Large Scale Biology Corporation previously investigated N. tabacum outdoor field-grown production of recombinant proteins and personnel involved in that work recommended pursuit of this agronomic approach, with special consideration of field condition variability on product consistency.40 N. tabacum is used instead of N. benthamiana for its increased resilience to agricultural pathogens and weather fluctuation.The upstream processing model is adapted from a techno-economic analysis of plant-made cellulase produced in the field.The upstream and downstream processing model flow sheets are graphically depicted in Supplementary Information, Figure S1 and Figure S2. A complete list of changes to the base case scenario assumptions can be viewed in Table S6.The base case manufacturing facility scenario produces 500 kg of AMP per year at 92% purity including a 42% loss in extraction, downstream processing, and formulation. This yearly production is achieved in 91 manufacturing batches, each with a 42.3-day duration, which process 1.22 million plants per batch with an expression level of 1 g AMP per kg plant FW for a yearly total of 111 million plants processed. The facility plant inventory is 14.7 million plants, which is divided into 12 concurrent batches of plant growth. Initialization of batches is staggered by 3.42 days. The AMP is produced and recovered through a series of manufacturing steps: plant growth, ethanol induction, incubation, harvest, extraction, clarification, concentration, chromatographic purification, vertical farming technology buffer exchange, and formulation. The upstream processing recipe cycle time is 41.4 days and has been designed as the production bottleneck; the downstream processing recipe cycle time is 0.91 days and is thus executed well within the allowable stagger time between plant harvest cycles.
To meet the yearly production demand of 500 kg AMP, upstream processing must produce 867 kg AMP to offset the 42% downstream processing loss. Each upstream processing batch yields 9,520 kg N. benthamiana plant FW containing 9.52 kg AMP, which represents 10% of the total soluble protein .This results in 866,000 kg N. benthamiana plant FW processed over the course of the 91 annual batches, grown in 111,000 units of soilless plant substrate with 1.30 million liters of plant nutrient. Of the annual plant nutrient volume, 436,000 L are sent to waste while the remainder is utilized during plant growth. A total of 7,410 L of 4% ethanol are consumed annually for induction.Manufacturing batches continue directly from upstream to downstream processing; batches are not pooled, and thus 91 downstream processing batches are executed annually. Each batch begins with the upstream production of 9,520 kg N. benthamiana plant FW and 9.52 kg AMP. Screw press extraction results in a stream mass flow of 11,200 kg per batch . After microfiltration and ultrafiltration the stream is considerably reduced to 476 kg per batch . The product stream is eluted from the cation exchange chromatography at 236 kg per batch . The product stream is then diafiltered for a buffer exchanged product stream of 230 kg per batch . The final spray dry formulation results in 9.06 kg formulated product per batch .The base case manufacturing facility requires $50.1 million CAPEX and $3.44 million/year OPEX. The AMPs’ cost of goods sold is calculated to be $6.88/g. Figure 3 shows an economic assessment of upstream and downstream processing. Upstream processing represents 58% of overall operating expenditures , and downstream processing makes up the remaining 42% of operating costs. Of the $2.01 million/year upstream OPEX, the seeding operation represents the majority of the cost. Chromatography and ultrafiltration/diafiltration operations represent the majority of downstream processing OPEX of $1.43 million/year. The downstream CAPEX accounts for 62% of the overall CAPEX with the clarification and UF/DF filtration units representing the largest portion of the downstream capital investment costs.To evaluate the impact of AMP expression level and facility AMP production level, we developed models for a 500 kg AMP/year production level with different AMP expression levels ranging from 0.5 to 5 g AMP/kg FW , and for an expression level of 1 g AMP/kg FW over a range of AMP production levels from 100 kg AMP/year to 1,000 kg AMP/year .
Note that in all cases, the unit operations were resized to meet the design requirements. COGS decreases with diminishing returns as a function of expression level, as can be seen in Figure 5. To illustrate this point, consider that an increase of expression level from 0.5 to 1 g/kg FW results in $4.43/g decrease in COGS, while an increase from 4 to 5 g/kg FW results in $0.22/g decrease in COGS. These changes are equivalent to 39% and 6% reductions, respectively. Also note that at low expression levels the upstream operating costs contribute more to the COGS, whereas at high expression levels downstream operating costs contribute more to the COGS. This is reasonable because the number of plants per batch will increase as expression level decreases, thus requiring more soilless growth media, seeds, and nutrients. CAPEX follows a similar trend with expression level; however, the downstream process is the main contributor to CAPEX, except for very low expression levels . The majority of COGS and CAPEX variation with expression level is attributable to upstream processing, with downstream process costs remaining fairly consistent over the range of expression levels considered. COGS also decreases with diminishing returns as a function of yearly production capacity. Downstream processing is the main contributor to COGS at low production levels while upstream processing is the main contributor at high production levels; at 100 kg/year, downstream processing represents 64% of the COGS, while at 1,000 kg/year the contribution is reduced to 35% of the COGS. Within the given parameter range for expression level and production capacity, COGS shows a higher sensitivity to expression level. Figure 6 shows N. benthamiana FW per batch as a function of expression level and yearly production demand. As expected, biomass requirements are reduced at higher expression levels and lower yearly production demand. At all yearly production levels, significant diminishing returns for increases to expression level are evident within the selected range expression level.
