The first target analyzed is human butyrylcholinesterase , an enzyme that can act as a bio-scavenger to counteract the effects of cholinesterase inhibitors such as sarin and that is a candidate for bio-defense countermeasures in several countries. While this product would encounter market dynamics that are different from other commercial products, it is nevertheless designed to satisfy an important component of public safety and merits review. Currently, BuChE is extracted from outdated human blood supplies, but it can also be made recombinantly in cell culture, transgenic animals, and plant systems. The second case study focuses on the cellulase complex, a mixture of 4–6 enzymes used to saccharify cellulosic feed stocks for the production of ethanol as a fuel extender. This target was selected for study because, for more than 30 years, the cost of cellulases has been a major impediment to the economic viability of cellulosic ethanol programs. Cellulases were also selected because they represent an extremely cost sensitive product class on which to conduct case studies. We reasoned that if plant-based manufacturing showed economic promise for this class, then the economically advantageous production of less cost-sensitive biotherapeutics and other products might also be anticipated. In contrast to BuChE, which consists of a purified molecule, the cellulase complex would be expressed in plants that are cultivated near the cellulosic feedstock and the bio-ethanol refinery and stored as silage without purification; the semidried catalyst biomass is mixed on demand with the cellulosic feed stock to initiate saccharification followed by fermentation. This approach varies significantly from previous approaches in which cellulase enzymes are produced via fermentation processes using native or engineered microorganisms.
For the cellulase case study, the plant-based cellulase production process is compared with a recent technoeconomic analysis of cellulase enzymes produced from Trichoderma reesei fer mentation using steam-exploded poplar as a nutrient source.The technoeconomic modeling for both case studies was performed using SuperPro Designer, Version 9.0 , a software tool for process simulation and flow sheet development that performs mass and energy balances,rolling bench equipment sizing, batch scheduling/debottlenecking, capital investment and operating cost analysis, and profitability analysis. This software has been used to estimate cost of goods in a variety of process industries including pharmaceuticals produced by fermentation and plant made pharmaceuticals. It is particularly useful at the early, conceptual plant design stage where detailed engineering designs are not available or warranted. Super Pro Designer was chosen because it has built-in process models and an equipment cost database for typical unit operations used in the biotechnology industry, such as bioreactors, tangential flow ultrafiltration and diafiltration, chromatog raphy, grinding/homogenization, and centrifugation. There are some unit operations and processes used in the case studies that are currently not included in SuperPro Designer, such as indoor or field plant cultivation, plant harvesting, vacuum agroinfiltration, and screw press/disintegrator. For the butyrylcholinesterase case study, SuperPro Designer’s “Generic Box” unit procedure was used to model these unit operations. For the cellulase case study, the indoor unit operations were modeled with the same software while the field production calculation and costs were tracked in Microsoft Excel spreadsheets. Unless otherwise noted, the costs of major equipment, unit operation-specific labor requirements and costs , pure components, stock mixtures, heat transfer agents, power and consumables used in the analyses were determined using the SuperPro Designer built-in equipment cost model and default databanks. For the cellulase case study, the program’s parameters such as water costs and total capital investment distributed cost factors were set to be the same as those used in the model described in Klein-Marcuschamer et al.; this SuperPro Designer model is also available at the Joint Bio-energy Institute technoeconomic analysis wiki site .
Additional case study specific design parameters were selected based on experimental data from journal articles, patent literature, the authors’ laboratory, interviews with scientists and technologists conducting the work cited, technical specification sheets or correlations, heuristics, or assumptions commonly used in the biotechnology and/or agricultural industry. The case study models were based on a new “greenfield” facility, operating in batch mode, although annual production costs neglecting the facility dependent costs were also determined to predict annual production costs using an existing facility. For the butyrylcholinesterase case study, annual operating time of 7920 hours for the facility was used with indoor grown Nicotiana benthamiana plants.For the cellulase case study, since the tobacco plants are grown in the field, it is assumed that plant growth occurs for 215 days of the year and the indoor facility is in operation for 127 days per year . For comparative purposes in the cellulase case study, the laboratory/QA/QC costs were neglected since they were neglected in the JBEI model and such costs are likely to be a minor component for the industrial enzyme case study.For the butyrylcholinesterase case study, the process flow sheet was split into separate modules to better understand the contributions of various process segments.Process flow and unit operations were derived from published methods and results from a number of sources as indicated in each case study, and from interviews with leading gene expression, agronomy, and manufacturing scientists and engineers who have participated in the development and scale-up of the processes described. On the basis of this information, the SuperPro Designer software was applied to calculate material inputs and outputs, bulk, and per-dose or per-unit costs.The two AI classes evaluated in these studies are produced in Nicotiana host plants. Nicotiana species, notably N. tabacum, N. excelciana, and N. benthamiana, are preferred hosts for PMB manufacture due to their metabolic versatility, permissive ness to the propagation of various viral replicons, and high expression yields achievable with a wide range of targets, as reviewed by Pogue et al., De Muynck et al., Thomas et al., Gleba et al., and others. Use of these hosts for production of clinical trial materials is also familiar to FDA and other regulatory agencies, thus facilitating Nicotiana’s acceptance in regulation-compliant manufacturing.
The enzyme is a globular, tetrameric serine esterase with a molecular mass of approximately 340 kDa and a plasma half-life of about 12 days; the plasma 1/2 is largely a function of correct sialylation. BuChE has several activities, including the ability to inactivate organophosphorus nerve agents before they can cause harm. 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,grow table hydroponic 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.. Goat milk 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, the plant route for BuChE manufacture is also the subject of continued DARPA interest and support .BuChE can be produced sta bly 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 Agrobacterium mediated expression systems, including Icon Genetics’ mag nICON 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 vacuum assisted infiltration has been described in detail by Klimyuk et al., Gleba et al., and others and is not further explained here.For BuChE, we mod eled the use of an N. benthamiana transgenic line modified to express the mammalian glycosylation pathway, beginning with a mutant host lacking the ability to posttranslationally 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 glycan engineered 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 bio-ethanol 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 biocatalyst 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 bio-fuel 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.