The original principle of using plants as a protein-manufacturing platform has been considerably extended through the subsequent development and application of a number of novel attributes, improvements, and ancillary technologies, as summarized in Table 3. Some of these developments such as enhanced expression levels, and ability to express multisubunit proteins, effectively increase the scope and appeal of the PMP approach. Others, such as customization of post-translational modifications in the plant, ability to accommodate genes for very large or very small proteins, and an emphasis on nonfood host plants, represent ways to address various limitations, concerns and criticisms that have been raised (conveniently summarized in Twyman et al., 2005). A third category of developments more directly addresses customer/client needs (Table 3).
PM(I)P application and performance enhancements
With expression levels of most PMP systems now attaining the ‘gold standard’ of 1–10% TSP and some systems substantially exceeding that enrichment (Table 1), it seems unlikely that further enhancements in this performance parameter will have a dramatic effect on prospects for successful commercialization. Nevertheless, to the extent that expression level influences the final yield of purified protein per plant, greenhouse or acre, its continued improvement (reviewed by Streatfield, 2007) may usefully contribute competitiveness within the industry, and in relation to the alternative production platforms. The demonstrated ability to express a wide range of product types, ranging from small peptides (e.g. Gil et al., 2001) to large molecules with complex quaternary structure such as secretory antibodies and collagen (Ma et al., 1995; Stein et al., 2009) has further expanded PMPs’ scope of competitive application. And optimization of host-plant germplasm and plant-production methods for responsiveness to the expression system and maximum biomass yield can also contribute to increased overall efficiency of the PM(I)P strategy (e.g. for tobacco: Davies, 2005; Sheludko et al., 2007).
A PMP ‘performance’ topic that has attracted considerable attention in relation to open-field production has been ‘containment’, i.e. the prevention of genetic and physical contamination of other, non-PM(I)P and nontransgenic crops. Genetic containment seeks to obviate any chance of transgene transfer via outcrossing to other plants, whether crops or naturalized ‘wild’ populations, while physical containment seeks to prevent accidental co-mingling of the harvested PM(I)P-plant material with other harvests. The frequent adoption and promotion of major food-plant species such as corn, rice, potatoes, alfalfa as the PM(I)P production hosts in the earlier years of commercialization was unfortunate in this regard. Although the use of fully developed agricultural crops offers many advantages this food-crop emphasis inevitably encountered the emerging opposition to ‘GM’ agriculture, to which it unwittingly added a further basis for objection, namely the imagined, unsettling prospect of contamination of the food supply with pharmaceuticals and industrial enzymes because of inadvertent co-mingling during production or harvesting. The concerns received further impetus from the much-publicized failure of PMP company ProdiGene to maintain adequate physical and genetic containment of its transgenic corn line that produced a veterinary vaccine, resulting in contamination of the next season’s (2002) non-PMP crop production on the same (and possibly adjacent) land (Fox, 2003). While this incident was intercepted long before any derived food products were affected, the resulting intensity of opposition to the use of food crops for PM(I)P applications began to include even pro-agricultural biotechnology observers (Editorial, 2004) and the food industry in general (GMA/FPA, 2007), effectively quenching future prospects for developing new PM(I)P applications in major food species like corn and soya beans.
Following those significant public objections to the perceived prospect of unintended contamination of food by pharmaceuticals and industrial proteins, the evolving PM(I)P industry has taken various steps to mitigate such possibilities (reviewed by Murphy, 2007). These actions have included selecting host-plant species that are not major food crops (e.g. SemBioSys Genetics’ emphasis on safflower, several companies’ interest in tobacco), growing the PMP crop in a region or location distant from that where the same plant is grown for food production (e.g. Ventria Bioscience’s location of transgenic rice production; Turner, 2006), exclusive use of indoor production (e.g. McCarthy, 2005; Bayer Innovation, 2008), and an emphasis on plastid-based and other gene-expression systems that reduce or obviate the chances of unintended transgene transfer to the same or related species through natural outcrossing events (e.g. Gleba et al., 2005; Grevich and Daniell, 2005). Even producers of nonfood crops may object to PMPs. For example, the traditional tobacco industry currently has a zero-tolerance standard for ‘GMO’ contamination in the crop (Dean Wallace, Council for Burley Tobacco, personal communication), and consequently we have examined the use of interspecific Nicotiana hybrids as alternatives to commercial tobacco varieties in PM(I)P applications (Davies, 2005). Such hybrids exhibit extremely low fertility, thereby reducing the chances of transgene transfer to conventional tobacco plants via pollination and of persistence of the transgenics in the environment through the setting and dispersal of seeds containing the transgene.
