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Ritala, Häkkinen & Schillberg Molecular in & cell cultures: a great future ahead?

2 Commentary Molecular pharming in plants and plant cell cultures: a great future ahead?

Pharm. Bioprocess. Keywords: biobetter • • • emergency • molecular farming • plant • plant • recombinant • transgenic • transient expression

Plant biotechnology may not be a famil- tion and genetic improvements. Both the Anneli Ritala*,1, Suvi iar concept to the general public, but it is US FDA and European Medicines Agency T Häkkinen1 & Stefan 2 a rapidly developing field of research that are familiar with these systems, and stan- Schillberg 1 involves the use of plants, plant tissues and dard protocols can be followed to ensure VTT Technical Research Centre of Finland, P.O.Box 1000, Tietotie 2, FI- plant cell cultures to make or modify prod- the approval of new products. However, 02044 VTT, Espoo, Finland ucts and processes. The versatility of plants equivalent protocols are only just emerg- 2Fraunhofer Institute for Molecular and plant cells can be harnessed to produce ing for plant-based production systems, and and Applied IME, diverse products, including valuable pro- only one plant-derived biopharmaceutical Forckenbeckstrasse 6, 52074 Aachen, teins. This is often described as ‘molecular protein is currently on the market. With Germany * Author for correspondence: anneli. farming’ and it requires the introduction of their established production infrastructure [email protected] foreign DNA into plants or plant cells, turn- and regulatory framework, microbial and ing them into factories for the production mammalian production systems have raced of specific recombinant protein products. far ahead of their plant-based counterparts. The term ‘molecular pharming’ is often No company will change their production 10.4155/PBP.14.21 used instead to highlight the production of host without a clear economic benefit, nor protein-based , which will they consider plants and plant cells for contributes to the sustainable production new products if there is no advantage over of drugs that promote human and animal their incumbent technology. Furthermore, wellbeing. Both terms also apply to the pro- new companies will not base their manufac- duction of valuable secondary metabolites turing on a second-best option. Therefore, 3 such as the anticancer drugs , vin- plant-based systems must begin to com- cristine and vinblastine, but we will focus pete head-to-head with the established sys- on recombinant and their use as tems and, on a technological basis, we can biopharmaceuticals in this article. already identify the areas where plant-based 2014 The biopharmaceutical markets have systems have the advantage, namely in terms expanded rapidly over the last 20 years, and of speed, improved product quality and scal- are projected to more than double in volume ability. over the next decade from US$200 billion The international success story of molec- in 2013 to at least US$500 billion in 2020. ular pharming began in 2006 with the US The two major biopharmaceutical produc- Department of approval of a tion systems are microbes (mainly Esch- poultry vaccine against Newcastle erichia coli and ) and mammalian cells developed by Dow AgroSciences (IN, USA) such as the Chinese hamster ovary platform. [1,2]. The vaccine was manufactured in trans- In both cases, has increased genic cell suspension cultures and substantially over the last decade due to was a benchmark for the regulatory accep- process optimization, platform standardiza- tance of plants as a manufacturing platform, part of

10.4155/PBP.14.21 © 2014 Future Ltd Pharm. Bioprocess. (2014) 2(3), 00–00 ISSN 2048-9145 1 Commentary Ritala, Häkkinen & Schillberg

