cell culture is become an enabling technology for biomanufacturing foreign proteins. In this article, the recent technological progress in bioreactor-based plant cell cultures for in vitro production of foreign proteins will be discussed.

2. Foreign protein production using in vitro plant cell culture Currently, foreign proteins including therapeutic proteins (antibodies, antigens, vaccines and human blood proteins, etc.), specialty proteins (e.g. gelatin and collagen for drug capsules), and industrial enzymes (such as cellulases and lipases for biofuel applications) can be expressed in plant cell cultures, including dedifferentiated plant cells (such as suspended tobacco, rice, and carrot cells, etc.) and differentiated plant tissue and organ cultures (such as moss and hairy root, etc.) (Holland et al., 2010). An ideal plant cell culture bioreactor system for large scale foreign protein production should exhibit the following features: 1) ease of genetic manipulation by either stable transformation or transient expression, 2) high protein expression level, 3) low endogenous proteolytic activity, 4) high product stability in the heterologous expression environment (inside and outside of the cells), 5) low concentration of secondary metabolites (may cause changes in protein structural and biological properties and/or complicate downstreaming processes), 6) posttranslational modication capability, uniform glycosylation pattern and proper protein folding, and 7) homogeneous dispersion in a bioreactor, etc. Table 1 shows examples of foreign protein expression by stably transformed plant cell culture systems. In this section, the current status and characteristics of different types of bioreactor-based plant cell cultures for foreign protein production will be discussed. 2.1. Foreign protein production in suspended dedifferentiated plant cells Dedifferentiated plant cell suspension cultures, driven by a variety of gene expression systems, are commonly used for foreign protein production (Huang and McDonald, 2009; Lico et al., 2008) due to the fact that they are more amenable to cGMP regulations and can be cultivated and optimized easily in large scale bioreactors (Shih and Doran, 2009). Plant cell suspension cultures are usually derived from stably transformed plant tissues by Agrobacterium-mediated transformation. Callus cells initiated from explants from transgenic plants can be grown in a chemically dened media to establish transgenic cell suspension cultures (Rao et al., 2009). Generally the addition of plant growth regulators is required in the medium to promote rapid cell growth and maintain cell morphology. Recently Protalix Biotherapeutics in Israel and Pzer in the US announced a collaboration to develop and market the plant-made recombinant glucocerebrosidase (prGCD) using a transgenic carrot suspension cell culture bioreactor platform as a biologic therapeutic protein drug for the treatment of Gaucher's disease in EU and USA (Ratner, 2010). This represents an exciting milestone for recognizing plant cell culture-based biomanufacturing as a bio-equivalent and economical alternative to mammalian production of human biopharmaceuticals, further suggesting the possibility of biosimilar products for existing protein drugs. In another promising development, a recombinant animal vaccine against Newcastle Disease Virus (NDV) produced in transgenic tobacco cell cultures by Dow Agrosciences was approved by USDA in February 2006 (Travis, 2008). Both of these represent breakthroughs for developing plant cell culture as a biomanufacturing platform for large scale production of foreign protein products. Protalix Biotherapeutics has developed a transgenic carrot suspension cell culture platform (called ProCellEx) for prGCD production for patients with the genetic disorder Gaucher's disease, a rare lysosomal disease, who are not able to degrade glucosylceramides in the body due to the fact that GCD is absent or nonfunctional (Shaaltiel et al., 2007). Currently, patients are treated with either Ceredase by Genzyme, a

400

Table 1 Selected examples of foreign protein productions by plant cell culture in various bioreactor systems (Huang and McDonald, 2009). Plant species Nicotiana benthamiana Nicotiana benthamiana N. benthamiana Culture type Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Suspension cells Hairy roots Protein product Human AAT Promoter system CaMV 35S (constitutive) Localization Bioreactor system STB, pitched blade impeller STB, pitched blade impeller STB, pitched blade impeller STB, pitched blade impeller STB, pitched blade impeller Membrane bioreactor (CELLine) STB, pitched blade impeller Bubble column Flask Flask Flask Flask Flask Flask Flask Disposable bubble column container Wave-mixed and Undertow bioreactor Wave-mixed bioreactor, fed-batch Flask; air-lift; mist reactor 28 m long tubular photoreactor STB, marine impeller, 500 rpm Operation conditions 25 C, 50 rpm, 40% DO, pH 6.4, batch Production level Ref. Huang et al. (2009) Huang et al. (2009) Huang et al. (2009) Huang et al. (2010) Trexler et al. (2005) McDonald et al. (2005) Schmale et al. (2006) Huang et al. (2005) Hong et al. (2002) Shin et al. (2003) Smith et al. (2002) Ganapathi et al. (2007) Sunil Kumar et al. (2006) Sharp and Doran (2001b) Sharp and Doran (2001b) Shaaltiel et al. (2007)) Girard et al. (2006) Ritala et al. (2008) Liu et al., 2009 (Lucumi and Posten, 2006) Jost et al. (2005)

Secreted, extracellular Human AAT Estradiol inducible XVE system Secreted, (chemical inducible) extracellular Human AAT Cucumber mosaic virus Secreted, inducible viral vector (CMViva) extracellular N. benthamiana Human AAT Cucumber mosaic virus Secreted, inducible viral vector (CMViva) extracellular Oryza sativa (rice) Human AAT RAmy3D (metabolic inducible) Secreted, extracellular O. sativa (rice) Human AAT RAmy3D Secreted, extracellular N. tabacum L. hSA CaMV 35S Secreted, cv. BY-2 extracellular O. sativa (rice) hSA RAmy3D Secreted, extracellular N. tabacum L. hGM-CSF CaMV 35S Secreted, extracellular O. sativa (rice) hGM-CSF RAmy3D Secreted, extracellular Glycine max HBsAg Chimeric (ocs)3mas Intracellular (soybean) (constitutive) G. max HBsAg Arabidopsis ubiquitin Intracellular, ER(soybean) (constitutive) retention Potato HBsAg Ethylene forming enzyme Intracellular (var. Kufri Bahar) gene promoter of banana N. tabacum cv NT-1 Hairy roots mouse mAb IgG CaMV 35S (pMON 530) Secreted, extracellular N. tabacum cv NT-1 Suspension mouse mAb IgG CaMV 35S (pMON 530) Secreted, extracellular Carrot Suspension Human GCD CaMV 35S Intracellular cells (vacuolar signal) N. tabacum L. cv. Suspension Human antirabies Heavy chain by CaMV 35S; Intracellular Xanthi virus mAb light chain by Pin2p Hordeum vulgare Suspension Human collagen I Arabidopsis ubiquitin Intracellular, ERalpha 1 chain retention (HDEL) N. tabacum cv Hairy roots Single chain Double-enhanced 35S Intracellular Xanthi murine IL-12 promoter Moss PhyscomitrProtonemal VEGF CaMV 35S Intracellular ella patens tissue Moss P. patens Protonemal VEGF CaMV 35S; beta-tubulin Intracellular tissue promoter from Physcomitrella

b 1 g-FAAT/L (no pH control), 100 g-FAAT/L (pH control) 25 C, 50 rpm, 40% DO, pH 6.4, batch, b 1 g-FAAT/L (no pH control), 10 M estradiol (single induction) 60 g-FAAT/L (pH control) 25 C, 50 rpm, 40% DO, pH 6.4, batch, 25 g-FAAT/L (without pH control), 10 M estradiol (single induction) 100 g-FAAT/L (pH control) 25 C, 50100 rpm, 40% DO, pH 6.4, SCC, 600 g-FAAT/L (pH control) 1 M estradiol (multiple induction) 75 rpm, 27 C, 70% DO, 0.10.2 vvm, 40110 mg/L, 312 mg/(L-day), two-stage culture 48 mg/(g-DCW-day) 25 C, 130 rpm, two-stage culture 100247 mg/L, 4-10% of TSP (sugar starvation) 26 C, 200 rpm, 20% DO, 0.1 vvm, batch 3.94.2 mg/L 27 C, pH 7, 1.6 vvm, repeated batch 110 rpm, 12 day, 25 C, batch 110 rpm, 13 day, 27 C, batch 120 rpm, 27 C, dark, batch 80 rpm, 16 h light/8 h dark, batch Gyratory shaker, dark for 10 days. 25 C, 100 rpm 25 C, 100 rpm 25 C, 0.25 vvm 25 C 22 C, 0.5 SLPM, 9 rocking angle, 10 rocks per minute. 25 C, 100 rpm (ask); 25 C, 0.1 vvm (AR and Mist) pH 5.8, 22 C, perfusion by CFF with D = 10 or 20/day; pH 4.57, 25 C, semiconscious with D at 0.57/day 76.4 mg/L 105 g/L 129 mg/L, 25% of TSP 22 mg/L 700 ng/(g-FCW) 97.1 ng/g-FCW 1.4 mg/L in 36 days, 1.1 mg/g-DCW 3.6 mg/L in 5 days, 1.2 mg/g-DCW Not available 30 g/g-DCW, 0.5 mg/L 2 to 9 g/L 31 g/L-day (ask), 14 g/L-day (AR), 22 g/L-day (Mist) 360 mg/g-DCW in 30 days 3-folds yield higher than obtained with the CaMV 35S promoter

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

AR: air-lift; BC: bubble column; DO: dissolved oxygen; DCW: dried cell weight; FCW: fresh cell weight; ER: endoplasmic reticulum; D: dilution rate (1/day). STB: stirred-tank bioreactor; CFF: cross-ow ltration; vvm: volume of gas per volume of culture per minute; SCC: Semicontinous culture. TSP: total soluble protein; AAT: alpha-1-antitrypsin; FAAT: functional alpha-1-antitrypsin; hSA: human serum albumin; HBsAg: Hepatitis B surface antigen. GCD: glucocerebrosidase; hGM-CSF: human granulocyte-macrophage colony stimulating factor; Pin2p: potato proteinase inhibitor II promoter.