The nicotine-free S. oleracea scenario produces 500 kg AMP/year at 1 g AMP/kg FW with 66% product recovery and 63% purity formulation . Manufacturing batches require ~10% fewer plants than the base case at 1.08 million SS. oleracea plants/batch, and a correspondingly lower plant inventory of 11.1 million plants. The upstream processing duration remains consistent with the base case, while the downstream processing time is reduced to 0.67 days after removal of the nicotine clearance chromatography step of the base case scenario. The S. oleracea manufacturing facility requires $46.5 million CAPEX and $2.50 million/year OPEX. In this scenario, AMPs are manufactured at a COGS of $4.92/g. The field-grown N. tabacum scenario produces 500 kg AMP/year at 1 g AMP/kg FW with 58% product recovery and 92% purity formulation . There are 63 manufacturing batches yearly of 13,900 N. tabacum plants per batch within the late March to late October growing season of the US Midwest/South. The lower number of plants is because of the much larger size of field grown N. tabacum plants compared with indoor grown N. benthamiana plants. The total inventory during steady-state operation is 619,000 plants. The upstream processing duration is 88.4 days, and the larger batches increase the downstream processing time to 1.08 days per batch. The N. tabacum manufacturing facility, including dedicated outdoor field equipment for transgenic handling, requires $27.5 million CAPEX and $1.51 million/- year OPEX. We have neglected labor costs associated with overseeing environmental release of transgenic material, the United States Department of Agriculture Biotechnology Regulatory Services regulatory application, and routine USDA Animal and Plant Health Inspection Service inspections. In this scenario, AMPs are manufactured at a COGS of $3.00/g. A comparison of the capital investment, production costs, and AMP COGS for the N. benthamiana base case, nicotine-free S. oleracea, and field-grown N. tabacum scenarios is shown in Table 2.A greenfield single-product bio-manufacturing facility was chosen to reflect the current whole plant protein bio-manufacturing environment in the United States. There is significant, yet limited, existing manufacturing capacity, most of which is positioned for pharmaceutical-grade production. For smaller annual production demands vertical tower planter, a single- or multi-product contract manufacturing organization model would also be viable. These trends are also reflected globally. The yearly production was determined to meet the demand of a projected market share anticipated for a product of this nature . The number of yearly batches was determined to fully utilize upstream plant growth capacity while leaving idle time for downstream equipment, which is likely to require more maintenance. Future work could include optimization of the plant inventory size, and thus batch size, to maximize the discounted cash flow rate of return over the project lifetime. The optimization will need to identify a balance in the fluctuation of equipment-associated CAPEX and labor- and utility-associated OPEX for both the upstream and downstream. The low-purity requirement of the AMP at 92% is associated with the selection of plant-based production and is a distinct advantage over traditional production platforms for food safety applications. Leafy plant extracts are safe for consumption and routinely consumed as a staple of human diet; when the impurities of the host organism are Generally Recognized as Safe for consumption, there is considerably lower burden on downstream processing. The major focus is redirected from product application safety to product stability and functionality in the presence of the host impurities. Depending on the application rate and consumer consumption, we expect that formulations of 50–95% purity could be employed. Therefore, this analysis represents an upper bound for the anticipated production costs.
Nicotiana benthamiana has been developed as an efficient recombinant protein expression platform. Except for nicotine and traces of anabasine, the N. benthamiana leaf constituents are considered safe for human consumption. Therefore, the processing and quality control are centered on host alkaloid reduction. Processing with a single cation exchange column can provide log reduction in the nicotine level in the product stream to <10 ng nicotine/mg TSP in the formulated product. Based on this reduction, the maximum daily intake of nicotine from the use of colicin as a food safety AMP would be much lower than is encountered in everyday consumption of Solanaceae plants such as peppers, tomatoes, or potatoes.15Vertical farming is just beginning to receive commercial interest as an agricultural solution for year-round, locally grown produce free of pesticides. As the vertical farming industry continues to gain traction, technological advances and process intensification will arise that substantially reduce manufacturing costs for both vertical farming of agricultural crops and plant molecular farming. For example, efficient capture and recirculation of water lost to transpiration will greatly reduce water requirements. Continued development of light emitting diode systems is expected to further improve growth rates, which should help reduce CAPEX, utility costs, and plant growth cycle time. Based on this current techno-economic analysis, advances in plant substrate processing strategies have particularly high potential for economic gain. Soilless plant substrate represents 41% of the overall OPEX in the N. benthamiana base case scenario. A single reuse of the soilless plant substrate prior to disposal would lower the overall OPEX by ~21% in the reduction of the cost of consumables. Reuse of the plant substrate can be achieved by either regrowth of harvested plants or a second round of seeding to generate new plants. In the former situation, manufacturing cost reductions would also include those associated with seeding and tray cleaning operations.Techno-economic analysis provides critical information at all stages of a project’s lifetime. Efficiency of internal research and development in biotechnology companies has suffered in recent years.Technoeconomic analysis is a useful tool for improving this efficiency through identification of key economic-influencing parameters and insights into the commercialization potential of the proposed technology. This preliminary analysis provides early indicators of success potential and reduces the risk of investment for key stakeholders. Furthermore, scenario analysis can guide research and development prioritization to maximize return on investment. In the base case model of this study, a change in the expression level from 0.5 to 1 g AMP/kg FW resulted in 20-fold greater COGS savings than from 4 to 5 g AMP/kg FW. This knowledge makes it clear that there is a significant economic incentive to improve expression levels, but only up to a point. Refinement of the analysis with pilot-scale data further strengthens the analysis and provides perspective to inform future scale-up work.