When transgenic plants are field-grown for PM(I)P applications, their developers will also need to incorporate other safety- or acceptance-related features which come to be expected or mandated for transgenic crops in general. The relevant technologies may have to be in-licensed by the PM(I)P company, potentially adding to the cost of development and affecting product price. As these aspects are not PM(I)P-specific they are not discussed in detail here, except to cite the drive to eliminate the use of antibiotic-resistance genes as a currently prominent example (technologies reviewed in Goldstein et al., 2005).
Governmental regulatory authorities in several countries have issued, and continue to develop, guidelines and regulations for the conduct of field trials and eventual production of PM(I)P crops (Spok, 2007; Chambers et al., 2008; Spok et al., 2008; USDA, 2008). The mandated regulatory requirements are equally applicable whether the new transgenic crops harbour products intended for medical or for industrial uses (USDA, 2008). The above-mentioned ‘containment’ technologies, together with appropriate growing and handling methods, contribute in the preparation of crop-production protocols that must be officially approved prior to any open-field planting. If there is reason to be concerned that the nonnative protein product may pose a health threat to organisms which are predatory on the plant species of choice, or which are consumers of plant-parts such as seeds or pollen, the regulatory authorities may also require an assessment or study of such ‘environmental’ risk. Such a study will take into account the potential toxicity and other properties of the pharmaceutical or industrial protein itself, and the physical and genetic containment prospects for the host plant and its cultivation. A convenient example of this process in the United States is provided by the ‘Finding of No Significant Impact’ (FONSI) issued by the USDA to PMP company Planet Biotechnology in relation to that company’s plans to field-grow tobacco plants expressing an antimicrobial antibody intended to treat dental decay (U.S. Federal Register, 2008).
It is reasonable to expect that stringent regulatory requirements will continue to exist for open-field PM(I)P deployment in the future. And this situation may not be limited to experimental plantings—the suggestion has been made that such crops may never be deregulated in the manner of today’s agronomically enhanced transgenics (Spok et al., 2008). It will, therefore, continue to benefit the industry to research and develop robust methods for both types of containment.
Optimization of PM(I)P production in relation to purpose and specifications of the product
In the opinion of this reviewer, it is the enhancements that directly address ‘fitness for purpose’, i.e. compatibility with the needs, standards, and expectations of the product’s end-user, that are most likely to influence the future commercial fortunes of the plant-made approach, especially for medical applications. Positioning the production platform as close as possible to the end-user’s requirements not only maximizes the chances of acceptance but also helps to contribute competitiveness with respect to the other protein-manufacturing systems. Currently, significant advances are being made in this category, both technologically (Table 3) and from a business perspective.
Naturally, production of authentic product, which exhibits the expected biological functionality and is not accompanied by improperly folded or assembled protein, is a basic requirement of any biomanufacturing system. Plants have proved themselves remarkably capable in this respect—for example, the accurate assembly of functional antibody molecules involves both post-translational disulphide bridging and noncovalent assembly of identical and different polypeptides, and this challenge has been met successfully for many different immunological products, including such complex molecules as pentameric secretory immunoglobulin A (Ma et al., 1995). But when biomanufacturing systems compete to make a pre-existing drug (as in the production of biosimilars or ‘follow-on’ protein drugs), the matter of product specification becomes especially critical. This is because the shortest, most economical route for regulatory approval of a pharmaceutical made by the new process will be the demonstration of product equivalence, i.e. showing that the product made in the new way is in all respects (structural, compositional, and functional) identical to the one made by the previously approved method (Woodcock, 2007; Schellekens, 2009). Authenticity of fundamental structure and biological activity may not be sufficient to achieve acceptance by the relevant pharmaceutical regulatory system; it will also be necessary to demonstrate that there are no plant-specific modifications such as covalent attachment of reactive secondary metabolites, species-specific glycosylation events etc., and to show that the molecule’s biological and pharmacological properties are sufficiently similar to those from the natural or previously approved source as to obviate any risks of unintended immunogenicity or toxicity (Woodcock, 2007; Schellekens, 2009). A recent example that illustrates a comprehensive structural and functional assessment of the plant-generated recombinant product is that of human collagen produced in tobacco plants (Stein et al., 2009). Authentic formation of collagen in a heterologous production system necessitates hydroxylation of lysyl and prolyl resides in the two different polypeptides, glycosylation of the hydroxylysyl resides with galactosyl and glucosyl substituents, formation of intrachain and interchain disulphide bonds, and assembly of the modified polypeptides into a triple helix. The PMP version of collagen was made by co-expression in the plant of the two collagen genes and two human enzymes to supplement the plant’s inherent mechanisms for post-translational modification of the polymer. Stein et al. (2009) employed a range of physical, biochemical and bioassay criteria to validate their plant-made collagen, and while their characterization does not appear to have been exhaustive, their emphasis on establishing product equivalence is both appropriate and timely.