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

401

form of the native GCD puried from human placental tissue, or Cerezyme by Genzyme, a recombinant human GCD produced in CHO cell culture (Ratner, 2010). However, the puried rGCD from CHO cell culture requires additional in vitro enzymatic reactions to expose the terminal mannose residues of its N-glycan chains to facilitate uptake of the GCD into macrophages. This complex processing makes GCD one of the most expensive therapeutics to date with an annual treatment cost of nearly US $200,000 per patient (Kaiser, 2008). The prGCD expressed in transgenic carrot cell culture from Protalix is fused to the N-terminal signal peptide from Arabidopsis thaliana basic endochitinase and to a Cterminal vacuolar targeting sequence from tobacco chitinase A. Because prGCD is retained in ER (endoplasmic reticulum) during glycosylation modication and then targeted to the vacuole, the N-glycans are trimmed to expose mannose residues and the correct mannose glycosylation pattern can be made within the plant cells. Therefore, the in vitro trimming of the glycans is eliminated in downstream processing, resulting in signicant cost reduction for in vitro protein modication (Shaaltiel et al., 2007). Furthermore, the half-life of prGCD was longer than that of Cerezyme by Genzyme. Currently, prGCD is undergoing a Phase III clinical trial study to evaluate its safety and efcacy in Gaucher patients. 2.2. Foreign protein production in differentiated plant organ and tissue culture Plant cell division can occur either in an unorganized pattern, as in callus growth, or in an organized pattern with the formation of meristematic centers directly in the explant tissues for developing into shoots, roots or somatic embryos. Plant tissue and organ cultures, such as moss and hairy root cultures, are commonly used for secondary metabolite (Zhong, 2001) and foreign protein production (Shih and Doran, 2009). Because the cells of the plant organ or tissue are fully differentiated, the genetic instability observed in dedifferentiated plant cell cultures has not been reported. However, the morphology of plant tissue and organ cultures in a bioreactor may complicate the bioreactor operation and make it difcult to achieve a homogeneous culture environment. 2.2.1. Moss and duckweed in vitro culture The lifecycle of the moss Physomitrella patens, belonging to the Bryophyta phylum, starts with the germination of a spore, followed by the growth of a branched cell lament, the protonema (Decker and Reski, 2004). Although in vitro cultivation of all stages of the moss lifecycle has been established, the protonema is the most vegetative stage for genetic engineering and large scale bioreactor operation for foreign protein production. The moss can be grown in a chemicallydened medium composed of inorganic salts with light and CO2 as the energy and carbon sources in a bioreactor system, providing unique features meeting the requirements for foreign protein production such as fast cell growth, ease of genetic manipulation and transformation, high genetic stability, capability of plant-made glycosylated protein production with non-immunogenic humanized glycan patterns (Gomord et al., 2010; Gorr and Wagner, 2008), and safe foreign protein production without the need for animal-derived compounds. In addition to Protalix using transgenic carrot cell culture for prGCD production, Biolex Therapeutics is another company having a plant culture-produced protein drug (interferon alpha-2b for the treatment of hepatitis C) in late Phase 2b clinical trial to date. Biolex has developed their LEX SystemSM, which uses transgenic aquatic Lemna culture in a bioreactor, for biomanufacturing of follow-on (biosimilar) biologics, hard-to-make therapeutic proteins and monoclonal antibodies that are expected to enhance their efcacy and potency compared to those made in mammalian cells (Ratner, 2010). 2.2.2. Hairy root in vitro cell culture Transformed hairy roots of many plant species have been investigated as an alternative plant cell bioreactor system to plant

cell suspension culture for both valuable secondary metabolites (Giri and Narasu, 2000) and foreign protein production (Georgiev et al., 2007; Shadwick and Doran, 2007). Hairy roots are neoplastic roots derived from plant roots transformed by the integration of the genes of Ri plasmid of Agrobacterium rhizogenes into the host plant genome, resulting in the active proliferation of hairy roots (Chilton et al., 1982). A unique advantage for large scale cultivation is that hairy roots can be propagated indenitely in liquid medium and retain their morphological integrity and stability in the absence of exogenous plant growth regulators. Studies have shown that hairy root culture has signicantly improved long-term genetic and biosynthetic stability compared to suspended plant cells for the production of foreign proteins (Sharp and Doran, 2001b). Shadwick and Doran (2007) used wild type hairy roots as an in vitro culture system for the propagation of plant viruses, suggesting the feasibility of transient plant viral-based expression in hairy roots. 2.3. Characteristics of in vitro plant cell cultures Plant cells exhibit attractive features for foreign protein production; however, their biological, morphological and cellular physiology characteristics, which are distinctive from microbial and mammalian cell cultures, need to be considered for their scalability in production. Table 2 summarizes the characteristics of in vitro plant cell cultures as practical considerations when developing a bioreactor-based operation for foreign protein production, including cell growth, oxygen demand, morphology and aggregation, culture rheological property and shear sensitivity of plant cell cultures, etc. The critical cell culture conditions, which impact foreign protein productivity and quality, and host cell growth, need to be optimized and controlled during bioreactor operation. 3. Bioreactor systems for in vitro plant cell cultures Different bioreactor designs offer unique features for providing an optimal environment for effective cell growth and foreign protein production under different circumstances. To design, select and operate a suitable bioreactor for foreign protein production, one needs to consider the characteristics of in vitro plant cell cultures (Table 2) and target protein properties including gene expression vector (constitutive or inducible promoter system), product localization and innate stability, etc. Bioreactor systems available for microbial and mammalian cell culture and for plant cell cultures for medicinal metabolite production and micropropagations can be implemented for the production of foreign proteins in plant cell cultures. In addition, the scalability and sustainable conditions of the bioreactor system which provide adequate mixing and mass transfer while minimizing the shear stress intensity to plant cells need to be considered for large scale foreign protein production. In this section, the technological progress in bioreactor systems for in vitro plant cell cultures will be discussed. Table 3 summarizes important features of various bioreactor systems for in vitro plant cell cultures. 3.1. Reusable bioreactor systems Reusable bioreactor systems, in which cultivation vessels are made of stainless steel with a standard or modied conguration, requiring sterilization-in-place (SIP) and clean-in-place (CIP) systems, are commonly utilized in the industry for large scale mammalian cell culture and microbial fermentation, with scales that range from 1,000 L to 25,000 L in working volume. 3.1.1. Stirred-tank bioreactors Stirred-tank bioreactors (STB) equipped with suitable impellers can provide high volumetric mass transfer coefcients and a homogeneous environment enabling suspended plant cell growth and foreign protein

402

Table 2 Important characteristics of in vitro plant cell cultures for bioreactor system selection and operation. Characteristics Plant cell growth Practical considerations for bioreactor selection and operation

