The genetic engineering of living organisms has offered an alternative to chemical synthesis of drugs1 and extraction of natural biopharmaceuticals with the production of complex therapeutic molecules in bacteria, yeast and animal cells. However, the biochemical, technical and economic limitations of existing prokaryotic and eukaryotic expression systems and the growing clinical demand for complex therapeutic proteins have created substantial interest in developing new expression systems for the production of therapeutic proteins. To that end, plant-based platforms have emerged in the past two decades that offer a suitable alternative to the current production systems for recombinant biopharmaceuticals.
Twenty years after the first production of an antibody in a plant expression system (Hiatt et al., 1989), this special issue of Plant Biotechnology Journal presents six reviews which synthesize two decades of active research on plant-made pharmaceuticals (PMPs) by academic and/or industrial research teams with at least 800 PMP-related publications in peer-reviewed journals (Figure 1) and about 100 PMP-related patent families (Thangaraj et al., 2009). Our bibliographic analysis also clearly shows that even after 20 years of intense research activity, there is no decline in publication activity related to PMPs, with an estimated number of 110 articles published in 2009 (Figure 1).
Interestingly, at least 25% of PMP-related research articles were published in Plant Biotechnology Journal in 2008 (Figure 2), which explains why we have decided that one of the first special issues of this journal will be dedicated to ‘Success stories in molecular farming’.
Thirty-four per cent of all PMPs-related articles from January 1992 to August 2009 are related to plant-based vaccines and 13% are related to plant-based antibodies (Figure 3) which represents 274 and 107 publications, respectively, after the production of a first monoclonal antibody (Hiatt et al., 1989) and a first subunit vaccine (Mason et al., 1992) in transgenic plants. This is why we have invited two leaders in these fields to sum up the state of the art and to highlight new perspectives on antibody (De Muynck et al., 2010) and vaccine (Ribicky et al., 2010) production in plants.
However, even after 20 years of active research, more than 600 original publications and about 100 PMP-related patent families, these two new reviews in addition to 250 previous ones (Figure 1) published on molecular farming are obviously not sufficient to claim a definitive success in molecular farming. Success is in the products, and a major criticism addressed to molecular farming is ‘After 20 years, where are the products?’
In fact, almost a dozen of biopharmaceuticals produced in plants are currently in clinical developments and at least two PMPs; a secretory IgA used for prevention of tooth decay (CaroRxTM-from Planet Biotechnology Inc, Ma et al., 1998, 2005) and a human intrinsic factor used as a dietary supplement for the treatment of vitamin B-12 deficiency (Cobento Biotech AS) have been approved for use in humans.
Several plant production platforms are presented in this special issue, ranging from seed- and leaf-based production in stable transgenic plant lines, to plant cell bioreactors, to viral or Agrobacterium-mediated transient expression systems. Here, we have chosen to present these platforms through companies exploiting these different plant expression systems.
Several companies are producing biopharmaceuticals in seeds, this is the case for instance for Ventria Bioscience which uses field-grown rice for production of human lactoferrin and human lysozyme (http://www.ventria.com, Huang et al., 2008; Zavaleta et al., 2007). ORF genetics (http://www.orfgenetics.com/) produces human growth factors and cytokines in seeds from barley grown in glasshouses. These products are currently commercialized for diagnostic, academic and private research that perfectly illustrates their batch-to-batch reproducibility and quality. In this issue, you will find the presentation of a seed-based platform developed by SemBioSys Genetics Inc (http://www.sembiosys.com/) and its most advanced product, a recombinant insulin now poised to enter phase III clinical trials, produced in seeds from safflower plants grown in open fields (Boothe et al., 2010). This platform perfectly illustrates that even with relatively low expression levels for therapeutic proteins (1% of TSP), the production is economically viable and that the capacity of biopharmaceuticals in transgenic plants is almost unlimited, as it only depends upon the surface dedicated to the plant culture. Currently, SemBioSys Genetics Inc is growing 100 ha of plants containing human Insulin, Apolipoprotein AI (Apo AI) and other proteins and enzymes. The technology developed by SemBioSys will allow the production of recombinant proteins up to 2–4 kg per hectare, depending on the molecular mass of the target protein.
Seed-based expression platforms are most competitive in applications that require large volume of recombinant proteins per annum. A second main advantage is that production of biopharmaceuticals in seeds enables crop production to be decoupled from extraction and purification processes because of the dormancy and storage properties of seeds.
