Current Pharmaceutical Design - Volume 19, Issue 31, 2013

Many different plant-based systems have been used to produce recombinant pharmaceutical proteins but only a small number have made the leap from an experimental platform to a viable commercial process. This reflects a combination of factors, principally the technical issues that must be addressed to achieve competitive performance, the economic principles that need to be satisfied to ensure manufacturing processes are financially viable and sustainable, and the regulatory demands that must be met to ensure that pharmaceuticals manufactured in plants are safe, efficacious and meet the quality standards demanded by the regulators. With the recent approval of the first plant-derived recombinant pharmaceutical protein designated for human use, we are now entering a new era in which plants not only meet all the demands of a commercial pharmaceutical manufacturing process but also provide unique benefits that allow the displacement of established platform technologies in niche markets. In this article, we consider the commercial aspects of molecular farming, specifically those required to make plants more competitive and attractive to industry.

Four major developments have taken place in the world of Molecular Pharming recently. In the USA, the DARPA initiative challenged plant biotechnology companies to develop strategies for the large-scale manufacture of influenza vaccines, resulting in a successful Phase I clinical trial; in Europe the Pharma-Planta academic consortium gained regulatory approval for a plant-derived monoclonal antibody and completed a first-in-human phase I clinical trial; the Dutch pharmaceutical company Synthon acquired the assets of Biolex Therapeutics, an established Molecular Pharming company with several clinical candidates produced in their proprietary LEX system based on aquatic plants; and finally, the Israeli biotechnology company Protalix Biotherapeutics won FDA approval for the commercial release of a recombinant form of the enzyme glucocerebrosidase produced in carrot cells, the first plant biotechnology-derived biopharmaceutical in the world approved for the market. Commercial momentum is gathering pace with additional candidates now undergoing or awaiting approval for phase III clinical trials. Filling the product pipeline is vital to establish commercial sustainability, and the selection of appropriate target products for Molecular Pharming will be a critical factor. An interesting feature of the four stories outlined above is that they span the use of very different platform technologies addressing different types of molecules which aim to satisfy distinct market demands. In each case, Molecular Pharming was an economically and technically suitable approach, but this decisionmaking process is not necessarily straightforward. Although the various technologies available to Molecular Pharming are broad ranging and flexible, competing technologies are better established, so there needs to be a compelling reason to move into plants. It is most unlikely that plant biotechnology will be the answer for the whole biologics field. In this article, we discuss the current plant biotechnology approaches that appear to hold the greatest promise and in doing so attempt to define the product areas that are most likely to benefit from different Molecular Pharming technologies.

The production of recombinant pharmaceutical proteins in plants is entering a new phase with the recent approval of recombinant glucocerebrosidase produced in carrot cells and the successful production of clinical-grade proteins in diverse plant-based production platforms. In the long journey from concept to product, the field of molecular farming has faced technical and economic hurdles, many reflecting the initially limited productivity of plants compared to established platforms such as mammalian cells. This challenge has been met by innovative research aiming to increase recombinant protein yields and maximize the economic benefits of plants. Research has focused on increasing the intrinsic yield capability of plants by optimizing expression construct design, and also on novel strategies to avoid epigenetic silencing and environmental effects on protein accumulation. In this article, we discuss the diverse approaches that have been used to increase the productivity of plant-based platforms for the production of recombinant pharmaceutical proteins and consider future opportunities to maximize the potential of plants and increase their competitiveness outside niche markets.

Many plant species and tissues have been investigated as production and delivery vehicles for recombinant pharmaceutical proteins. Examples include cultured cells, whole aquatic plants and transgenic plants expressing recombinant proteins in their leaves, seeds, fruits or tubers/roots. Each platform has unique properties in terms of production time, environmental containment, scalability and overall costs. Plant tissues also differ in their abilities to sort, modify and accumulate proteins. Seeds are naturally adapted for protein accumulation and possess specialized storage organelles that may be exploited to accumulate recombinant proteins, offering stability both in planta and after harvest. Generally, the post-harvest stabilizing effect offered by storage tissues is advantageous for pharmaceuticals, allowing them to be delivered via the mucosal route because they are better able to withstand the harsh microenvironment when protected by the plant matrix. Native storage organelles such as starch granules, protein storage vacuoles and protein bodies thus offer interesting possibilities for the delivery of vaccines and antibodies, as well as novel storage organelles that can be induced ectopically in non-storage tissues. The specialization and distinct intracellular organization of storage tissues also affect the trafficking and modification of recombinant proteins. The N-glycosylation of recombinant glycoproteins often differs subtly depending on the plant species and tissue, reflecting both the availability of different sets of glycan-modifying enzymes and the compartmentalization of the proteins. Where a specific glycan structure is required, it is therefore important to choose the appropriate plant system as a production platform.

Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex glycosylation is well known. Moreover, the targeted manipulation of the plant N-glycosylation pathway allows the production of proteins carrying largely homogeneous, human-type oligosaccharides. These outstanding results have placed plants in a favourable position compared to other eukaryotic expression systems. This review provides a comprehensive summary of the N-glycosylation of plant-produced recombinant proteins, the possible impact of plant-specific N-glycans on the human immune system, and recent advances in engineering the plant N-glycosylation pathway towards the synthesis of (complex) human-type glycan structures, highlighting challenges and achievements in the application of these powerful technologies.

In this article, the general principles of genetically modified (GM) plant risk assessment and the regulatory framework for contained use and open field production of plant-made pharmaceuticals/plant-made industrials (PMP/PMI) are described. While significant progress has been made for the containment grown (plant cell culture) production of PMPs, with the first regulatory approval made by the FDA in 2012, the commercialization of medicinal or industrial products produced in the field has yet to emerge in either Europe or the US. In the current paper, we discribe the regulatory environment in Europe and the US surrounding GM crops, and provide case studies for experimental field releases of PMP and PMI producing plants in both regions. Suggestions for reducing the regulatory burden for GM plants will be discussed, also in light of the emerging new technologies to modify the genetics of plants. Since regulations surrounding the commercialization of GM crops are very costly and not appropriate for most of the PMP/PMI applications in Europe, we propose that amendments to the EU Directive 2001/18/EC are necessary to allow for the commercialization of products from GM plants without the need of an ‘authorization’. To fully acknowledge the overall outcome of adopting plants to produce PMP/PMI, the conclusion is that broader and more balanced legislative oversight is needed in Europe; while specific legislation is not needed in the US.

Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sustainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the product and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production platforms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in containment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower compared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process optimization, helping to increase yields and scalability.

The production of recombinant proteins in seeds is achieved by driving transgene expression using promoters and protein targeting sequences derived from genes encoding abundant seed storage proteins. This approach is advantageous because high yields, stability and containment are conferred by the accumulation of recombinant proteins in specialized storage compartments such as protein bodies and protein storage vacuoles. Seeds are particularly suitable for the production of pharmaceutical proteins in developing country settings because they reduce the costs of production and distribution by avoiding the need for fermenter-based production capacity and a cold chain for storage and distribution, thus increasing access to critical medicines for the poor in rural areas. Seeds are also ideal for the production of oral vaccine antigens, because encapsulation within the seed provides protection that prolongs exposure to the gastric immune system and thus increases the potency of the immune response. In this review we discuss the current state of the art in seed-based molecular pharming and the future potential of production platforms based on seeds.

Seeds are organs specialised in accumulating proteins, and they may provide a potential economically viable platform for the large-scale production and storage of many molecules for pharmaceutical and other productive sectors. Soybean [Glycine max (L.) Merrill] has a high seed protein content and represents an excellent source of abundant and cheap biomass. Under greenhouse conditions and a daily photoperiod of 23 h of light, the soybean plant’s vegetative growth can be significantly extended by inducing more than a tenfold increase in seed production when compared with plants cultivated under field conditions. Some factors involved in the production of different recombinant proteins in soybean seeds are discussed in this review. These include transgenic system, regulatory sequences and the use of Mass Spectrometry as a new tool for molecular characterisation of seed produced recombinant proteins. The important intrinsic characteristics and possibility of genetically engineering soybean seeds, using current advances in recombinant DNA technology including metabolic engineering and synthetic biology, should form the foundation for large-scale and more precise genome modification, making this crop an important candidate as bioreactor for production of recombinant molecules.

Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.

Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of “edible” vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the M2e-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.

In the last two decades the development of efficient plant-based expression strategies and new concepts for the purification of recombinant proteins prompted the application of plant-derived vaccines for veterinary purposes. The expression of recombinant proteins in plants possesses advantages over conventional eukaryotic expression systems and therefore represents a versatile tool for the production of “edible” and “seasonal” vaccines. This review aims to provide an overview about the expression of vaccines using transgenic plants for veterinary medicine with the focus on increasing the amount of the recombinant proteins as well as concepts for their efficient purification. ELPylation of recombinant proteins is one strategy for on one side boosting the amount of the recombinant protein and on the other side simplifying its purification. This up-and-coming tool was applied for the development of effective production and purification strategies for antigens against Avian Flu, a very important animal disease with a strong economic impact. Future perspectives of plant-based veterinary vaccines in the context of purification and economy are also discussed within this article.

Molecular farming is a technology that is very well suited to being applied in developing countries, given the reasonably high level of expertise in recombinant plant development in many centers. In addition, there is an urgent need for products such as inexpensive vaccines and therapeutics for livestock and for some human diseases – and especially those that do not occur or are rare in developed regions. South Africa and Argentina have been at the fore in this area among developing nations, as researchers have been able to use plants to produce experimental therapeutics such as nanoantibodies against rotavirus and vaccines against a wide variety of diseases, including Rabbit haemorrhagic disease virus, Foot and mouth disease virus, Bovine viral diarrhoea virus, bovine rotaviruses, Newcastle disease virus, rabbit and human papillomaviruses, Bluetongue virus, and Beak and feather disease virus of psittacines. A combination of fortuitous scientific expertise in both places, coupled with association with veterinary and human disease research centers, has enabled the growth of research groups that have managed to compete successfully with others in Europe and the USA and elsewhere, to advance this field. This review will cover relevant work from both South Africa and Argentina, as well as a discussion about the perspectives in this field for developing nations.

Secondary products are small molecular weight compounds produced by secondary metabolic pathways in plants. They are regarded as non-essential for normal growth and development but often confer benefits such as defense against pathogens, pests and herbivores or the attraction of pollinators. Many secondary products affect the survival and/or behavior of microbes, insects and mammals and they often have useful pharmacological effects in humans. Most secondary products can only be obtained as extracts from medicinal plants, many of which grow slowly and are difficult to cultivate. Chemical synthesis, although possible in principle, is often impractical or uneconomical due to the complexity of their molecular structures. The large scale production of secondary products by metabolic engineering has therefore been investigated in a number of heterologous systems including microbes, plant cell/organ cultures, and intact plants. In this critical review of production platforms for plant secondary products, we discuss the advantages and constraints of different approaches and the impact of post-genomics technologies on gene discovery and metabolite analysis. We highlight bottlenecks that remain to be overcome before the routine exploitation of secondary products can be achieved for the benefit of mankind.

Molecules derived from plants make up a sizeable proportion of the drugs currently available on the market. These include a number of secondary metabolite compounds the monetary value of which is very high. New pharmaceuticals often originate in nature. Approximately 50% of new drug entities against cancer or microbial infections are derived from plants or micro-organisms. However, these compounds are structurally often too complex to be economically manufactured by chemical synthesis, and frequently isolation from naturally grown or cultivated plants is not a sustainable option. Therefore the biotechnological production of high-value plant secondary metabolites in cultivated cells is potentially an attractive alternative. Compared to microbial systems eukaryotic organisms such as plants are far more complex, and our understanding of the metabolic pathways in plants and their regulation at the systems level has been rather poor until recently. However, metabolic engineering including advanced multigene transformation techniques and state-of-art metabolomics platforms has given us entirely new tools to exploit plants as Green Factories. Single step engineering may be successful on occasion but in complex pathways, intermediate gene interventions most often do not affect the end product accumulation. In this review we discuss recent developments towards elucidation of complex plant biosynthetic pathways and the production of a number of highvalue pharmaceuticals including paclitaxel, tropane, morphine and terpenoid indole alkaloids in plants and cell cultures.