Plant cell oxygen demand

Plant cell aggregation and size distribution

Rheological properties

Shear stress sensitivity

Foaming and wall growth

pH for cell growth Temperature for cell growth

Shows varied cell growth kinetics in bioreactor (doubling time, td, of 1296 h). Tobacco BY-2 cells exhibit a shorter td (12 h), N. benthamiana and rice cells expressing AAT in a STB have td of 3 days (Huang et al., 2010) and 2 days (Trexler et al., 2002), respectively. Potato hairy root expressing HBsAg exhibits td of 2.5 days (Sunil Kumar et al., 2006). Typical OUR is about 210 mmol-O2/(L-h). Specic OUR is reported as 0.8 mmol-O2/(g-DCW-h) for rice cell cultures expressing human AAT and 0.30.5 mmolO2/(g-DCW-h) for NT-1 cell cultures expressing GUS. Typical volumetric oxygen mass transfer coefcient (kLa) required is between 10 and 50 h 1 (Curtis and Tuerk, 2006), suggesting a critical dissolved oxygen concentration of 1.31.6 g/m 3 (i.e. 20% of air saturation) (Doran, 1999). Inadequate oxygen mass transfer may inhibit cell growth and reduce foreign protein production (Sharp and Doran, 2001a). Dependent on plant species, inoculum preparation, growth stage, medium composition, bioreactor types and operations. Cell aggregates promote cellular organization and differentiation, leading to oxygen, nutrient or inducer inhomogeneities inside large cell aggregates and resulting in adverse effects on foreign protein production (Kieran, 2001). Cell aggregates complicate the bioreactor operation and make cell aggregates susceptible to shear stress, resulting in cell damage, attributed to aggregate surface attrition (Kieran et al., 2000) and aggregate shattering (Namdev and Dunlop, 1995). Affected by cell aggregate size and morphology, biomass concentration, cell growth stage and culture conditions. Plant cells tend to transition from spherical to elongated shapes when cell division is terminated (Cosgrove, 1997), leading to high PCV at a given DCW and exhibit non-Newtonian uid behavior during cultivation (Su and Arias, 2003). Plant cells exhibit Newtonian rheological behavior and do not elongate (cells remained nearly spherical in shape) when growing in semicontinuous culture. Typical apparent viscosity of cell culture broth is 4150 cP. Susceptible to shear stress when the cells are of relatively large size and contain large vacuoles (Dunlop et al., 1994). The cellular responses to stress include changes in cell viability and metabolism (OUR, mitochondrial activity, ATP level, cell wall composition, calcium ions in cytoplasm) and changes in morphology and aggregate sizes (Kieran et al., 2000). Reducing stress by lower impeller speed leads to poor mixing and oxygen supply in high viscosity cell culture and enhances the clumping of cells into aggregates of varying sizes. Critical shear stress is between 50 and 200 N/m 2 (Kieran et al., 1997). Cells entrapped in the foam are subjected to nutrient and oxygen deciency, resulting in cell growth and productivity reduction, and generate a thick layer that adheres to the reactor wall, impeller shaft and sensors, hindering the bulk mixing. Reducing the foaming by 1) reducing agitation speed and aeration rate without impacting mixing intensity and mass transfer, 2) adding antifoam reagents, 3) applying surface aeration or bubble free-aeration, and 4) using a foam breaker. Medium pH is adjusted to the range of 5.0 to 5.9 before sterilization and extremes of pH are avoided in cell culture. The effects of pH on the stability of secreted foreign protein in extracellular medium need to be investigated. Temperature is maintained between 20 and 28 C for induction of callus tissues and growth of plant cells. Different temperature setpoints during the in cell growth and protein production phase can be applied to achieve higher product titers. Temperature and pH gradients and variations due to inhomogeneous mixing in bioreactor may impact product quality.

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409 Table 3 Comparisons of bioreactor systems for foreign protein production by plant cell cultures (relative evaluation between systems). Bioreactor Features Stirred-tank bioreactor (STB) Bubble column Air-lift bioreactor Fix-bed or TIB Single-use STB Wave-mixed bioreactor Membrane bioreactor Microbioreactor

403

kLa (OTR) Cell damage by agitation Cell damage by aeration Mixing time Operation Flexibility CIP/SIP Scale size Scale-up Power consumption Culture type Cell density Productivity Monitoring and control Operation cost Capital cost Ease of GMP compliance

High High Medium Short Medium High Yes Commercial Medium High Susension; microcarrier Low/medium Medium Direct, easy, multiple High High Yes

Low Low High Long Simple High Yes Commercial Easy Low Suspension; microcarrier Medium Low Direct, easy, multiple Medium Medium Yes

Medium Low High Medium Simple High Yes Commercial Easy Low Suspension; microcarrier Medium Medium Direct, easy, multiple Medium Medium Yes

Low Low Low Long-medium Complex Low Yes Pilot Complex Medium Immobilized; immersion High Low-medium Indirect, complex, limited Medium Medium Difcult

Low-medium Medium Medium Medium Medium Medium No Pilot (2,000 L) Medium Medium Suspension; microcarrier Low/medium Medium Direct, easy, limited Low Low Yes

Medium Low Low Medium Medium Medium No Pilot (1,000 L) Complex Medium Suspension; microcarrier Medium Medium Direct, medium, limited Low Low Yes

Low Low Low Medium Medium Low No Pilot Complex Low Immobilized; immersion High High Indirect, complex, limited Low Low Difcult

Medium Low Low Short Simple Low No Lab. Complex Low Suspension Medium Medium Direct, complex, limited Low Low Not available

production to be controlled consistently. Although Rushton turbines (resulting in a radial ow pattern) can provide complete plant cells and gas dispersion, they also induce high turbulence around the impeller region and exhibit higher specic power input than other impellers with axial ow patterns (such as marine impellers, helical ribbon impellers, paddle impellers, and pitched-blade impellers), resulting in higher shear damage to plant cells. The pitched-blade or marine impeller with the upward-pumping mode offers similar capabilities, compared to the Rushton impeller, for cell aggregate dispersion while reducing shear stress to plant cells when the power input needs to be restricted due to cell damage considerations (Doran, 1999). However, the oxygen mass transfer performance of an upward-pumping pitch-blade impeller was poor in highly viscous cultures (Kieran, 2001), compared with that of the same impeller operated in the downward-pumping mode (Junker et al., 1998). Generally, impeller systems exhibiting axial ow patterns with low tip speed (up to 2.5 m/s) are considered suitable for plant cell cultures (Amanullah et al., 2004). Recent advances continue to reduce cell damage from hydrodynamic shear stress by agitation and from gas bubble rupture as it enters the headspace through 1) developing new impellers and modifying existing impellers to provide more efcient mixing at lower impeller tip speeds, 2) designing new aeration systems (such as bubble-free aeration, gas basket, and cage-aeration) to reduce shear caused by bubbles, and 3) addition of appropriate protective agents, etc. New types of impellers have been developed for shear-sensitive cell culture processes including curved-blade disk turbine, hydrofoil impeller, centrifugal impellers and cell-lift (Doran, 1999). A number of lowpower-number impellers such as Intermig, Prochem Maxow and Scaba designed for mammalian cell culture can be implemented for plant cell culture (Varley and Birch, 1999). In addition, the bioreactor geometric specications such as impeller diameter, spacing between impellers, impeller off-bottom clearance, bafes and their width, sparger type and position, ratio of liquid height to tank diameter, and impeller number are also critical for large-scale bioreactor performance to provide sufcient mixing and adequate mass transfer.

Phyton Biotech in Germany operates the world's largest cGMP plant cell culture facility using stainless stirred-tank bioreactors up to 75,000 L to produce the taxanes and paclitaxel (up to 880,000 L/year production capacity for taxanes) and has been an API (active pharmaceutical ingredient) supplier for Bristol-Myers Squibb's Taxol oncology product. Phyton Biotech has an agreement with Insmed, Inc., in which Phyton Biotech utilizes their cGMP plant cell culture facility to manufacture the IPLEX (mecasermin rinfabate), approved by FDA in December 2005 for the treatment of growth failure in children with primary insulin growth factor-1 deciency or with growth hormone gene deletion. Phyton Biotech has made promising progress toward achieving a product yield of 2 g protein/(L-broth) (Lukjan et al., 2007). 3.1.2. Pneumatic bioreactors The pneumatic bioreactor (bubble column and air-lift) consists of a cylindrical vessel in which compressed air or gas mixture is introduced at the bottom of the vessel through a sparger for aeration and mixing. The features of low capital and operational cost, no moving mechanical parts and sealing elements, and ease of scale-up are advantageous for large scale plant cell cultures. The low shear stress in pneumatic bioreactors is desirable for shear-sensitive plant cells (Eibl and Eibl, 2008). Bubble columns are often used for organogenic micropropagations, however, they are unable to provide homogenous mixing and are less applicable to high biomass cultures due to the lower gas-liquid interfacial area resulting from bubble coalescence in the viscous cultures and lack of mechanical break-up of bubbles. The performance of a bubble column can be improved by installing multiple spargers in different segments for delivering oxygen into the zones of high cell density hairy root cultures in the column (Mishra and Ranjan, 2008). Air-lift or modied air-lift bioreactors containing a draft tube (internal or external loop) exhibit improved features: 1) preventing bubble coalescence by directing them in one direction; 2) enhancing oxygen mass transfer by increasing the number of bubbles and gasliquid interfacial area for mass transfer; 3) distributing shear stress more evenly and lowering shear stress (Mishra and Ranjan, 2008); and 4) promoting

