Pharmaceutical Review

Pharmaceutical Review 2 Review On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect Pharm. Bioprocess. Plant cell culture is emerging as an alternative bioproduction system for recombinant Jianfeng Xu*,1,2 pharmaceuticals. Growing plant cells in vitro under controlled environmental & Ningning Zhang1 conditions allows for precise control over cell growth and protein production, batch- 1Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401, to-batch product consistency and a production process aligned with current good USA manufacturing practices. With the recent US FDA approval and commercialization 2College of Agriculture & Technology, of the world’s first plant cell-based recombinant pharmaceutical for human use, Arkansas State University, Jonesboro, β-glucocerebrosidase for treatment of Gaucher’s disease, a new era has come in AR 72401, USA which plant cell culture shows high potential to displace some established platform *Author for correspondence: Tel.: +1 870 680 4812 technologies in niche markets. This review updates the progress in plant cell culture Fax: +1 870 972 2026 processing technology, highlights recent commercial successes and discusses the [email protected] challenges that must be overcome to make this platform commercially viable. The term ‘biopharmaceuticals’ refers to thera- and mammalian cell-based production. It peutic proteins produced by modern biotech- has been estimated that 45% of recombinant nological techniques [1] . Biopharmaceuticals proteins in the USA and Europe are made in have revolutionized modern medicine and mammalian cells (35% in Chinese hamster represent the fastest growing sector within the ovary or CHO cells, and 10% in others), pharmaceutical industry. There are over 200 40% in bacteria (39% in Escherichia coli and protein biopharmaceuticals currently on the 1% in others) and 15% in yeasts [6]. These market [2], used for the treatment of diabetes, established production platforms will con- anemia, hepatitis, cancer and cardiovascu- tinue to be the focus of most biopharmaceu- lar diseases [3,4], and in excess of 400 under tical companies who may not look beyond development. Included among this group these for regulatory reasons or simply due to of protein therapeutics are mainly antibod- inertia borne from unfamiliarity. However, 6 ies and antibody derivatives, vaccines and there are limitations associated with these some serum-derived proteins, for example, systems in terms of cost, scalability, safety cytokines, growth hormones, interleukins and quality/authenticity of proteins. For and interferon. The world market for bio- example, the mammalian cell-based system 2014 pharmaceuticals was valued at approximately suffers from limitation in culture scalability, US$199.7 billion in 2013, and is estimated to high production cost and risk of contamina- reach US$497.9 billion by 2020, representing tion with human pathogens. The prokary- a compound annual growth rate of 13.5% [5]. otic nature of bacteria (e.g., E. coli) limits Monoclonal antibodies constitute the largest the complexity of the proteins (cannot be segment in the biopharmaceuticals market, correctly processed, such as glycosylation), accounting for an estimated share of 25.6% and the appearance of inclusion bodies in 2013. In terms of therapeutic areas, neurol- increases the cost of production. For yeast- ogy applications is the largest market with an based systems, low product yields, inefficient estimated share of 28.2% in 2013 [5]. protein secretion and hyperglycosylation of Currently, the biopharmaceutical indus- proteins (addition of a large number of man- try relies mainly on microbial fermentation nose residues) are common problems. These part of 10.4155/PBP.14.32 © 2014 Future Science Ltd Pharm. Bioprocess. (2014) 2(6), 499–518 ISSN 2048-9145 499 Review Xu & Zhang Key terms based biopharmaceutical company (Karmiel, Israel), Molecular farming: A new biotechnology that uses and marketed by Pfizer. plants as a host to produce recombinant therapeutics, Before Protalix’s landmark success, Dow AgroSci- such as vaccines and antibodies, as well as industrial ences (IN, USA; [12]) received in 2006 the world’s first proteins in large quantities. regulatory approval by the US Department of Agricul- Plant cell culture: Techniques used to maintain or grow ture (USDA) for a tobacco cell-based vaccine against undifferentiated plant cells under sterile conditions on Newcastle disease virus [13] . The commercial success a nutrient culture medium of known composition. Plant by these two companies undoubtedly ushers in a new cell culture has been used to produce valuable secondary metabolites and recombinant proteins. era in the biopharmaceutical industry that promises to provide growth