With the recent use of chemical nerve agents such as sarin, there is continued interest on the part of many governments in stockpiling BuChE as a countermeasure. Currently BuChE is purified from outdated blood supplies; however, the high cost of this route and its low supply limit its utility. It has been estimated that extraction of BuChE from plasma to produce 1 kg of enzyme, which would yield small stockpile of 2,500 400- mg doses, might require extraction of the entire US blood supply. Large amounts of the enzyme are required for effective prophylaxis because of the 1 : 1 enzyme/substrate stoichiometry needed for protection against OP agents. Not surprisingly, recombinant routes have been explored and the enzyme can in fact be produced by microbial fermentation, animal cell culture, and transgenic goats and stably or transiently expressed in Nicotiana, albeit at modest levels of 20–200 mg/kg fresh weight biomass, with yield improvements being the target of ongoing research. The bacterial product is nonfunctional and the mammalian cell culture products do not have the plasma 1/2 needed for prophylaxis and may be difficult and expensive to scale, as discussed by Huang et al.. Goatmilk produced BuChE can be obtained at 1–5 g/L milk, but consists mostly of dimers, is undersialylated and has short plasma 1/2. While expression yields are impressive, transgenic animal sources face challenges of herd expansion to satisfy emergency demand, as well as potential adventitious agent issues, and these challenges need further definition. Furthermore, of these options, only plant-based bio-synthesisyields an enzyme that is sialylated and appears to reproduce the correct tetrameric structure of the native human form in sufficient yield to be commercially attractive; hence, the plant-based route became the basis for our modeling exercise. Not surprisingly,grow hydroponic the plant route for BuChE manufacture is also the subject of continued DARPA interest and support .

BuChE can be produced stably in recombinant plants or transiently in nonrecombinant plants by viral replicons delivered by agrobacterial vectors introduced into the plants via vacuum-assisted infiltration. Relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility, and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many PMP . In our analysis of BuChE, we used expression yields from several sources that evaluated various Agrobacteriummediated expression systems, including Icon Genetics’ magnICON expression technology. Magnifection should be familiar to most readers of this volume as it has been applied in R&D programs throughout the world and its features have been the topic of multiple original studies and reviews ; therefore, the method is not described here in further detail. Likewise, the process of vacuumassisted infiltration has been described in detail by Klimyuk et al., Gleba et al., and others and is not further explained here.For BuChE, we modeled the use of an N. benthamiana transgenic line modified to express the mammalian glycosylation pathway, beginning with a mutant host lacking the ability to post translationally add plant-specific pentoses but with the ability to add galactosyl and sialic acid residues to polypeptides, based on work recently reported by Schneider et al.. Use of this host obviates the need to enzymatically modify the plant-made polypeptide in vitro after recovery to ensure the presence of correct mammalian glycan, a procedure that could substantially increase the cost of the AI. A glycanengineered host can be produced in two ways, by stable transformation or via use of multi-gene agrobacterial vectors. The feasibility of sialylation via the latter approach was shown recently by Schneider et al.for BuChE. Although there is an extra element of time required to develop a stable transgenic host compared to the transient modification of a pathway, the availability of a transgenic plant obviates the need to manufacture several Agrobacterium vectors carrying the genes for the product and two binary vectors carrying genes for the sialylation pathway; a procedure that would require additional capital and operational investments to generate multiple inocula in large scale.

Therefore, for modeling upstream processes, we assumed that transgenic seed was available and that the resultant BuChE would have mammalian glycans and form tetrameric structures, and hence its biological activity and plasma half-life would be comparable to the native human enzyme.To model downstream purification of BuChE, we assumed harvest and extraction at 7 days after inoculation. Biomass disruption was by homogenization, followed by filtration and clarification, as generally described, but with modifications required for scale-up as indicated in Results and Discussion. Purification of the enzyme was by procainamide affinity chromatography. In the overall process, plant growth, inoculation, and product accumulation steps occur indoors in controlled environments, and extraction, clarification, and final purification of BuChE take place in classified suites, so that manufacturing and release of the enzyme can be compliant with FDA cGMP guidance for human therapeutics. Design premises for this process, specific assumptions used in modeling, and resultant cost calculations are presented .Cellulases currently under evaluation in bioethanol programs are all produced by microbial fermentation. Despite decades of research on lowering cellulase manufacturing costs, these enzymes still account for 20–40% of cellulosic ethanol production costs. Hence, lowering the cost of the bio-catalyst is critical to the eventual adoption of bio-fuel processes that utilize renewable plant biomass feed stocks without competing with food or feed supplies. An alternative to fermentation produced cellulases is the production of these enzymes in crop plants, with the ultimate goal of producing cellulases at commodity agricultural prices. This process concept was modeled to estimate enzyme and ethanol costs produced by this approach. Should such a process for cellulases prove economically viable, it might encourage the production of other cost-sensitive PMB as well as bio-materials, food additives, and industrial reagents.Scale requirements and cost limitations of cellulases for biofuel applications constrained us to model production to open fields, with minimal indoor operations. We initially surveyed two scenarios for inducing production of cellulases in field-grown plants. The first was adaptation of the typical agroinfiltration method. Nomad Bioscience has reported successful substitution of the agroinfiltration step with “agrospray,” a technique in which a suspension containing the Agrobacterium inoculant is admixed with a small amount of surfactant and sprayed onto the leaves of host plants. This approach eliminates the necessity to grow plants in containers , a requirement imposed by the mechanics of the vacuum infiltration treatment in current procedures.