404

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

the cyclical movement of the medium and cells resulting in shorter mixing times (Huang et al., 2001b; Wang et al., 2002). For high cell density cultures, however, the issues of inadequate oxygen mass transfer and poor mixing leading to inhomogeneities in biomass, nutrient, oxygen and pH, and extensive foaming (resulting from polysaccharides, proteins, fatty acids, and high supercial gas velocity) are still limiting factors in bioreactor operation (Tanaka, 2000). Furthermore, uneven distribution of high cell density cell cultures or hairy root tissues at certain positions and excessive gas-phase channeling due to high aeration rate may cause clumping of root tissue and blockage of liquid ow. 3.1.3. Fixed-bed bioreactors Fixed-bed (or packed-bed) bioreactors are frequently utilized for immobilized plant cell, differentiated plant tissue or organ systems. For the application of cell cultures, cells are immobilized in appropriate microcarriers (Gilleta et al., 2000; Medina-Bolivar and Cramer, 2004), which are packed in the xed zones, resulting in high solidliquid specic interfacial contact areas. A medium reservoir is used to circulate the oxygenated nutrient medium through the bed and the spent medium containing the product that returns to the reservoir can be harvested in batch or continuous mode. The major disadvantages of the xed-bed bioreactors are their relatively poor mass and heat transfer capability due to low liquid velocities. Fixed-bed bioreactors have shown applications in perfusion animal cell cultures (Meuwly et al., 2007), where cells are immobilized within microcarriers and therefore they are less sensitive to shear force than in suspension culture. Although the microenvironment created for the immobilized cells within the carrier matrix are different from that in the bulk liquid, with the appropriate selection of carriers for cell immobilization, high cell density and high productivity can be achieved. 3.1.4. Rotary drum bioreactors The rotary drum bioreactor, having moderate surface area to volume ratio compared with other bioreactor types, can provide adequate oxygen mass transfer with less power consumption and shear stress compared with STB. For plant cell cultures, the rotary drum bioreactor has shown advantages in terms of suspension homogeneity, low shear environment and reduced wall growth, over either air-lift or STB (Sajc et al., 2000). However, the limitation is their comparatively high energy consumption in large scale operations and difculty in scale-up. 3.2. Disposable bioreactor systems (single-use technology) Disposable bioreactors, in which the cultivation vessel is made of a single-use plastic bag or container, provide very attractive features for the biomanufacturing production of foreign proteins especially for timeand cost-savings from exibility for small to medium scale operation, easy handling, not requiring CIP and SIP (pre-sterile disposable bioreactor bags will be discarded after product harvest), simple facility design and set-up (modular manufacturing design), reduced WFI requirements, ease of validation, less capital investment for stainless steel vessels, reduced turnover time between each run, and the potential use of integrated processes for more robust processes with shorter development time and increased throughput (Eibl et al., 2009b; Eibl et al., 2010a; Hacker et al., 2009). The chance of cross-contamination between each process run can also be minimized by using disposable bioreactor systems, suggesting the possibility of multi-product manufacturing in one suite. The recent commercialization of disposable bioreactors, such as the Sartorius Stedim Biostat CultiBag STR, Xcellerex XDR, Hyclone SUB (Single-Use Bioreactor), and GE WAVE bioreactor, etc., offer the prospect of reducing the use of bench- and pilot-scale stainless steel bioreactors and have been successfully implemented into preclinical, clinical, and production-scale biomanufacturing facilities (Eibl et al., 2010b). The current limitations on the use of disposable bioreactors include the insufcient plastic material strength in scalability and the availability of reliable, disposable sensors and peripheral

elements such as valves and sampling systems, etc.(Eibl et al., 2010b). In addition, the most discussed concern for using disposable bioreactors in biopharmaceuticals production are the extractables and leachables which may interact with or contaminate the product for human therapy (Eibl et al., 2010a). 3.2.1. Disposable standard bioreactors To date, disposable stirred-tank bioreactors are available from companies such as Sartorius, HyClone, and Xcellerex that manufacture single-use bioreactors with stirred-tank congurations, although these bioreactors are still limited in volume and mostly used for seed expansion and inoculation of large conventional bioreactors. The disposable stirred bioreactor bag, up to 2,000 L, equipped with gas inlet and exhaust lter and sensor ports for pH, DO, and temperature are contained within a stainless steel outer shell with cooling and heater jacket to providing the temperature control. The mixing and mass transfer capacity is achieved by a sterile, disposable impeller which is built into each plastic bag vessel. However, oxygen mass transfer limitation, wall growth and cell otation are observed in high cell density plant cell suspension cultures in disposable stirred bioreactors, partly due to increased culture broth viscosity, stable foam layers and the microsparger used for aeration (Raven et al., 2010). Disposable pneumatic bioreactors, such as bubble columns, are commonly used for biomass propagation for plant regeneration for transfer to the greenhouse (Raven et al., 2010). Protalix utilizes a disposable, bubble column-type bioreactor (400 L working volume), which consists of a sterilizable polyethylene bag lled with plant cells and medium, for the prGCD production in transgenic carrot cell culture (Shaaltiel et al., 2008). In addition, the Slug Bubble bioreactor, designed to generate intermittent large and long cylindrical single bubbles from the bottom of the bag by varying the air inlet pressure, valve opening time and bubble frequency, has been demonstrated to exhibit comparable kLa to values in STB and has been implemented for the cultivation of tobacco BY-2 and soy suspension cells (Ducos et al., 2009; Terrier et al., 2007). The orbitally shaken single-use bioreactor (such as the Disposable Shaken Bioreactor from Nalgene) consists of a cylindrical vessel mounted on a circular moving shaker platform and a disposable, sterile plastic bag with appropriate connection tubes for seeding, feeding, gas supply and harvesting (Micheletti et al., 2006b). The shaking diameter, shaking speed, working volume and smoothness of the cultivation container can be adjusted to achieve adequate oxygen mass transfer. Studies have demonstrated that disposable shaken bioreactors can provide sufcient oxygen mass transfer, negligible foaming and lower shear stress (due to homogeneous distribution of the power consumption) for the growth of plant and animal cells (Eibl et al., 2010c). 3.2.2. Wave-mixed bioreactors Wave-mixed bioreactors possessing the advantages of low-cost and low shear stress utilize a non-gas permeable sterile bag composed of a plastic lm which contains host cells and medium. The bags are lled with medium and cell suspension up to 5060% of their total capacity (Eibl et al., 2009a). The mixing, mass and heat transfer in the wave-mixed bioreactor are characterized by rocking rate, rocking angle, bag type and its geometry and culture working volume. Oxygen is supplied from the air or gas mixture continuously through headspace aeration. While the wave-mixed bioreactor is rocking, the liquid surface of the medium in the bag is continuously renewed and bubble-free surface aeration takes place resulting in oxygenation and bulk mixing with less shear stress to cultivated cells. Additional advantages include reduced foaming, easy operation and low risk of contamination. Notably, the shear stress is highest in the wave-mixed bioreactor when the bag is rocking with minimum lling level at maximum rocking angle and rocking rate. The wave-mixed bioreactor can be operated in different culture modes, including batch, fed-batch and perfusion when combined with different cell-retention devices such as oating lters. On-line measurements of pH, DO and other sensing technology (Read et al., 2009) make the wave-