although ultimately, the product was not marketed floodgates for the clinical development of additional because the company withdrew from animal vaccine products in the future. research. Currently, the brightest star in the molecu- There are many different plant-based production lar pharming sky is Elelyso™ () pro- systems in development and one can evaluate and duced in carrot cells by the Israeli company Protalix select the most suitable system for a given target prod- Biotherapeutics (Carmiel, Israel) [2] and licensed to uct to achieve the optimal characteristics. However, Pfizer Inc. (NY, USA). This is a recombinant form this has diluted efforts to establish a standardized reg- of the human enzyme glucocerebrosidase, which is ulatory process and may delay overall progress in the used for the treatment of the lysosomal storage dis- field and fulfilment of industrial standards. Neverthe- order Gaucher disease. The recombinant product met less, none of the plant-based systems under develop- the primary end points in successful Phase III clinical ment are ideal for all target molecules, so the parallel trials in September 2009 and gained FDA approval development of different platforms is advantageous in 2012. The product is currently on the US and for the progress of molecular pharming. The poten- Israeli markets, but the European Medicines Agency tial of different plant-based production platforms is granted 10-year European marketing exclusivity discussed in more detail below. to another product in 2010 and thus Elelyso can- The greatest advantage of intact plants that are sta- not receive approval for Gaucher disease until 2020. bly transformed to produce a target protein is their Elelyso has a longer serum half-life than its Chinese unparalleled scalability. For biopharmaceutical prod- hamster ovary-derived counterpart Cerezyme® (Gen- ucts, manufacturing will probably be restricted to zyme, MA, USA) [3,4] and is produced by targeting greenhouses and other closed environments to ensure the protein to the plant vacuole, which exposes ter- product safety and batch-to-batch consistency when minal mannose residues on the glycan chains that are production is carried out under controlled condi- required for binding. This avoids the need tions. For example, ORF (Kopavogur, Ice- to trim the terminal sugars in vitro, which is part of land) [8] uses barley plants grown in greenhouses to the production process for Cerezyme. The long serum produce recombinant growth factors, and half-life has a strong impact on patient compliance interleukins in the cereal for research purposes. because fewer doses are required. In addition, the Similarly, the Canadian company SemBioSys (AB, disposable bioreactor production platform ProCel- Canada) developed a -based production lEx® (Protalix Biotherapeutics) can easily be scaled system for and completed Phase I/II clinical up to address market needs, and thousands of liters trials in 2009 before filing for bankruptcy in 2012. of cell suspension culture can be harvested weekly. The SemBioSys platform was so efficient that theo- From the traceability and cross-contamination point retically 16 mid-sized Canadian farms could have of view, disposable are the best option for produced enough insulin to meet the entire global biopharmaceutical production. demand. Although the current E. coli platform also Several plant-derived biopharmaceutical products meets this capacity, it is more expensive to establish in are currently undergoing clinical trials. The EU FP6- developing countries, which would therefore benefit funded academic consortium Pharma-Planta [5] pio- most from the production of inexpensive medicines neered the regulatory process for the entire European in plants. molecular pharming community by taking a tobacco- The use of plant cell suspension cultures for derived HIV-neutralizing monoclonal from molecular pharming is advantageous owing to the initial vector construction and transfer through high product quality and scalability. Since the with- all phases of development and manufacturing to drawal of Dow AgroSciences from the animal vaccine launch a Phase I clinical trials, which concluded in market, Fraunhofer Institute for 2011. The consortium worked closely with EU and and Applied Ecology [9] has continued to develop national regulatory authorities to ensure the safety of the tobacco BY-2 cell platform in the context of the the antibody and promote the acceptance of plant- EU-FP7-funded project CoMoFarm [10] combined based production platforms. In January 2014, a col- with orbitally shaken bioreactor technology from laboration between Icon Genetics GmbH (Halle, Ger- Kühner (Basel, Switzerland) [11] . The 200 l OrbShake many) [6] and Innovation GmbH (Düsseldorf, device was used for the large-scale cultivation of 100- Germany) [7] resulted in the completion of a Phase liter BY-2 cell suspension cultures, resulting in cell I clinical trial for a personalized plant-derived vac- growth and target protein yield comparable to stan- cine for the treatment of non-Hodgkin’s lymphoma. dard cultivation using shake flasks, thus achieving a These examples represent important milestones in the several 100-fold scale up without loss of productivity history of plant molecular pharming and open the [12] . VTT Technical Research Centre of Finland used

