To ensure consistency of the nutrient solution, all water was assumed to be treated by reverse osmosis with solution-monitoring for proper pH and dissolved solids content. The three phases of plant growth require a total batch time of 38 days in the upstream portion of the facility. Due to the protracted and continuous nature of plant cultivation, the upstream portion of the facility contains multiple concurrent batches staggered at different stages of growth. When one batch graduates to the next step of production , the trays containing the batch’s biomass are cycled out and the corresponding rack space is immediately filled with a new rotation of trays. We divided the 38-day growth period into 11 concurrent batch periods, with one batch ready to enter downstream purification every 3.44 days. Table 1 is a summary of the number of plants, trays and batches that comprise the upstream facility at any given moment. For model building, batch schedules were calculated under the initial assumption of 24/7 operation for 330 days per year. Plant uptake of nutrients and growth were assumed to be linear reaching 15 g FW per plant at viral inoculation and then increasing in mass to reach 40 g FW per plant at harvest. A 5% failure rate of TMV inoculation was assumed . The Griffithsin expression rate was fixed at 0.52 g/kg FW of harvested biomass, with a downstream recovery of 70%, based on pilot-scale results. Additionally, nutrient solution demand was assumed to match observed biomass growth rates assuming that for each kilogram of nutrient solutions, 0.5 kilogram goes into biomass and the remainder is considered aqueous waste. The materials used, quantities and source are summarized in Supplementary Table 1 in Supplementary Material, plastic growing bag together with clarifying comments and references that were used to assist in the calculations. Using the inputs shown in Supplementary Table 1, the upstream and downstream processes were modeled in SuperPro.
The results generated by the software for the upstream operations are shown in Figure 1, with scheduling shown in the equipment occupancy chart in Figure 2. The following descriptions elaborate on the schema presented in each figure. Griffithsin recovery and purification was modeled as a batch process in a facility with an available operating time of 330 days a year for 24 h a day and 7 days a week. In each year, there are 95 batches total to produce 20 kg of purified Griffithsin API. Since the recovery and purification process only takes 1.6 days, the downstream facility has a significant down time of 2.78 days between batches. Overall, each batch requires 39.6 days from seed planting to formulating the final product, with 38 days upstream and 1.6 days downstream. In Figure 1, the upstream processes are dictated by 11 concurrent batches with each batch being 3.44 days apart from each other. A batch basis of 3.44 days was chosen to decrease equipment idle time and thereby increase downstream equipment utilization efficiency. Despite the 39.6-day batch period and a 332-day operating year, in the model the batch time upstream was reduced to approximately 38 days and the operating year was increased to 365 days to reach the desired 95 batches per year. This was done because SuperPro reproduces uniform results for each year. The goal of the upstream process operations is to produce sufficient biomass to enable isolation of 20 kg Griffithsin per annum. The modeling results show that each batch would produce 578 kg of biomass containing 300 g of Griffithsin, assuming an expression yield 0.52 g API/kg FW biomass . Because induction was modeled using infection with recombinant TMV vector, the three main phases in upstream are germination, pre-inoculation, and post inoculation. The duration of the phases in the model are 21 days, 3 days, and 14 days, respectively. Each batch of N. benthamiana plants goes through a germination phase of 21 days and the germination room is designed with a capacity to grow the 86,700 plants necessary to reach the production goal.
This step of the process uses 90 germination trays, each holding about 960 plants, distributed among 6 batches in the germination room. After 21 days post germination, the N. benthamiana plants are transplanted to a lower density to enable further growth. Thus, seedlings from one germination tray are transplanted into three grow trays , meaning that there are three times the number of trays in pre- and post-inoculation, individually, than in germination. The plant density is 646 plants per m2 in the germination trays and 215 plants per m2 after transplantation. In practice, during transplantation each plant will spend only a few minutes away from its growth environment to minimize transplant shock and undue stress. In the model, the overall time was overestimated to be 3 h to accommodate other necessary procedures, such as moving the plants back to the tray stacks. The transplanted trays are relocated to pre-inoculation rooms that are designed to accommodate the increased area from transplanting for ∼3 days. The pre-inoculation room contains 1 batch, each containing 45 trays with 320 plants per tray. Recombinant TMV for inoculation is produced in and isolated from N. benthamiana. The plant growth model is the same as the rest of N. benthamiana plants. By using infected plants and the purification model defined by Leberman , 4 mg of pure TMV per gram of infected plant material can be recovered . Each batch is equivalent to 14,450 plants distributed on 45 trays. Less than 1 microgram of TMV virion is needed to inoculate each plant . Thus, approximately 14.5 mg of TMV is needed per batch and the necessary amount of TMV to inoculate a batch can be produced from a single N. benthamiana plant. Multiple batches of TMV solution can be made simultaneously and stored at −20◦C . TMV production can be done at lab scale and equipment, labor and material costs are negligible compared to the overall cost of plant maintenance. The isolated TMV is incorporated in diatomaceous earth buffer solution at a concentration of 10 micrograms per 2.5 mL of diatomaceous earth buffer solution, which contains 1% by volume diatomaceous earth and 2% by volume of sodium/potassium-based buffer .