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

405

mixed bioreactor and other disposable bioreactors highly attractive for plant cell cultures (Eibl et al., 2009a). Three commercial wave-mixed bioreactors are currently used including WAVE Bioreactor (GE Healthcare, up to 1,000 L total volume), BIOSTAT CultiBag RM (Sartorius Stedim, up to 100 L working volume) and AppliFlex (Applikon, up to 25 L working volume). Investigations have demonstrated the application of the WAVE bioreactor for cultivating tobacco, grape and apple suspension cells up to 500 L working volume (Raven et al., 2010). Eibl et al. achieved high plant cell (V. vinifera) biomass productivities of 40 g-FCW/(L-day) with a doubling time of 2 days and observed that there was no signicant change in cell morphology when compared to cultivations in STB (Eibl and Eibl, 2006). The wave-mixed bioreactors have been implemented for the production of human collagen (Ritala et al., 2008) and human antirabies virus mAb (Girard et al., 2006). However, the increase in culture broth viscosity (more than 70 times) and high PCV (N 60%) during suspended plant cell cultivation may lead to inhomogeneous mixing in wave-mixed bioreactor. Other wave-mixed bioreactors, such as Wave and Undertow bioreactor (Terrier et al., 2007) and the ebb-andow bioreactor (EFBR) (Mishra and Ranjan, 2008), designed for micropropagations and secondary metabolite production can be implemented for foreign protein production. In addition, a culture bag integrated with matrix supporting the plant tissue has been used for hairy root-based cultivation (Eibl and Eibl, 2006). 3.2.3. Immersion bioreactors The temporary immersion bioreactor (TIB), consisting of two vessels (one for holding the plant tissue cultures and another for the liquid medium), was developed to allow cycling of the culture medium by using air pressure or a pump to push the medium from one vessel to the other to immerse the plant tissues and using gravity to withdraw the medium, thus the plant tissues or immobilized plant cells are exposed to the medium intermittently rather than continuously. A separate air or gas mixture is introduced through a sparger to aerate the plant cell or tissue cultures. TIB provides attractive advantages including adequate oxygen transfer and low shear stress to plant tissues (such as hairy root culture) due to the lack of mechanical agitation, although some limitations need to be addressed including vessel size at commercial scale, disposability, and insufcient mixing leading to the accumulation of inhibitory metabolites that can affect cell growth (Ducos et al., 2009). In addition, a modied TIB, consisting of a rigid box placed inside a transparent plastic bag, called a box-in-bag TIB provides culture headspace between the immersion periods and allows horizontal distribution of biomass for better oxygenation and illumination than that in TIB or other type of immersion bioreactor (Ducos et al., 2009). Ports located above the bag are used for inoculation and outlet gas and ports positioned below the bag are used for inlet air and medium exchange. The box-in-bag TIB also offers the mass cultivation of in vitro plant cell culture under photoautrophic conditions. TIB are considered an appropriate bioreactor system for large scale hairy root cultures. 3.2.4. Membrane bioreactors Membrane bioreactors are designed to retain host cells and foreign protein products in a cell compartment by utilizing specialized membranes with specic molecular weight cut-off (MWCO) for either in situ aeration, nutrient supply, or product separation (Qi et al., 2003). The culture medium is circulated in the membrane bioreactor for bringing oxygen and nutrients to the cells and removing the waste metabolites. Membranes can be packed into different geometries including plate-and-sheet, tubular, spiral-wound, and hollow-ber modules. The main features of using membrane bioreactors in plant cell cultures are high cell density and high protein volumetric productivity, resulting from the use of membranes that retain the secreted foreign protein in the cell compartment, concentrating the product before harvest. Another feature is that the shear stress-induced cell damage found in STB can be minimized in a membrane bioreactor because the

cells are retained in a relatively quiescent region in which cells are protected from mechanical damage and are not in direct contact with gas bubbles. McDonald et al. (2005) applied a membrane bioreactor, CELLine CL 350, for the production of human alpha-1-antitrypsin (AAT) using transgenic rice cell culture with a rice alpha amylase promoter, Ramy3D, which is activated under sugar starvation conditions, resulting in extracellular product titer up to 250 mg/L and 410% of the extracellular total soluble protein. However, large scale operation in membrane bioreactors may cause operation problems resulting in poor cell viability, poor process stability, product heterogeneity, membrane fouling, and diffusion gradients (heat and mass transfer). Therefore the membrane bioreactor is mainly implemented for small scale processes and is difcult to scale up for large scale applications, although it may be useful for smaller scale applications such as patient-speci c therapeutics. 3.2.5. Microbioreactors The microbioreactor is designed as a high-throughput platform for cell line selection and evaluation, bioprocess characterization (design space determination), media design and optimization (Betts and Baganz, 2006; Diao et al., 2008), and as a scaled-down model to represent the production bioreactor for bioprocess scaling-up purposes (Micheletti et al., 2006a). Microbioreactor platforms including microtiter plates (6, 12, 24, 96, with up to 384 wells with a few microliter to milliliter volumes), spin tubes (5 50 mL), shake asks (251000 mL), and parallel miniature stirred and bubble column bioreactor systems (Betts and Baganz, 2006) have been implemented for cultivation of many different host cell lines. Feeding, sampling, and harvesting can be automated by using a liquid handling system with an automation control system that can be programmed. Recently the optical sensing systems based on noninvasive process analytical technology have been used for on-line measurements of pH, dissolved oxygen, and optical density in a microbioreactor (Zhiyu et al., 2007). Applikon 24-microreactor system is a type of 24 deep-well plates with on-line pH, DO and temperature monitoring and control options. However, the scaled-down microbioreactor may not be able to accurately mimic the large-scale bioreactor process conditions (such as gradients of dissolved oxygen concentration, temperature, pH, nutrients, and shear stress) due to the differences in heterogeneous microenvironments across varied scale bioreactors. The limitations of oxygen transfer and process control of the operational parameters inherent to the microbioreactor typically only allow for small- or medium-scale process development. 3.3. Special considerations for moss bioreactor systems Moss can grow in a simple medium of inorganic salts in constant low light or daynight cycles, therefore, a photobioreactor is the most common vessel for moss in vitro cultivation. Small scale moss cultivation can be performed in a glass ask or a controllable photobioreactor, either in a stirred-tank glass bioreactor or tubular glass bioreactor, with working volumes up to 30 L. Although glass stirred-tank and air-lift bioreactors have been modied for moss in vitro cultivation, they have been limited to small scale production because of light limitation due to mutual cell shading at moderate and high cell density, resulting in scale-up complexity. Currently, the modular 100 L tubular photobioreactor and 200 L disposable wave-mixed bioreactor systems have been established for foreign protein production by moss cell culture by Greenovation Biotech GmbH in Germany (Decker and Reski, 2007). The modular structure of bioreactors for moss in vitro culture allows easy scale up by operating several bioreactors in parallel. Moss cell differentiation and growth can be tightly controlled by distinct parameters in a photobioreactor for high cell density cultures in batch, semicontinuous and continuous operation (Lucumi and Posten, 2006). Adequate mechanical stress to the cell laments may prevent plants from developing adult tissues in a bioreactor. Although the simple growth medium makes moss in vitro culture a very cost-effective bioprocess compared to other mammalian cell cultures, the scalability of

406

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

photobioreactors and relatively complex bioreactor operation (critical considerations including light intensity, darklight cycles, circulation rate in tubular photobioreactor, etc.), are currently a challenge that limits the use of moss cultures for large scale production of foreign proteins.

systems for hairy root cultures at a small scale (b 10 L) were also discussed previously (Mishra and Ranjan, 2008). 4. Bioreactor operation mode optimization The advanced bioreactor operations developed for mammalian and microbial systems can be implemented for plant cell cultures. One important aspect in developing a robust plant cell culture process is to establish bioreactor operating conditions and operation ranges while considering the characteristics of the in vitro plant cell culture. Although the optimal conditions and operation mode will vary for different plant species and culture media, important considerations are discussed in this session. 4.1. Constitutive expression of foreign proteins in plant cell culture bioreactors For the growth-associated foreign protein production (secreted or intracellular product) driven by a constitutive promoter, target protein productivity can be enhanced by increasing cell density in the culture and prolonging the exponential active cell growth phase. Fed-batch cultures have been applied to achieve high cell density when utilizing an effective substrate feeding strategy (Suehara et al., 1996). However, the accumulation of toxic or inhibitory metabolites during fed-batch cultures might limit foreign protein productivity. Therefore, a perfusion culture with a cell retention device (such as an acoustic cell lter, ATF (alternative tangential ow), and spin lter, etc.) can be an alternative to obtain high cell density culture and to continuously withdraw cultured medium (De Dobbeleer et al., 2006; Lucumi and Posten, 2006; Su and Arias, 2003). On the other hand, the cell growth rate in a batch or fed-batch plant cell culture is usually retarded when the PCV reaches about 6070%, resulting in a reduction of cellular metabolic activity (Maccarthy et al., 1980). Therefore, although fed-batch and perfusion culture can be applied to increase cell density, semi-continuous culture or perfusion culture with a bleed stream has been demonstrated to be more appropriate for high cell density culture due to plant cell aggregation, surface adhesion, and the high apparent viscosity observed at high biomass concentration (De Dobbeleer et al., 2006). 4.2. Inducible expression of foreign proteins in plant cell culture bioreactors For foreign protein production driven by an inducible promoter, twostage cultures are typically implemented to allow the cell growth and protein production phases to be independently optimized. The bioreactor operations for the induction phase (protein production phase) are highly dependent on the type of inducible promoter used (Huang and McDonald, 2009). The sugar-starvation Ramy3D promoter system (metabolically regulated) has been investigated to express foreign proteins including human AAT (Huang et al., 2001a) and hGM-CSF (Shin et al., 2003) in transgenic rice cell cultures. In these studies, a media exchange to a sugarfree medium or medium containing an alternative carbon source (Terashima et al., 2001) for inducing foreign protein production was applied at an appropriate time in the growth phase. During the induction phase without carbon source supplementation, however, the cell viability was signicantly decreased, resulting in increased protease activity. A cyclical semi-continuous process, which alternates between growth and production phases, has been developed to reuse the transgenic rice cells for long-term operation (Trexler et al., 2005). For a chemically inducible system, the timing of induction and concentration and mode of inducer addition (single, multiple or continuous induction) applied to plant cell cultures are important for optimizing inducible plant cell culture bioreactor operation. This will need to be investigated based on the nature of the inducer (inducer stability and toxicity) and plant species (cell growth rate and aggregation tendency) for enhancing foreign protein expression. Higher inducer

