In the cost-constrained markets of food additives and processing aids, these new biotic approaches to food sanitation will need to be accessible at the low selling prices that the food industry is accustomed to, or gain market entrance as a luxury good on the basis of their differentiating features, including worker safety in the preparation and handling of the products, environmentally friendly disposal, non-impact on the organoleptic properties of food, and no or minimal food matrix alteration.Strategies to meet low cost of use can be broadly classified as either pertaining to molecular engineering of the treatment agent or manufacturing science and technology. Substantial research has been done to employ genetic engineering to alter the action of native antimicrobial proteins.For example, the modular structure of the bacteriophage class of enzymes known as endolysins provides a perfect “Lego® block”-like molecular engineering platform to swap the N-terminal catalytic domain or the C-terminal binding domain to create novel hybrid moieties.Although molecular engineering approaches possess substantial potential for human therapeutics, changes to the native structure of antimicrobial proteins for food safety applications bar them from taking advantage of the expedited GRAS marketing allowance pathway. For antimicrobials that are novel, or altered, and hence not “generally recognized” as safe, the alternative marketing approval route requires a full preclinical safety data package, which is a costly and time-consuming process that creates a significant barrier to entry for new food safety interventions, given the above mentioned current pricing structures, regulations, and public perception. Consequently,how to make a vertical garden biotic food safety approaches are more amenable to cost containment through manufacturing science and technology.

The cost sensitivity of the food industry is the most significant barrier to the adoption of new food sanitizing treatments, such as antimicrobial protein preparations. Plant-based platforms have the potential for producing market-relevant volumes of AMPs at competitive costs, because they do not require expensive bioreactors and culture media. In recent studies, we have shown that plants such as Nicotiana benthamiana, spinach, and leafy beets are an attractive and scalable production platform for production of AMPs, including antibacterial colicins, salmocins, and bacteriophage endolysins. We have previously reported expression levels as high as 3 g/kg plant fresh weight .In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. We used laboratory scale results and working process knowledge from pilot and commercial processes to develop a process simulation model using SuperPro Designer® to assess the commercial viability of the production platform and to identify economic “hotspots” to help guide future research and development. A selection of recently published studies on the techno-economics of N. benthamiana plant-based production of a variety of recombinant proteins is summarized in Table 1.To our knowledge, this study is the first techno-economic analysis of a plant-based production platform for AMPs as food safety additives.Nicotiana benthamiana is used as the plant host organism in the base case scenario. Nicotiana benthamiana is used extensively for indoor plant molecular farming applications based on its rapid growth, genetic tractability, susceptibility to agrobacterium transformation, and high expression levels of recombinant proteins.The species is used in the commercial scale production of therapeutics and vaccines by companies such as Kentucky BioProcessing Inc. ,Medicago Inc. ,and iBio CMO.The modeled facility is designed to accommodate a previously reported process using transgenic N. benthamiana featuring a doubleinducible viral vector, developed by Icon Genetics GmbH.

Published results demonstrate minimal background expression of recombinant protein until the induction of deconstructed viral RNA replicons from stable DNA proreplicons is triggered by 1–20% ethanol applied as a spray on the leaves and/or a drenching of the roots, to achieve expression levels as high as 4.3 g/kg plant FW.Although the more common Agrobacterium-mediated transient expression production platform enables rapid production of recombinant target molecules,this transgenic system obviates the need for additional expenses associated with Agrobacterium tumefaciens preparation, vacuum infiltration, and agrobacterium-introduced endotoxin removal.The simulated manufacturing facility is composed of two separate process models/flow sheets: the upstream processing models the plant growth, ethanol induction, and product generation, which feeds into the downstream processing model for purification of the product from the process and product impurities to meet food processing aid specification. Quality assurance , quality control , and laboratory costs associated with good agricultural and collection practices for upstream processing and FDA food industry current good manufacturing practice for downstream processing are included in the design. Equipment, materials of construction, and prices are also modeled on food cGMP standards.The location of commercial-scale plant molecular farming operations of Kentucky Bio-Processing Inc. was selected as the basis for location-dependent costs. Location-dependent costs are based on values obtained from publicly available Owensboro, Kentucky municipal pricing charts . The simulated manufacturing facility is assumed to be a greenfield single-product bio-manufacturing facility that is operational 24 hr per day and 7 days per week with an annual operating time of 90% or 329 days per year. Independent market analyses project a reasonable base case facility production level of 500 kg AMP per year for food safety applications of interest . To meet this demand, the proposed facility employs three-layer vertically stacked indoor plant cultivation stages designed for hydroponic host plant growth in a soilless substrate to support the plant and its roots. The cultivation stages are equipped with a light-emitting diode lighting system and a recirculating ebb and flow hydroponic water supply. The cultivation stage plant growth is divided into a series of trays that advance unidirectionally across the plant cultivation room toward automated plant harvesters and further downstream processing.