The selected inoculation volume of 2.5 mL is a safe middle value from the range suggested in the literature . In the model, the estimated mixing and transfer time for the solution is 1 h, which starts at the beginning of post-inoculation, so the plants and solution enter the same stage together. A forklift is used to transport the plants into the inoculation room. The plants are inoculated with the diatomaceous earth buffer solution described above with a high velocity spray. Inoculation machines are often custom made and consist of a conveyor traveling through an enclosed cylinder equipped with high pressure spray nozzles aimed at the plants’ aerial structures. Once the inoculation is complete, the trays are conveyed to the post-inoculation growth room,wholesale grow bags which is similar in design to the pre-inoculation growth room; the main difference being its size. The post-inoculation room contains 4 batches at any given time for a total of 180 trays with 320 plants per tray. The scheduling of 3 batches is summarized in the equipment occupancy chart in Figure 2. As shown, seeding, germination, transplant, pre-inoculation, inoculation, and post-inoculation occur sequentially, and the batches are staggered by 3.44 days. The downstream unit operations developed in SuperPro are shown in Figure 3, with scheduling summarized in the equipment occupancy chart shown in Figure 4. The following descriptions elaborate on the schema presented in each figure. At the end of each 3.44 day growing rotation cycle upstream, one batch of N. benthamiana plants is ready to be transferred to downstream processing. This is done by placing each tray of plants onto a conveyor system which leads them to the first phase of downstream operations. The matured plants are first harvested for the green biomass from which the majority of Griffithsin can be recovered with a single extraction. Additional Griffithsin could be recovered from fibrous material by reprocessing and from roots ; however, reprocessing was not included in this model. The automated harvester processes the 578 kilograms of biomass at a rate of 193 kilograms of biomass per hour. With an operational buffer time of 1 h, this process is thus expected to take 4 h. As the biomass is processed by the harvester, it is directly fed into a shredder which further comminutes the biomass to improve Griffithsin recovery. The shredder operates at a capacity of 193 kg of harvested biomass per hour for 2.8 h. The shredded biomass is then mixed with an extraction buffer in a buffer addition tank. For every kilogram of plant material, 1 L of extraction buffer is added. Thus, for 578 kg of N. benthamiana in a batch, approximately 578 L of extraction buffer are added. The resultant solid-liquid mixture has a total volume of about 1,135 L and is sent through a screw press, which is represented as a generic box in the model. The screw press separates the solidliquid slurry leaving a main process fluid stream of plant extract and a waste stream of biomass. A loss about 12% of the original starting Griffithsin was modeled assuming it to be non-liberated from the homogenized biomass. The removal of the biomass leaves a main process stream that contains about 585 L . To facilitate the aggregation of proteinaceous impurities, the extract solution is transferred into a mixing tank and heated to 55◦C for 15 min. The mixture is passively cooled and simultaneously transferred out of the tank and fed into the first 0.3µm plate-and-frame filter. The extract solution is filter-pressed at 25–30 psig to remove the aggregated protein impurities.
Filtering has a process time of 1 h and requires a filter area of 3 m2 to handle the 590 kg/batch of the process stream. At this stage, the process loses a further 8% of the Griffithsin but removes all the RuBisCO and 87% of the TMV coat protein impurities. The filtrate from this step is transferred to a second mixing and storage tank, mixed with bentonite clay and magnesium chloride, and stored at 4◦C for a 12-h period. This stage is the bottleneck operation for the downstream process. After the 12-h incubation, the solution is filtered through a second 0.3µm filter press and a 0.2µm inline sterilizing filter. These operations remove the remaining protein impurities leaving a Griffithsin extract with greater than 99% purity but at the cost of losing 6% of the Griffithsin. The second plate-and-frame filter has a filter area of about 3 m2 and will process all of the extract in 1 h. There is approximately 222 g of Griffithsin per batch at the end of the filtration phase. Following the filtrations steps, the Griffithsin extract solution is collected in a storage tank and further purified using an AxiChrom column with Capto MMC resin to remove residual color and potential non-proteinaceous impurities. To accommodate the 222 g of Griffithsin in solution, 4.9 L of MMC bed resin is needed at a 45 mg/mL binding capacity . The order of the operations for this chromatography step are: Equilibrate, load, wash, elute, and regenerate. In total, chromatography requires 10 h with the load step taking the longest, at 8 h, because approximately 600 L of solution are processed. Chromatography is necessary to decolorize the extract at the expense of losing 4% of the Griffithsin, giving a remaining Griffithsin mass of 210 g per batch. The 10 L of eluant process fluid is sent through a viral clearance filter and transferred into a pool/storage tank. Subsequently, the extract is sent through an ultrafiltration/diafiltration cycle to remove salts introduced in the chromatography column. After ultrafiltration, the product is transferred into a storage tank to be mixed with the final formulation components. The concentrated Griffithsin is diluted to give a concentration of 10 g/L Griffithsin in 10 mM Na2HPO4, 2.0 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl at pH 7.4. The final volume of the DS is 21 L per batch. As shown by Figure 4, each batch in the downstream requires 39 h of process time which includes all SIP and CIP operations.