3.4. Special considerations for hairy root bioreactor systems The morphology and culture characteristics of hairy root cultures grown in a bioreactor are quite different from those of suspended plant cells. The root thickness, root length, number of root hairs and root branching frequency are affected by plant species and the Agrobacterium strain used for hairy root induction. These characteristics have led to the design of alternative bioreactor congurations. Special considerations need to be taken into account for bioreactor selection for hairy root in vitro culture such as a low shear stress environment, adequate oxygen mass transfer and nutrient availability in high dense packed root cultures. The growth of hairy roots in liquid medium results in dense and packed root biomass, inducing the formation of stagnation and becoming an inhibitory role in uid circulation and limiting oxygen availability. Furthermore, the non-homogeneous cell growth and product production of hairy roots in a bioreactor may complicate the process optimization. Georgiev et al. indicated that the tendency to form clumps composed of primary and bridged roots is the main concern for hairy roots grown in a bioreactor (Georgiev et al., 2007). The traditional STB is not applicable for the mass production of hairy roots since high shear stress is generated by the impeller, causing wound response and callus formation. Standard pneumatic and liquid-dispersed bioreactors (such as trickle-bed and mist-trickling reactor) are modied for improving hairy root cultures because of the low shear stress and the simplicity of their design and construction (Mishra and Ranjan, 2008), although some limitations are still observed. Immobilization of hairy roots on anchor matrices (such as metal trays and meshes) have been demonstrated to promote root growth in modied STB, bubble column and rotary drum bioreactors, where the roots are immersed in the culture medium (Shadwick et al., 2005). To prevent root damage from shear stress by the impeller, the isolation of roots from the impellers in STB is necessary. However, insufcient mass transfer for oxygen and nutrients due to gentle mixing may inhibit root growth. To overcome the oxygen mass transfer limitation, hairy roots can be grown predominantly in spray or droplet bioreactors and mist bioreactors, where the roots are immobilized on mesh support and exposed to humidied air or a gas mixture and nutrients are supplied as small droplets by spray nozzle, resulting in high growth rate and biomass (Gupta et al., 2006). Liu et al. (Liu et al., 2009) compared the root growth and murine interleukin 12 (mIL-12) production by N. tabacum hairy roots in three different bioreactors including shake asks, air-lift bioreactors, and mist bioreactors, and found that roots are not homogeneously distributed and formed a dense ring around the wall of the air-lift reactor. Liu et al. indicated that the total mIL-12 production in the mist bioreactor was about 50% more than that in the air-lift bioreactor. However, the challenge of uniform loading of the root anchor matrices may limit the scalability of hairy root culture in mist or spray bioreactors. The roots can be mechanically chopped and pumped as a slurry into the reactor to eliminate the need for manual labor for cutting, inoculation and homogenously loading in large scale operations. Gupta et al. introduced a commercial ginsenoside production process using hairy roots in a 20,000 L balloon-type bioreactor as a model for developing hairy root based manufacturing process for foreign protein production (Gupta et al., 2006). Disposable bioreactors, such as wave-mixed bioreactor or TIB, are also suitable for growing hairy roots with stable morphological characteristics in large-scale operation. Furthermore, bioreactor systems integrated with hairy root biomass harvest systems for following downstream processing need to be considered. Other bioreactor

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409

407

concentrations and multiple or continuous application may benet high cell density operational modes. Semi-continuous/continuous or perfusion bioreactor operation at high cell density with slower specic growth rate for a prolonged protein production phase can be benecial for inducible production of the foreign protein and the secreted foreign protein can be continuously harvested from the cell culture broth. Our results suggested that oxygen uptake rate (OUR) is an important physiological parameter to determine the optimal timing of induction (TOI). In the case of a chemically inducible, estrogen receptor-based promoter (XVE) system in tobacco cell culture, the optimal TOI occurs at the maximum OUR which occurs at the end of the exponential phase (Huang et al., 2010). We further developed the semicontinuous culture production of human AAT using a chemically inducible plant viral vector in transgenic tobacco cell culture, leading to ve-fold increase in volumetric productivity of biologically functional AAT compared with batch operation (Huang et al., 2010). 4.3. Strategies to enhance foreign protein stability Foreign protein stability in a heterogeneous expression environment is a critical consideration. Foreign proteins expressed in plant cell cultures can be secreted to the extracellular culture medium or retained in an intracellular compartment such as ER, cytoplasm, or vacuole. From a costeffective bioprocessing standpoint, secreted products offer advantages over intracellular products. However, secreted foreign proteins are commonly degraded by proteolytic enzymes that are produced during plant cell cultivation or results from cell death/lysis (Doran, 2006a) and/or may be unstable in the simple plant cell culture medium (Tsoi and Doran, 2002). To improve the stability of secreted foreign proteins in plant cell culture process, investigations (Benchabane et al., 2008; Doran, 2006b) have used protein stabilization agents or protease inhibitors, such as gelatin, BSA and other low-value proteins (for protein-based stabilization), mannitol (to regulate the osmotic pressure of the medium to minimize cell lysis), PEG, Pluronic F-68 and other polymers (as stabilizing agents for protection of the protein product from denaturing agents liberated from the plant cells into the medium). These approaches have been shown to decrease the degradation or loss of the foreign protein through different mechanisms, however, protease inhibitors have short lifetimes and are expensive for large-scale application, and the use of stabilizing agents could exhibit negative effects on cell growth and increase downstream processing costs. Huang et al. (Huang et al., 2009) proposed a bioreactor strategy involving pH control for improving functional foreign protein production in transgenic tobacco cell culture, which can enhance foreign protein stability and minimize proteolysis effects in cell cultures, demonstrating as an effective alternative to adding protease inhibitors or protein stabilizing agents to plant cell culture. Other approaches for reducing proteolytic effects on foreign proteins include 1) co-expression of protease inhibitors hindering endogenous protease activities along the cell secretory pathway or released into the culture medium (Komarnytsky et al., 2006), 2) suppression of protease gene expression using RNAi (Kim et al., 2008), 3) the development of specic protease-decient host cells (Schiermeyer et al., 2005), 4) insitu protein recovery (by adding resins into the culture or circulating the culture through a chromatography column external to the bioreactor) (James et al., 2002; Sharp and Doran, 2001a), and 5) immobilization of plant cells to facilitate recovery of secreted protein from the culture broth (Osuna et al., 2008). The capability of strategies for minimizing foreign protein degradation in plant cell cultures has to be evaluated on a case-by-case basis. 5. Opportunities for improving foreign protein production in plant cell culture Plant cell cultures as a bioproduction platform provide unique advantages for foreign protein production. Future directions for improving foreign protein yield and quality in plant cell culture