If problematic issues such as spurious derivatization or proteolysis should occur in the plant or upon extraction of the product, the possibility theoretically exists to engineer the plant to eliminate the problem. As a hypothetical example, the activity of polyphenol oxidase, which by catalysing oxidation of phenols to reactive quinones can seriously compromise proteins during purification from some plant species, can be reduced through gene-suppression (Bachem et al., 1994). A variety of techniques for mitigating proteolysis were recently summarized (Benchabane et al., 2008), including the use of host plants with minimal protease activities against the product, subcellular targeting of the product to reduce its accessibility to proteases, ectopic expression of protease inhibitors, etc.
There is no more salient example of customization of the host-plant for generation of application-acceptable product than that of protein glycosylation. This topic is important because the carbohydrate moieties of glycoprotein drugs may contribute significantly to tolerance by the patient’s immune system, and to the proper functioning of antibodies (Wright and Morrison, 1997). Moreover, there has been some concern regarding the suitability of plants for the biomanufacturing of mammalian glycoproteins, as the glycosylation pathways in plants are sufficiently different from those in animals that mammalian-authentic glycan structures are not achieved, and the resulting ‘novel’ plant-made glycan moieties can be immunogenic (for a recent example of this issue see Jin et al., 2008). For example, after some debate on the matter it seems likely that most plants do not elaborate sialic acid, a common residue in animal glycoprotein oligosaccharides (Zeleny et al., 2006). Many other detail, but significant, differences occur in linkages and arrangements of the monosaccharide residues in plants vs. animals, as well as in overall heterogeneity of the protein-linked glycans (reviewed in Gomord et al., 2005). But, fortunately, the glycosylation sequence-motifs in protein primary structure are the same in plants and animals, as is much of the fundamental post-translational glycosylation mechanism, so that carbohydrate attachment tends to occur at the correct locations in the protein. Thus, the challenge of making a ‘humanized’ glycoprotein in a plant is principally one of adjusting the composition and structure of the oligosaccharide substituents.
Significant progress is being made towards this fascinating if ambitious metabolic engineering goal, as recently illustrated by Frey et al. (2009) and Castilho et al. (2010) and reviewed by Karg and Kallio (2009). The recent accomplishment of Castilho et al. (2010) is particularly striking; six mammalian genes were introduced into Nicotiana benthamiana to enable that plant to make and utilize sialic acid, with subsequent successful incorporation of sialate residues at the termini of the glycan moieties of a monoclonal antibody that was co-expressed with them. Meanwhile, an interesting nearer-term and less demanding alternative that may be applicable to some pharmaceutical products is to modify the glycan moieties until they are no longer immunogenic (Gomord et al., 2005) or to eliminate them altogether (Nuttall et al., 2005). It remains to be seen whether such modified molecules, or ‘humanized’ ones that still exhibit a percentage of variant structures as in the incompletely sialylated forms observed by Castilho et al. (2010), will be accepted as ‘biosimilars’ by regulatory authorities or regarded as sufficiently novel to require the same approval process as entirely new drug candidates.
Another aspect of the PMP strategy that can influence product quality and standards is postharvest bioprocessing. Given the considerable extent to which product-purification can be influenced and controlled in fermentation systems, e.g. ensuring that the product is secreted from the cells into the culture medium, engineering the cells to eliminate problem host constituents, etc., the challenge of purification from the protein complexity represented by whole-plant tissues and organs might seem to be a disadvantage, especially when one considers the very high standards of purity required of medical products. But this topic has received considerable attention (Menkhaus et al., 2004), and many strategies that facilitate efficient purification from other protein biomanufacturing systems have also been employed successfully with plants, such as those based on biological affinity between the product and molecules that can be immobilized on inert supports (e.g. protein A recognition by antibodies; Hober et al., 2007), or on expression of the product fused to an affinity-matrix-recognized protein or peptide ‘tag’ that can easily be removed after the chromatography step has been completed (e.g. Desai et al., 2002; Leelavathi and Reddy, 2003). Such sophisticated and potentially expensive methods may not be obligatory, despite the relative complexity of plant extracts; Platis et al. (2008) demonstrated that even the simplest, traditional techniques such as solvent partitioning and ion-exchange chromatography have potential in the quest to purify monoclonal antibodies from tobacco leaves in a minimum number of economical steps. And recently attention has been drawn to new strategies that employ fusion of the recombinant protein product with elastin so that purification can take advantage of the thermally triggered aggregation and precipitation of the latter (Floss et al., 2010), and with fungal hydrophobins that enable the fusion product to be separated from other proteins by solvent partitioning (Joensuu et al., 2010). These methods effectively achieve the level of specificity which is obtained with affinity chromatography but without the high cost incurred with the latter media and systems.