Concomitantly, it also eliminates the cost of setting up and operating commercial-scale vacuum chambers, robotic tray manipulators, biomass conveyer systems, and so forth. Thus, this new approach should enable large-scale field inoculation of plants with agrobacteria and the production of biologics with more favorable economics. While we modeled the costs of producing cellulases via the agrospray approach, the sheer volume of enzymes needed for commercial-scale cellulosic ethanol processes necessitated a large investment in inoculum production infrastructure, including multiple fermentation trains and associated processing equipment. Further, the most efficient method of inoculating large areas was by aerial spraying, a procedure that not only entailed higher cost but that would also face regulatory uncertainties over spraying GM bacteria. We opted instead for an alternative model using transgenic N. tabacum plants, each line of which carries an ethanol-inducible gene for one component enzyme of the cellulase complex. Synthesis of the cellulase is triggered by application of a dilute solution of ethanol onto the leaves, a process that has been demonstrated in small scale using a double-inducible viral vector.We assumed that the dilute ethanol solution would be applied via ground irrigation systems that are currently used in agricultural practices,mobile grow rack instead of aerial tankers. It was also assumed that the ethanol would be taken off as a side stream from the associated ethanol production facility that uses the cellulase enzymes. In so doing, we obviated the need to produce multiple inocula of GM bacteria and deliver them via aerial spraying. We were also able to model higher biomass density as well as higher expression yields of the enzymes in planta. These changes resulted in multiple economic benefits and were therefore adopted in our calculations.Issues that are important in PMP, such as mammalian-like glycosylation or other post translational modifications, high purity, or specific formulation, are not relevant in the manufacture of cellulases and hence we modeled the use of conventional Nicotiana species in the production of the several enzymes necessary for complete saccharification of feed stock.The use of agricultural crops to produce enzymes at low cost has been suggested. In this case study, we modeled the use of stable transgenic N. tabacum varieties, each modified to express one cellulase protein upon induction with dilute ethanol. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the non-induced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. In greenhouse studies, recombinant proteins have been expressed at up to 4.3 g/kg FW leaf biomass in the ethanol-inducible hosts, but seed lines for field application have yet to be developed. In our modeling, we assumed that each transgenic line would have been already field tested and available for implementation. We also assumed that large-scale stocks of each transgenic seed would need to be produced and have included this unit operation in our cost calculations. Because cellulases are needed in different ratios to effect saccharification of different feed stocks, we assumed that seeds would be mixed at the appropriate ratios and that the seed mixtures would be planted directly in the field. At maturity, what one would expect is a field of plants representing all the needed cellulase classes in the appropriate ratio for the intended feed stock.

The current method of hydroponic cultivation of seedlings for transplantation to open fields, a common commercial tobacco cultivation practice to ensure germination and plants with good leaf size and quality, was substituted by direct seeding for more favorable economics. For example, traditionally tobacco may be grown at 12,000–16,000 plants/ha depending on variety. Higher-density seedling production for nontraditional uses of tobacco has been reported, targeting planting densities of over 86,000 plants/ha. While transplanting ensures germination and quality, there is an economic limit to the scale at which it can be deployed with highly cost-sensitive AI, leading to interest in direct seeding practices. Experimental high density cultivation studies via direct seeding have reported 400,000 to over 2 million plants/ha and biomass yields exceeding 150 mt/ha. Our modeling included these higher-density practices to determine economic impact.In contrast to typical PMP products, the cellulases would not be extracted after accumulation; rather, the plants would be mechanically harvested and transported to a centralized facility for silaging and storage. Since the cellulase enzymes need to be continuously supplied to the saccharification process in the bio-ethanol plant and the harvested tobacco is only available for a limited period during the year, the silage inventory would increase during the tobacco-harvesting period and would decrease during the fall/winter. Cellulase activity in the ensilaged biomass is expected to be stable during the off-season storage. For feedstock conversion, cellulase-containing biomass would be mixed with pretreated lignocellulosic feed stock under controlled conditions to effect saccharification. Although not considered in this economic analysis, this feed stock replacement could also reduce corn stover feed stock requirements and associated costs. After separation of solids, the sugar solution would be fermented conventionally into ethanol, followed by distillation. The overall process we modeled is based on the US National Renewable Energy Laboratory process described by Humbird et al, with substitution of fungal cellulase production in the NREL model by the cellulases stored as silage described herein. Design premises for this process, specific assumptions used in modeling, and the resultant cost calculations are presented .Process flow sheets for rBuChE production are shown. The seeding and indoor growth of N. benthamiana is shown in Figure 1.Figure 2 shows the agrobacterial seed train and production fermentor , the vacuum infiltration system , and the plant incubation facility for the infiltrated plants .