bioreactor system include: 1) selection of plant hosts (plant species and dedifferentiated or differentiated plant cells), gene expression system (constitutive promoter, inducible promoter, viral amplicon or inducible viral amplicon) and gene of interest, 2) generation and selection of the most productive cell lines, 3) medium design and optimization, 4) enhancing foreign protein stability and preventing proteolytic degradation, 5) selection of bioreactor systems and scale-up strategies (standard stainless steel or disposable bioreactors) according to the interactions between host cells, product formation and bioreactor design, and economical considerations to meet the market demand, 6) optimization of bioreactor conditions for cell growth and protein production, 7) incorporation of gene silencing suppressors, 8) development of scalable transient expression in plant cell culture, 9) adaptation of single-use technology, 10) long term cryopreservation for master cell and working cell banks of the production line, and 11) engineering humanized plant-made glycosylated proteins. 6. Conclusions The technological progress in bioreactor systems and operations for foreign protein production using in vitro plant cell cultures has been discussed. The promising progresses of Protalix and Biolex both provide opportunities of using in vitro plant cell culture bioreactor systems for biomanufacturing of specialty proteins, orphan drugs for rare genetic diseases, and biosimilars or biobetter therapeutics, to lower the cost of goods while maintaining or improving the plantmade protein quality. Furthermore, bioreactor-based plant cell cultures exhibit the advantage of producing plant-made therapeutics in a similar fashion to microbial and mammalian cells which have met regulatory requirements set by the FDA and EMEA over the past 20 years. Although lower protein production yields have been observed in plant cell cultures, implying a 10100 fold increase required to reach commercial levels, strategies for making the bioreactor-based plant cell culture a practical and economical platform is not unrealistic considering the impressive improvements in recombinant microbial fermentation and mammalian cell culture over the past 20 years. References
Amanullah A, Buckland BC, Nienow AW. Mixing in the fermentation and cell culture industries. In: Paul EL, Atiemo-Obeng VA, Kresta SM, editors. Handbook of industrial mixing: Science and Practice. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2004. doi:10.1002/0471451452.ch18. Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D. Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnol J 2008;6: 63348. Betts JI, Baganz F. Miniature bioreactors: current practices and future opportunities. Microb Cell Fact 2006;5:2134. Chilton M-D, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempe J. Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 1982;295:4324. Cosgrove DJ. Relaxation in a high-stress environment: The molecular bases of extensible cell walls and cell enlargement. Plant Cell 1997;9:103141. Curtis WR, Tuerk AL. Oxygen transport in plant tissue culture systems. Plant tissue culture engineering 2006:17386. Davies HM. Review article: commercialization of whole-plant systems for biomanufacturing of protein products: evolution and prospects. Plant Biotechnol J 2010;8:84561. De Dobbeleer C, Cloutier M, Fouilland M, Legros R, Jolicoeur M. A high-rate perfusion bioreactor for plant cells. Biotechnol Bioeng 2006;95:112637. De Muynck B, Navarre C, Boutry M. Production of antibodies in plants: status after twenty years. Plant Biotechnol J 2010;8:52963. Decker EL, Reski R. The moss bioreactor. Curr Opin Plant Biol 2004;7:16670. Decker EL, Reski R. Moss bioreactors producing improved biopharmaceuticals. Curr Opin Biotechnol 2007;18:3938. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 2009;27:297306. Desai PN, Shrivastava N, Padh H. Production of heterologous proteins in plants: strategies for optimal expression. Biotechnol Adv 2010;28:42735. Diao JP, Young L, Zhou P, Shuler ML. An actively mixed mini-bioreactor for protein production from suspended animal cells. Biotechnol Bioeng 2008;100:7281. Doran PM. Design of mixing systems for plant cell suspensions in stirred reactors. Biotechnol Prog 1999;15:31935.

408

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409 Karg SR, Kallio PT. The production of biopharmaceuticals in plant systems. Biotechnol Adv 2009;27:87994. Kieran PM. Bioreactor design for plant cell suspension cultures. In: Tramper JMSCMMJ, editor. Multiphase Bioreactor Design. Routledge, USA: Taylor & Francis Ltd; 2001. p. 391426. Kieran PM, MacLoughlin PF, Malone DM. Plant cell suspension cultures: some engineering considerations. J Biotechnol 1997;59:3952. Kieran P, Malone D, MacLoughlin P. Effects of hydrodynamic and interfacial forces on plant cell suspension systems. Inuence of stress on cell growth and product formation; 2000. p. 13977. Kim NS, Kim TG, Kim OH, Ko EM, Jang YS, Jung ES, 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;68:26375. Komarnytsky S, Borisjuk N, Yakoby N, Garvey A, Raskin I. Cosecretion of protease inhibitor stabilizes antibodies produced by plant roots. Plant Physiol 2006;141:118593. Lico C, Chen Q, Santi L. Viral vectors for production of recombinant proteins in plants. J Cell Physiol 2008;216:36677. Lienard D, Dinh OT, van Oort E, Van Overtvelt L, Bonneau C, Wambre E, et al. Suspension-cultured BY-2 tobacco cells produce and mature immunologically active house dust mite allergens. Plant Biotechnol J 2007;5:93108. Liu C, Towler MJ, Medrano G, Cramer CL, Weathers PJ. Production of mouse interleukin12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor. Biotechnol Bioeng 2009;102:107486. Lucumi A, Posten C. Establishment of long-term perfusion cultures of recombinant moss in a pilot tubular photobioreactor. Process Biochem 2006;41:21807. Lukjan S, Srinivasan V, Tous G, Parekh S, Swindell C, Horn M. Large-scale production of pharmaceutical proteins using plant cell suspension cultures. In Vitro Cell Dev Biol Anim 2007;43:S501. Maccarthy JJ, Ratcliffe D, Street HE. The effect of nutrient medium composition on the growth-cycle of Catharanthus roseus G. Don cells grown in batch culture. J Exp Bot 1980;31:131526. McDonald KA, Hong LM, Trombly DM, Xie Q, Jackman AP. Production of human alpha-1antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnol Prog 2005;21:72834. Medina-Bolivar F, Cramer C. Production of recombinant proteins by hairy roots cultured in plastic sleeve bioreactors. Methods Mol Biol 2004;267:35163. Meuwly F, Rufeux PA, Kadouri A, von Stockar U. Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications. Biotechnol Adv 2007;25:4556. Micheletti M, Barrett T, Doig SD, Baganz F, Levy MS, Woodley JM, et al. Fluid mixing in shaken bioreactors: implications for scale-up predictions from microlitre-scale microbial and mammalian cell cultures. Chem Eng Sci 2006a;61:293949. Micheletti M, Barrett T, Doig SD, Baganz F, Levy MS, Woodley JM, et al. Fluid mixing in shaken bioreactors: implications for scale-up predictions from microlitre-scale microbial and mammalian cell cultures; 2006b. p. 293949. Mishra BN, Ranjan R. Growth of hairy-root cultures in various bioreactors for the production of secondary metabolites. Biotechnol Appl Biochem 2008;49:110. Namdev PK, Dunlop EH. Shear sensitivity of plant-cells in suspensions present and future. Appl Biochem Biotechnol 1995;54:10931. Osuna L, Moyano E, Mangas S, Bonll M, Cusido RM, Pinol MT, et al. Immobilization of Galphimia glauca plant cell suspensions for the production of enhanced amounts of Galphimine-B. Planta Med 2008;74:949. Qi HN, Goudar CT, Michaels JD, Henzler HJ, Jovanovic GN, Konstantinov KB. Experimental and theoretical analysis of tubular membrane aeration for mammalian cell bioreactors. Biotechnol Prog 2003;19:11839. Rao AQ, Bakhsh A, Kiani S, Shahzad K, Shahid AA, Husnain T, et al. The myth of plant transformation. Biotechnol Adv 2009;27:75363. Ratner M. Pzer stakes a claim in plant cell-made biopharmaceuticals. Nat Biotechol 2010;28:1078. Raven N, Schillberg S, Kirchhoff J, Brndli J, Imseng N, Eibl R. Growth of BY-2 suspension cells and plantibody production in single-use bioreactors. John Wiley & Sons, Inc; 2010. Rawel HM, Kroll J, Kulling S. Effect of non-protein components on the degradability of proteins. Biotechnol Adv 2007;25:6113. Read EK, Park JT, Shah RB, Riley BS, Brorson KA, Rathore AS. Process analytical technology (PAT) for biopharmaceutical products: Part I. Concepts and applications. Biotechnol Bioeng 2009;105:27684. Ritala A, Wahlstrm EH, Holkeri H, Hafren A, Mkelinen K, Baez J, et al. Production of a recombinant industrial protein using barley cell cultures. Protein Expr Purif 2008;59:27481. Sajc L, Grubisic D, Vunjak-Novakovic G. Bioreactors for plant engineering: an outlook for further research. Biochemical Engineering Journal 2000;4:8999. Schiermeyer A, Schinkel H, Apel S, Fischer R, Schillberg S. Production of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAalpha1) in tobacco is hampered by proteolysis. Biotechnol Bioeng 2005;89:84858. Schmale K, Rademacher T, Fischer R, Hellwig S. Towards industrial usefulness cryocell-banking of transgenic BY-2 cell cultures. J Biotechnol 2006;124:30211. Shaaltiel Y, Baum G, Bartfeld D, Hashmueli S, Lewkowicz A. Production of high mannose proteins in plant culture. United States Patent 20080038232. 2008. Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, et al. Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J 2007;5:57990. Shadwick FS, Doran PM. Propagation of plant viruses in hairy root cultures: a potential method for in vitro production of epitope vaccines and foreign proteins. Biotechnol Bioeng 2007;96:57083. Shadwick FS, Doran PM. Foreign protein expression using plant cell suspension and hairy root cultures, in molecular farming: plant-made pharmaceuticals and technical proteins. In: Fischer R, Schillberg S, editors. Wiley-CH Verlag GmbH & Co. KGaA, Weinheim, FRG; 2005. doi:10.1002/3527603638.ch2.