Automated belts convey harvested plant tissue to the double-stack disintegrator and further downstream processing.The upstream processing model flow sheet is graphically depicted in Figure 1. Transgenic N. benthamiana seeds consumed in upstream processing are generated in-house from validated Working Seed Banks, which were in turn generated from validated Master Seed Banks. The seed bank release testing includes germination efficiency >95%, confirmation of growth kinetics, and viral testing. CAPEX related to seed generation are excluded, but associated seed production costs are included in the estimate of $9.50/g seed . The seeds are set in soilless plant substrate at a density of 94 N. benthamiana seeds per 30 × 50 cm tray. The seedlings are cultivated hydroponically during the plant growth phase to reach manufacturing maturity by 35 days. Nutrient solution for plant growth is recirculated with minimal waste and routinely monitored and adjusted for consistent quality based on pH and conductivity. At manufacturing maturity, the plants are transferred to an induction space, complete with a separate hydroponic reservoir, curtains for temporary enclosure,vertical planters for vegetables and double rail spray booms. Recombinant expression of AMP is induced over the course of 1 hr via root drenching and aerial tissue spraying with a combined 0.01 L of 4% ethanol per kg FW plant tissue. The plants are then moved to the incubation phase. Post-induction plants are expressing recombinant AMP, and so the nutrient solution is circulated via a separate feed tank and hydroponic reservoir. The nutrient solution in the incubation phase may contain trace levels of ethanol, which may prematurely initiate AMP production and impair plant growth kinetics. AMP accumulates in the N. benthamiana tissue over the course of 6 days. The nutrient solution in the incubation phase is not recirculated between batches, but sent to bio-waste instead, amounting to an overall 23% plant uptake of the nutrient solution. The spent nutrient solution in the incubation phase is treated as bio-waste to address trace amounts of the viral expression vector that may be present in solution.The downstream processing model flow sheet is graphically depicted in Figure 2. Downstream processing begins with plant harvest. This starts with automated harvester collection of aerial N. benthamiana plant tissue. The spent soilless plant substrate is sent to waste along with the remaining N. benthamiana root matrix. The disposal costs for this step are considered negligible and are not explicitly calculated in the model. There are several routes possible for disposal of plant growth substrate such as composting on site, using it for mulch on facility landscape, collection by farmers for spreading on agricultural land, and, as a last resort, sending it to a landfill. It may be possible, and more cost effective, to sterilize and reuse the growth media but this was not considered in the model. The harvested trays are cleaned in an automated washer with 0.1 L of water per tray. The harvested plant tissue is conveyed via automated belts to extraction, which starts with a double-stack disintegrator to reduce plant biomass particle size.

The disintegrated tissue is then sent to a screw press with an extraction ratio of 0.5 extraction buffer:plant FW for acidic extraction. The extraction buffer and conditions for efficient N. benthamiana extraction have been reported.39 All buffer compositions can be viewed in Table S5. A plant-made AMP purification protocol uses similar acidic extraction to remove N. benthamiana host proteins.The plant extract is clarified using tangential flow microfiltration. The clarified stream is then ultra filtered with additional tangential flow filtration using a 10 kDa molecular weight cutoff to a concentration factor of 20. The AMP in the retentate stream is then purified with cation exchange column chromatography in a bind-and-elute mode of operation. The AMP is eluted isocratically in elution buffer . The purified stream is subjected to one final tangential flow filtration procedure for buffer exchange into phosphate-buffered saline with a diafiltration factor of 3. The purified formulation is spray dried and filled in 1-L plastic bags to obtain the final bulk AMP. All downstream processing water in direct contact with the product stream is reverse osmosis water. All equipment from extraction to formulation are sanitized post processing with a clean-in-place procedure consisting of a prerinse with municipal water, caustic wash with 0.5 M NaOH, postrinse with municipal water, acid wash with 0.5% HNO3, and a final rinse with RO water.Base case scenario outputs were used to identify parameters with significant impact on process economics. We focused the scenario analysis on two different classes of parameters: facility performance parameters and resource purchase costs. Facility performance parameters are defined as inputs that directly impact the physical outputs of the model. Typical biotechnology facility performance parameters include host organism expression level, unit operation recovery, and yearly production level. We chose to investigate expression level and yearly production level. To analyze the impact of facility performance parameters, we set a parameter range based on working process knowledge and then developed a model derived from the base case scenario for each parameter increment within the range. Facility performance parameter changes result in a cascade of changes to the model inputs and outputs; each model is adapted to the resulting stream composition and throughput of the given parameter value while maintaining the constraints of the fixed base case scenario process inputs. Resource purchase costs are defined as inputs that directly control the economic impact of resource utilization for outputs of the model. For the purpose of this analysis, purchase price parameters are contained to cost items within OPEX.Alternative facility design scenarios were developed as comparative models to more broadly explore the context of the base case scenario process economics. The alternative scenario models were designed in alignment with base case scenario inputs unless otherwise noted; each alternative scenario was chosen to isolate the impact of a key facility design assumption. The first scenario investigates an alternative transgenic leafy plant host organism, spinach cultivar Industra, for the base case scenario indoor growth and ethanol-inducible expression. Some colicins have been successfully expressed in S. oleracea plants; however, their expression levels were approximately 10 times lower than that in N. benthamiana so additional research is needed to increase production levels.Several salmocins and lysins can be expressed at high levels in spinach, which is comparable to expression levels in N. benthamiana.The primary distinction in this alternative plant host organism is the lack of nicotine, the major alkaloid in Nicotiana species. In the base case scenario, significant downstream processing emphasis is placed upon nicotine removal. The upstream and downstream processing model flow sheets are graphically depicted in Figure 1 and Figure 2. A complete list of changes to the base case scenario inputs can be observed in Table S4. The second scenario investigates outdoor field-grown transgenic ethanol-inducible Nicotiana tabacum as an alternative to an indoor plant growth facility.