In contrast, seed-based expression systems are less suited to certain applications such as the production of influenza vaccines, which change each year, because of the amount of time needed to produce sufficient quantities of seed for processing.
Interestingly, the recent emergence of pandemic influenza strains (H5N1 and H1N1) has illustrated that conventional antiquated egg-based production process requiring months of development for vaccine production cannot meet global demand. As an alternative to current production methods, a rapid vaccine development and manufacturing technology was developed that uses plant transient expression systems for the production of virus-like particles (http://www.medicago.com).
This, together with the successful completion of phase I clinical trial of H5N1 pandemic vaccine candidate, illustrates that plant-based transient expression of antigens is perfectly adapted to the unpredictable emergence of a pandemic as presented in the present issue by D’Aoust et al. (2010).
The rapid production of individualized scFv vaccines for the treatment of non-Hodgkin’s lymphoma and the FDA approval of these plant-made injectable purified antibody fragments further illustrate the advantages provided by transient expression in plants over any other expression systems (McCormick et al., 2008). The current development of platforms specialized in transient plant expression to produce a wide range of biopharmaceutical products at high purity and activity such as Kentucky BioProcessing (http://www.kbpllc.com, Pogue et al., 2010) will help exploiting the flexibility and power of transient plant expression systems to provide recombinant protein products of the quality and quantity required for clinical development. This company is already selling a recombinant aprotinin for nonclinical use.
Plant cell cultures have been used over the past 50 years primarily for production of medicinal secondary metabolites such as paclitaxel, shikonin and berberine. More recently, plant cells have proven to be an important alternative system to mammalian cells for production of biopharmaceuticals. This was illustrated with a first success story when a purified injectable Newcastle disease virus vaccine for chickens produced in suspension-cultured tobacco cells by Dow Agroscience was approved by USDA Center for Veterinary Biologics. This company is currently developing other animal vaccines produced in tobacco cell culture (http://www.dowagro.com/animalhealth). Recent developments in biopharmaceutical production in plant cell cultures are also illustrated through the production of a recombinant human glucocerebrosidase for the treatment of Gaucher’s disease in genetically engineered carrot cells (Shaaltiel et al., 2007). The successful completion of phase III clinical studies for this plant-made glucocerebrosidase was announced at the end of 2009 almost simultaneously with the agreement of Pfizer and Protalix to develop and commercialize this PMP (http://www.protalix.com).
Through two decades it has been shown that plant expression systems can produce, for a lower cost, large amounts of biopharmaceuticals free of human infective viruses and prions, and free of bacterial contaminants such as endotoxins. Unlike microbial fermentation, plants are also capable of carrying out post-translational modifications (PTMs) often required for functionality of therapeutic proteins. Despite this, the pharmaceutical industry still shows some reluctance to integrate plant expression platforms into its production strategy. The most recent success stories in molecular farming show that in addition to most classical advantages, transient expression in plants can produce high amounts of biopharmaceuticals faster than any other expression system. The in vivo efficiency of plant-made recombinant glucocerebrosidase without the need of in vitro N-glycan processing as it is the case with the commercial product produced in Chinese Hamster Ovary cells (CHO cells) (Fabrazyme made by Genzyme) provides an interesting illustration that plant-specific PTMs offer the opportunity to produce not only biosimilars but also ‘biobetters2’. More generally, a major advantage of transgenic plants over other production systems available for large-scale and low-cost production, such as Escherichia coli or yeasts, is their ability to perform most PTMs required for therapeutic protein bioactivity and pharmacokinetics. This is illustrated by their capacity to produce many therapeutic proteins requiring proteolytic cleavage(s), oligomerization and glycosylation for their bioactivity, pharmacokinetics, stability and solubility. In this issue, Gomord et al. (2010) describe plant-specific protein N- and O-glycosylation and the considerable progress that have been made towards humanization of N-glycosylation in plants. In addition to their well-described advantages for fast, high-yield, low-cost and contamination-free production of pharmaceutical proteins, recent progress made in the engineering of PTMs making plants a powerful expression system for the production of optimized plant-made therapeutic proteins showing similar, or even higher, biological activity than their homologues expressed in cultured mammalian cells.
All of these advances illustrate the ‘coming of age’ of plant-based biopharmaceutical manufacturing. As platforms are matched to products, we shall see a number of novel plant-derived products move through the clinic and into approval. Clearly, there is nothing about plant-based systems that poses insurmountable obstacles at the regulatory level. It is now necessary for plant scientists to demonstrate the power of plant systems in the many applications described in this issue.