Doran PM. Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 2006a;24:42632. Doran PM. Loss of secreted antibody from transgenic plant tissue cultures due to surface adsorption. J Biotechnol 2006b;122:3954. Ducos J-P, Terrier B, Courtois D. Disposable bioreactors for plant micropropagation and mass plant cell culture. Disposable bioreactors. Berlin/Heidelberg: Springer; 2009. p. 89115. Dunlop EH, Namdev PK, Rosenberg MZ. Effect of uid shear forces on plant-cell suspensions. Chem Eng Sci 1994;49:226376. Durocher Y, Butler M. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 2009;20:7007. Eibl R, Eibl D. Design and use of the wave bioreactor for plant cell culture. Plant tissue culture engineering. Springer; 2006. p. 20327. Eibl R, Eibl D. Design of bioreactors suitable for plant cell and tissue cultures. Phytochem Rev 2008;7:5938. Eibl R, Werner S, Eibl D. Disposable bioreactors for plant liquid cultures at litre-scale. Eng Life Sci 2009a;9:15664. Eibl R, Eibl D, Eibl R, Werner S, Eibl D. Bag bioreactor based on wave-induced motion: characteristics and applications. Disposable bioreactors. Berlin/Heidelberg: Springer; 2009b. p. 5587. Eibl R, Kaiser S, Lombriser R, Eibl D. Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology. Appl Microbiol Biotechnol 2010a;86:419. Eibl D, Peuker T, Eibl R. Single-use equipment in biopharmaceutical manufacture: a brief introduction. John Wiley & Sons, Inc; 2010b. Eibl R, Lffelholz C, Eibl D. Single-use bioreactorsan overview. John Wiley & Sons, Inc; 2010c. Franconi R, Demurtas OC, Massa S. Plant-derived vaccines and other therapeutics produced in contained systems; 2010. p. 87792. Ganapathi TR, Kumar GBS, Srinivas L, Revathi CJ, Bapat VA. Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures. Plant Cell Rep 2007;26:157584. Georgiev M, Pavlov A, Bley T. Hairy root type plant in vitro systems as sources of bioactive substances. Appl Microbiol Biotechnol 2007;74:117585. Gilleta F, Roisin C, Fliniaux MA, Jacquin-Dubreuil A, Barbotin JN, Nava-Saucedo JE. Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin. Enzyme Microb Technol 2000;26:22934. Girard LS, Fabis MJ, Bastin M, Courtois D, Petiard V, Koprowski H. Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture. Biochem Biophys Res Commun 2006;345:6027. Giri A, Narasu ML. Transgenic hairy roots: recent trends and applications. Biotechnol Adv 2000;18:122. Gomord V, Fitchette A-C, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D, et al. Plant-specic glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol J 2010;8:56487. Gorr G, Wagner S. Humanized glycosylation: production of biopharmaceuticals in a moss bioreactor. Wiley-VCH Verlag GmbH; 2008. Gupta SD, Ibaraki Y, Choi Y-E, Kim Y-S, Paek K-Y. Types and designs of bioreactors for hairy root culture. In: Hofman M, Ann J, Nedovi V, Willaert R, editors. Plan tissue culture engineering. Netherlands: Springer; 2006. p. 16172. Hacker DL, De Jesus M, Wurm FM. 25 years of recombinant proteins from reactorgrown cells where do we go from here? Biotechnol Adv 2009;27:10237. Hellwig S, Drossard J, Twyman RM, Fischer R. Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 2004;22:141522. Holland T, Sack M, Rademacher T, Schmale K, Altmann F, Stadlmann J, 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: 27889. Hong SY, Kwon TH, Lee JH, Jang YS, Yang MS. Production of biologically active hG-CSF by transgenic plant cell suspension culture. Enzyme Microb Technol 2002;30:7637. Huang T-K, McDonald KA. Bioreactor engineering for recombinant protein production in plant cell suspension cultures. Biochem Eng J 2009;45:16884. Huang J, Sutliff TD, Wu L, Nandi S, Benge K, Terashima M, et al. Expression and purication of functional human alpha-1-antitrypsin from cultured plant cells. Biotechnol Prog 2001a;17:12633. Huang TK, Wang PM, Wu WT. Cultivation of Bacillus thuringiensis in an airlift reactor with wire mesh draft tubes. Biochem Eng J 2001b;7:359. Huang LF, Liu YK, Lu CA, Hsieh SL, Yu SM. Production of human serum albumin by sugar starvation induced promoter and rice cell culture. Transgenic Res 2005;14:56981. Huang TK, Plesha MA, Falk BW, Dandekar AM, McDonald KA. Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures. Biotechnol Bioeng 2009;102:50820. Huang T-K, Plesha MA, McDonald KA. 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 2010;106:40821. James E, Mills DR, Lee JM. Increased production and recovery of secreted foreign proteins from plant cell cultures using an afnity chromatography bioreactor. Biochem Eng J 2002;12:20513. Jost W, Link S, Horstmann V, Decker EL, Reski R, Gorr G. Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression. Curr Genet 2005;47:11120. Junker BH, Stanik M, Barna C, Salmon P, Buckland BC. Inuence of impeller type on mass transfer in fermentation vessels. Bioprocess Eng 1998;19:40313. Kaiser J. Is the drought over for pharming? Science 2008;320:4735.

T.-K. Huang, K.A. McDonald / Biotechnology Advances 30 (2012) 398409 Sharma AK, Sharma MK. Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 2009;27:81132. Sharp JM, Doran PM. Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol Bioeng 2001a;73:33846. Sharp JM, Doran PM. Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures. Biotechnol Prog 2001b;17:97992. Shih SMH, Doran PM. Foreign protein production using plant cell and organ cultures: advantages and limitations. Biotechnol Adv 2009;27:103642. Shin YJ, Hong SY, Kwon TH, Jang YS, Yang MS. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol Bioeng 2003;82:77883. Smith ML, Mason HS, Shuler ML. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form. Biotechnol Bioeng 2002;80:81222. Streateld SJ. Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 2007;5:215. Su WW, Arias R. Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production. J Biosci Bioeng 2003;95:1320. Suehara KI, Takao S, Nakamura K, Uozumi N, Kobayashi T. Optimal expression of GUS gene from methyl jasmonate-inducible promoter in high density culture of transformed tobacco cell line BY-2. J Ferment Bioeng 1996;82:515. Sunil Kumar GB, Ganapathi TR, Srinivas L, Revathi CJ, Bapat VA. Expression of hepatitis B surface antigen in potato hairy roots. Plant Sci 2006;170:91825. Tanaka H. Technological problems in cultivation of plant cells at high density (reprinted from Biotechnology and Bioengineering, vol 23, pg 12031218, 1981). Biotechnol Bioeng 2000;67:77590.

409

Terashima M, Ejiri Y, Hashikawa N, Yoshida H. Utilization of an alternative carbon source for efcient production of human alpha(1)-antitrypsin by genetically engineered rice cell culture. Biotechnol Prog 2001;17:4036. Terrier B, Courtois D, Henault N, Cuvier A, Bastin M, Aknin A, et al. Two new disposable bioreactors for plant cell culture: the wave and undertow bioreactor and the slug bubble bioreactor. Biotechnol Bioeng 2007;96:91423. Travis J. Is the drought over for pharming? Science 2008;320:4737. Trexler MM, McDonald KA, Jackman AP. Bioreactor production of human 1-antitrypsin using metabolically regulated plant cell cultures. Biotechnol Prog 2002;18:5018. Trexler MM, McDonald KA, Jackman AP. A cyclical semicontinuous process for production of human alpha(1)-antitrypsin using metabolically induced plant cell suspension cultures. Biotechnol Prog 2005;21:3218. Tsoi BMY, Doran PM. Effect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures. Biotechnol Appl Biochem 2002;35:17180. Varley J, Birch J. Reactor design for large scale suspension animal cell culture. Cytotechnology 1999;29:177205. Wang PM, Huang TK, Cheng HP, Chien YH, Wu WT. A modied airlift reactor with high capabilities of liquid mixing and mass transfer. J Chem Eng Jpn 2002;35:3549. Yin JC, Li GX, Ren XF, Herrler G. Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 2007;127:33547. Zhiyu Z, Gerardo P, Paolo B, Anthony JS, Oliver G, Klavs FJ. Microbioreactors for Bioprocess Dev 2007;12:14351. Zhong JJ. Biochemical engineering of the production of plant-specic secondary metabolites by cell suspension cultures. Adv Biochem Eng Biotechnol 2001;72:126.