The PMP host and expression system may also be chosen and designed specially to facilitate downstream bioprocessing. Targeting the gene product to specific plant organs such as seeds and tubers from which purification will be easier because of a less complicated native-protein composition, and/or the absence of interfering secondary metabolites, is the most basic approach (examples listed in Horn et al., 2004; reviewed in Lau and Sun, 2009). SemBioSys’ PMP system directs the transgene product to the oil body of an oilseed plant, effecting an even closer integration with the bioprocessing method because the oil body is an organelle that offers the combined benefits of a simple native-protein composition and facile extraction from the seed (Markley et al., 2006). Similarly, Hassan et al. (2008) have explored the relationship between product localization within leaf tissue and ease of subsequent extraction. Recently, another in-cell sequestration system appears to be in development (Erabiotech, 2009). Other strategies that enable the product to be separated from the bulk of the host-plant constituents quickly and efficiently include fusion to virus particles (e.g. Smith et al., 2009), and natural secretion from roots into the surrounding medium (Borisjuk et al., 1999; Xuejun et al., 2002).
The development of economical postharvest bioprocessing also requires construction of pilot facilities which represent significant scale-up from laboratory-based demonstrations, and their use to research procedures for efficient isolation of product from substantial quantities of plant material to the required final specifications. To date, a few PM(I)P-oriented commercial ventures have made this investment, e.g. Kentucky Bioprocessing (USA), Medicago (Canada), Meristem Therapeutics (France), SemBioSys (Canada), Sigma-Aldrich Fine Chemicals (USA), Ventria Biosciences (USA). For pharmaceutical products, the bioprocessing pathway itself will have to meet government-mandated standards, and to do so in a completely reproducible and reliable manner. Establishment of the stringent quality control and quality assurance procedures that meet these requirements for plant-based production can only be achieved by working at the final production scale. Thus, an emphasis on postharvest bioprocessing is increasingly important, and the pioneering work that has been done on this topic so far is undoubtedly money well-spent.
Another topic of considerable importance in relation to postharvest bioprocessing of PMPs, and assurance of product quality, is the need to obviate ‘adventitious presence’. This term is commonly used in reference to inadvertent mixing of transgenic and nontransgenic foodstuffs, but in the PMP context it can mean the (unintended) presence in the harvest of biological or nonbiological materials that are not taken into account in the design of the bioprocessing protocol which isolates the pharmaceutical product. Insect parts, bird excrement, and pesticide drift from other crops are some of the many examples of materials which might contribute substances that could perhaps work their way through the purification sequence and thus contaminate the final protein product. Obviously, production platforms that use higher organisms face a greater challenge with this issue than do the fully contained fermentation systems. One way in which some PMP companies are addressing it is to eschew field-based production in favour of indoor growing (e.g. Bayer Innovation, 2008; Drummond, 2010; other companies via personal communication), a change that also eases the earlier-mentioned concerns about containment and public acceptance. A further advantage of indoor planting is that it enables year-round production, as opposed to the seasonal format of the field (production year-round may also be achieved outdoors in regions of the world having suitable climates, but this option will not be acceptable for production of certain nationally strategic agents such as vaccines to combat bioterrorism). Because growing plants in greenhouses may be significantly more expensive than open-field agriculture, additional research will be necessary to optimize germplasm and production methods for maximum efficiency in the enclosed environment. Economies may be realized through the use of very small plants that can be grown in a highly mechanized, automated manner, such as Biolex’s choice of the aquatic plant Lemna (McCarthy, 2005). Whatever the eventual solutions, the PMP industry today is cognizant of the issue of adventitious presence, and this is a welcome contrast to the early years when the concept of making pharmaceuticals in the open field seemed to be promoted with little or no attention to this important topic.
The continuing development of technology enhancements will become increasingly important not only to strengthen the capability and applicability of the PM(I)P approach but also from a business perspective; as the lifetimes of patents covering the fundamental PM(I)P gene-expression technologies come to an end this ‘add-on’ intellectual property will help to sustain company competitiveness and investor interest. Examination of the scientific literature describing many of these innovations will show the considerable and continuing role of universities and other nonprofit organizations in conducting the underlying inventive research. Given the under-resourced status of the PM(I)P industry compared with the large multinational companies that market the ‘GM’ mainstream crops, this public-domain research is likely to remain critical to the commercial prospects for PM(I)Ps for some time to come.