Plant Biotechnology Journal (2015) 13, pp. 1191–1198 doi: 10.1111/pbi.12401 Review article Moss-made pharmaceuticals: from bench to bedside Ralf Reski1,2,3,*, Juliana Parsons1 and Eva L. Decker1 1Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany 2FRIAS - Freiburg Institute for Advanced Studies, Freiburg, Germany 3BIOSS - Centre for Biological Signalling Studies, Freiburg, Germany Received 13 March 2015; Summary revised 16 April 2015; Over the past two decades, the moss Physcomitrella patens has been developed from scratch to accepted 17 April 2015. a model species in basic research and in biotechnology. A fully sequenced genome, outstanding *Correspondence (Tel +49 761 2036969; possibilities for precise genome-engineering via homologous recombination (knockout moss), a fax +49 761 2036967, email ralf. certified GMP production in moss bioreactors, successful upscaling to 500 L wave reactors, [email protected]) excellent homogeneity of protein glycosylation, remarkable batch-to-batch stability and a safe cryopreservation for master cell banking are some of the key features of the moss system. Several human proteins are being produced in this system as potential biopharmaceuticals. Among the products are tumour-directed monoclonal antibodies with enhanced antibody-dependent cytotoxicity (ADCC), vascular endothelial growth factor (VEGF), complement factor H (FH), keratinocyte growth factor (FGF7/KGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), asialo-erythropoietin (asialo-EPO, AEPO), alpha-galactosidase (aGal) and beta- glucocerebrosidase (GBA). Further, an Env-derived multi-epitope HIV protein as a candidate vaccine was produced, and first steps for a metabolic engineering of P. patens have been made. Some of the recombinant biopharmaceuticals from moss bioreactors are not only Keywords: ADCC, biobetter, similar to those produced in mammalian systems such as CHO cells, but are of superior quality knockout moss, Morbus Fabry, moss (biobetters). The first moss-made pharmaceutical, aGal to treat Morbus Fabry, is in clinical bioreactor, Physcomitrella patens. trials. Introduction which requires production in self-contained systems such as bioreactors. Most recombinant biopharmaceuticals are complex human gly- Within this special issue on Molecular Farming, we concentrate coproteins. Therefore, the benchmarks for any production system on the production of PMPs utilizing the moss Physcomitrella are mammalian cell factories, especially CHO cells, which were patens as a production host. Broader information on specific derived from Chinese hamster ovaries (Beck et al., 2008; aspects of this topic can be found in previous reviews. The basic Durocher and Butler, 2009). The use of plants as alternative concept was described in Decker and Reski (2004), different expression hosts is on the rise because their cultivation may be aspects of glycoprotein production were discussed in Decker and easier, less expensive and per se excludes possible contaminations Reski (2007), and the production process is reviewed in Decker of the product with infectious agents deleterious to the patient, and Reski (2008). Detailed reviews on glyco-engineering aspects which should make downstream processing and safety tests more can be found in Decker and Reski (2012) and in Decker et al. straightforward and thus less expensive (Fischer et al., 2004; (2014). Instead, we aim here at providing a concise review of the other articles in this special issue). Consequently, several plant- system, the established enabling technologies, and the most made pharmaceuticals (PMPs) are already in clinical studies (Paul current update on the status of produced potential biopharma- and Ma, 2011; Sabalza et al., 2014), and the first product, ceuticals. Taliglucerase alfa, a beta-glucocerebrosidase to treat Morbus Gaucher (Shaaltiel et al., 2007), was released to the market in The moss system 2012 by Pfizer/Protalix. In comparison with the benchmarks, three major challenges Bryophyte (comprising mosses, hornworts and liverworts) remain before plants can be widely used as alternative cell research has a long history with several important contributions factories: to basic science (reviewed in Reski, 1998a). From an evolutionary 1. The amounts of recombinant products need to be enhanced point of view, mosses are situated halfway between single-celled by several techniques. algae and complex seed plants, which are divergent by an 2. Although the core di-antennary N-glycan-structure on glyco- evolutionary distance of about one billion years (Lang et al., proteins is the same between human and plants, specific 2008). As in algae, the dominant phase of moss development is differences do exist and can affect stability, efficacy and tolerance haploid (gametophytic), while the dominant phase in all seed by the patient’s immune system. plants is at least diploid (sporophytic). Mosses can be grown in 3. The prerequisite for approval is a production of the drug self-contained systems like Petri dishes, Erlenmeyer flasks or according to good manufacturing practice (GMP) guidelines, bioreactors in pure mineral media without any organic additions ª 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. 