opportunity for this new platform. Protalix: An Israel-based biopharmaceutical company that Other plant cell-made pharmaceuticals, including uses plant cell cultures as a bioproduction platform for recombinant therapeutic proteins. In May 2012, Protalix monoclonal antibodies [14–16], vaccines [17–20], growth partnered with Pfizer to commercialize the world’s first factors [21] and cytokines [22–25] that are in pre- and early plant cell-based recombinant pharmaceutical for human clinical stages, as well as more therapeutic enzymes in use, taliglucerase alpha for treatment of Gaucher’s disease. the Protalix’s development pipeline [11], are expected to enter into the marketplace in the future. Plant cell limitations have prompted research into alternative culture is now reaching the stage at which it may chal- bioproduction platforms. lenge those established bioproduction systems that use ‘Molecular farming’ in plants is emerging as a bacterial, yeast and mammalian cells, though major promising approach for the production of recombinant problems with plant cell culture still exist with regards pharmaceuticals; its potential for low-cost production to low product yields, inherent production variability of high quality, safe and biologically active mamma- and nonmammalian glycosylation. This review high- lian proteins has been well reported [7]. Unlike other lights the recent advancements and commercial suc- expression systems, molecular farming has different cess of the plant cell-based bioproduction platform, forms, including cultivation of whole plants in fields, and discusses the prospect and challenges that must be transient expression by agroinfiltration of plants or overcome to make this platform commercially viable. virus-infected plants, in vitro culture of plant tissues or organs and plant cell suspension culture. All of them Plant cell culture as an attractive have been investigated as economical alternative bio- bioproduction platform production platforms in the past two to three decades Basics of plant cell culture system [7,8]. Attention is now shifting from basic research Similar to the microbial and mammalian cells, undif- toward commercial exploitation of the molecular ferentiated plant calli can be dispersed in liquid media farming system. and propagated in perpetuity under a sterile and con- Compared with cultivation of whole plants or plant trolled environment. Plant cell suspension culture was tissues or organs, plant cell suspension culture has originally developed for production of valuable sec- more immediate potential for industrial application, ondary metabolites, such as paclitaxel, shikonin, arte- as it is analogous to traditional microbial fermenta- misinin, digoxin, ginsenosides and ajmalicine with a tion and mammalian cell culture with less regulatory few commercial successes [26–29]. Only in the last two and environmental concerns. In fact, the plant cell decades has the production potential of the plant cell culture system has long been exploited for its unique culture for heterologous proteins been recognized and biosynthetic potential for secondary metabolites or it has now become a viable alternative bioproduction therapeutic proteins, but with limited success [9]. platform for pharmaceutical proteins. This is mainly because the characteristics of growth The most widely used plant cell lines for recombi- and metabolism of plant cells differ considerably from nant biopharmaceutical production are those derived those of microbial and mammalian cells. An impor- from tobacco (Nicotiana tabacum), such as cultivars tant breakthrough was achieved in May 2012, when BY-2 (N. tabacum cv. Bright Yellow 2) cells (Figure 1) the carrot cell-produced therapeutic enzyme, taliglu- and NT-1 (N. tabacum-1) cells. They have appealing cerase alfa (commercially known as ELELYSO™, a features, including being fast-growing, robust and able hydrolytic lysosomal glucocerebrosidase for intrave- to readily undergo Agrobacterium-mediated transfor- nous infusion) was finally approved by the US FDA mation and cell cycle synchronization [30–32]. Other as an orphan drug for treatment of Gaucher’s disease, commonly used plant cell lines are those derived from and thereby became the world’s first plant-made phar- common edible crop species, such as rice (Oriza sativa), maceutical used in humans [10] . Taliglucerase alfa was soybean (Glycine max), alfalfa (Medicago sativa), car- developed by Protalix Biotherapeutics [11], an Israel- rot (Daucus carota) and tomato (Lycopersicon esculen- 500 Pharm. Bioprocess. (2014) 2(6) future science group On the way to commercializing plant cell culture platform for biopharmaceuticals: present status & prospect Review tum). In fact, these

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