2 Pharm. Bioprocess. (2014) 2(3) future science group Molecular pharming in plants & plant cell cultures: a great future ahead? Commentary traditional microbial bioreactors to cultivate the same One perceived drawback that is often discussed in BY-2 cells at the 600 l scale [13] . The growth kinet- the context of molecular pharming is the presence of ics of BY-2 cells in bioreactors was again strikingly plant-specific glycans on recombinant human proteins. similar to shake flasks, although the productivity was Although it is now accepted that plant glycans pose little variable. This must be addressed by generating mono- risk of immunogenicity (e.g., Elelyso, discussed above, clonal cultures to achieve batch-to-batch consistency contains typical plant glycans but no adverse effects sufficient for industrial standards[14] . have been reported even after multiple injections) a Transient expression systems have been developed raft of technologies has been developed to remove plant to complement stably-transformed transgenic plants, glycans and add human-like counterparts, which have and they benefit from the rapid onset of recombi- facilitated the development of ‘biobetter’ products with nant protein production. This is particularly useful customized glycan profiles that enhance their activi- for rapid response situations, such as the production ties. A recent breakthrough is the use of mutant plants of against epidemic or bioterror- lacking plant-specific N-glycan residues but express- ist threats. The plant material is propagated before ing 11 human proteins acting in different subcellular the introduction of foreign DNA, allowing plants to compartments at different stages of the be grown in the open if necessary (although green- pathway, resulting in the synthesis of a highly complex house conditions are preferred from the perspective mammalian oligosaccharide structure [17] . of GMP). The plants are moved into contained, GMP-compliant facilities for protein production. Conclusion The Defense Advanced Research Projects Agency Molecular pharming provides a safe and sustainable program in the USA has invested substantially into platform for the production of valuable recombinant the development of this manufacturing technology proteins. However, the economic prospects of this and, for example, Medicago [15] has the capacity to technology depend heavily on the selection of suitable manufacture 10 million vaccine doses per month and target proteins that can be produced more rapidly, less their pandemic and seasonal influenza vaccines are expensively or at a higher quality in plants or plant currently undergoing Phase I/II clinical development, cell cultures compared with established microbial and and Kentucky Bioprocessing [16] has successfully pro- mammalian systems. If the targets and platforms are duced vaccine-grade recombinant proteins with their chosen with care, the forecast for molecular pharming manufacturing capacity of several hundred kilograms is indeed very promising. per day. In the future, perhaps the greatest promise of Information resources molecular pharming is the use of edible host tissues The International Society for Plant Molecular Farm- as a vehicle for recombinant proteins such as oral vac- ing (ISPMF) was established in February 2014. Mem- cines and prophylactic . For example, Pro- bership is open to everybody and the idea is to pro- talix Biotherapeutics has completed the Phase I clini- mote and support excellent research, scholarship and cal testing of a carrot cell line expressing recombinant practice in the field of plant molecular pharming. We glucocerebrosidase, which is orally administered are happy to invite all readers of Pharmaceutical Bio- rather than injected, and thus preferable in terms of processing to join us at the frontier of plant molecu- patient compliance. Other companies are develop- lar pharming. The ISPMF has regular meetings; for ing products expressed in cereal seeds, thus benefit- more information, please visit the ISPMF website [18]. ing from natural forms of bioencapsulation. As stated above, transient expression is emerging as the ideal Financial & competing interest disclosure strategy to deploy emergency vaccines, particularly A Ritala and ST Häkkinen are employees of VTT Technical Re- for zoonotic diseases such as influenza, but molecu- search Centre of Finland, and S Schillberg is an employee of lar pharming in general appears to well suited for the Fraunhofer Institute for Molecular Biology and Applied Ecol- deployment of veterinary vaccines, for example, by ogy and Honorary Professor at the Justus-Liebig University adding plants or plant tissues expressing vaccine anti- of Gießen (Germany). The authors have no other relevant gens to animal feed. In large-scale industries such as affiliations or financial involvement with any organization or fish and poultry farming, oral vaccination saves the entity with a financial interest in or financial conflict with the labor and stress involved in isolating and manually subject matter or materials discussed in the manuscript. injecting thousands of animals. The plant-based pro- The authors wish to thank Richard M Twyman for manu- duction of vaccines is also ideal for companion ani- script editing which was funded by the European Union Sev- mals, offering a robust and cost-effective platform for enth Framework Programme under the grant agreement no. this sector. 227420 (CoMoFarm).

future science group www.future-science.com 3 Commentary Ritala, Häkkinen & Schillberg

References 11 Kühner. www.kuhner.com/en/product/shakers/single-use/sb200-x. 1 Dow AgroSciences. html www.dowagrosciences.com 12 Schillberg S, Raven N, Fischer R, Twyman RM, Schiermeyer 2 Katsnelson A, Ransom J, Vermij P, WaltzIn E. Brief. Nature A. Molecular farming of pharmaceutical proteins using plant Biotechnology 24, 233–234 (2006). suspension cell and cultures. Curr. Pharm. Des. 19(31), 3 Protalix Biotherapeutics. 5531–5542 (2013). www.protalix.com/products/elelyso-taliglucerase-alfa_ 13 VTT Technical Research Centre of Finland. uplyso-alfataliglicerase.asp www.vtt.fi 4 . 14 Reuter LJ, Bailey MJ, Joensuu JJ, Ritala A. Scale-up of www.genzyme.com/Products/Product-Information.aspx hydrophobin-assisted recombinant protein production 5 EU Framework 6 Pharma-Planta project. in tobacco BY-2 suspension cells. Plant Biotech. J. www.pharma-planta.net 12(4), 402–410 (2014). 6 Icon Genetics GmbH. 15 Medicago. www.icongenetics.com/html/5975.htm www.medicago.com/English/Manufacturing/Facility- 7 Bayer Innovation GmbH. Overview/default.aspx www.bayer.com 16 Kentucky Bioprocessing. 8 ORF Genetics. www.kbpllc.com/News/March2010.aspx www.orfgenetics.com 17 Castilho A, Neumann L, Gattinger P et al. Generation of 9 Fraunhofer Institute for Molecular Biology and Applied biologically active multi-sialylated recombinant human Ecology IME. EPOFc in plants. PLoS ONE 8(1), e54836 (2013). www.ime.fraunhofer.de 18 The International Society for Plant Molecular Farming 10 EU Framework 7 CoMoFarm project (ISPMF). www.societyformolecularfarming.org www.comofarm.org

4 Pharm. Bioprocess. (2014) 2(3) future science group