1191 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1192 Ralf Reski et al. such as antibiotics, carbon sources or growth regulators and information is freely available via www.cosmoss.org and is under highly controlled environmental conditions (Cerff and constantly improved (Zimmer et al., 2013). A variety of whole Posten, 2012; Hohe and Reski, 2005; Hohe et al., 2002a). Even genome transcription profiling data sets were published (e.g. under these conditions, the moss P. patens can complete its life Beike et al., 2015; Richardt et al., 2010), which have been cycle with the release of persistent spores. Sexual reproduction, integrated into a transcriptomic platform allowing analysis of however, is only initiated under low temperature and short day transcript abundances, and thus indirectly promoter activity, conditions (Hohe et al., 2002b). Therefore, no spores are formed under different culture conditions (Hiss et al., 2014). under production conditions. For the production of biopharmaceuticals, large-scale pro- cesses have to be implemented. Bioreactors have been used for Enabling technologies plant cell cultures (Kieran et al., 1997) and transgenic hairy root cultures (Giri and Narasu, 2000) as they provide containment and Vector-free genetic transformation of P. patens has been are easy to control, thus facilitating production under GMP established by conferring antibiotic resistance to wild-type conditions. Different kinds and sizes of photobioreactors moss (Schaefer et al., 1991). Shortly afterwards, it became have been established for the controlled large-scale culture of evident that this moss is suitable for precise gene targeting P. patens: the first production was achieved in a 2-L foil-reactor (GT) via homologous recombination (HR) (Kammerer and Cove, (Reutter and Reski, 1996), subsequently followed by 5 L,10 L and 1996; Schaefer and Zr€yd, 1997). Because of its outstanding 20 L stirred glass tanks (Hohe et al., 2002a), which are still the high rate of HR and the haploid nature of its dominant growth ‘working horses’ in the laboratory. A further upscale to 100 L was phase, GT was subsequently used to disrupt genes of interest achieved by the development of tubular photobioreactors (Pern- and infer gene functions from these knockout mosses in a er-Nochta et al., 2007). Currently, for commercial production reverse genetics approach without complex backcrosses (Girke under GMP-certified conditions, 100 L and 500 L disposable et al., 1998; Strepp et al., 1998) and even allowing high- wave reactors are in use (Niederkruger€ et al., 2014; Figure 1). throughput analyses (Egener et al., 2002). Protocols for culture While plant cell cultures show a high degree of genetic instability, media, protoplast isolation and regeneration, and for PEG- the so-called somaclonal variation (Larkin and Scowcroft, 1981; mediated transformation have been optimized, allowing for the Phillips et al., 1994), moss tissue culture is genetically stable over simultaneous disruption of multiple different genes in one long periods of time (von Stackelberg et al., 2006), because it experiment (Hohe and Reski, 2002; Hohe et al., 2004; relies on differentiated moss plants instead of undifferentiated Schween et al., 2003, 2005). The high rate of GT is a clear plant cell cultures. advantage for glyco-engineering of moss when compared to An important issue in GMP regulations is the molecular similar approaches in seed plants (Reski, 1998b; Schaefer and characterization of the producing cell factories. Once character- Zr€yd, 2001). However, as alternatives to the complete destruc- ized and approved, subsequent production has to rely on identical tion of genes via GT (Kamisugi et al., 2006), methods for base- clones that have to be stored in master cell banks (MCBs). This specific genome alterations and for creating fusion proteins in can easily be achieved for clonal moss tissues, as they can be the original genomic context (Mosquna et al., 2009; Mueller stored for years over liquid nitrogen and survive this cryopreser-