Griffithsin also effectively inhibits transmission of HSV-2 , HCV , SARS-CoV , Ebola , and possibly other viruses yet to be studied. Importantly, Griffithsin appears devoid of cellular toxicity that is associated with other lectins. O’Keefe et al. conducted studies with explants of macaque and rabbit vaginal tissues ex vivo and showed that Griffithsin did not induce changes in the levels of cytokines or chemokines, nor did it alter lymphocyte levels in human cervical tissue nor elicit inflammatory responses in rabbit tissue . The combination of extremely wide viral target range and demonstrated preclinical safety makes Griffithsin potentially useful as a prophylactic and/or therapeutic in multiple and diverse antiviral indications. The potential indications for Griffithsin as a human prophylactic or therapeutic include its use as an active pharmaceutical ingredient in vaginal and rectal microbicides. In spite of the value shown by pre-exposure prophylaxis drugs to prevent HIV transmission, issues of cost, side effects, the potential for development of viral resistance through chronic use of antiretrovirals as prevention modalities, and access to PrEP drugs by under resourced populations remain. These unmet needs could be met by the availability of affordable, safe and effective “on demand” antivirals, especially with Griffithsin as the API and its potential to control co-transmitted viruses such as HIV-1, HSV-2 and HCV during intercourse. Adoption of Griffithsin as a new biologic drug, especially in cost-constrained products such as microbicides, is predicated on the feasibility of a scalable manufacturing process that can supply market-relevant volumes of the API at an acceptable cost of goods sold . Previously, we showed that recombinant Griffithsin can be expressed and isolated with high efficiency using transient gene expression in green plants . Although the process described can be further optimized,growing bags the achieved pilot-scale expression yields of >0.5 g Griffithsin per kg of fresh green biomass , recovery efficiencies of 60–90% overall, and Griffithsin purity of >99% of total soluble protein are already impressive.

In this study, we developed a technoeconomic model for Griffithsin manufacturing using a plant-based system with the goal of estimating API manufacturing cost and determined the factors that have the greatest impact on COGS. The output of our study should serve as a basis for additional process improvements, selection of a commercial-scale manufacturer, and should assist in the identification of future product targets for cost-sensitive markets such as prophylactic microbicides as well as those for less cost-constrained therapeutic indications. Technoeconomic modeling was performed with the widely used SuperPro Designer modeling software . The main analysis in this study was conducted using data available from pilot-scale manufacturing of Griffithsin in Nicotiana benthamiana plants using tobacco mosaic virus -induced transient gene expression, and assuming that manufacturing would take place in an existing and fully equipped state-of-the-art plant-based bio-manufacturing facility. Modeling costs based on existing resources of a contract manufacturing organization instead of a “greenfield” build of a new facility was seen as the most likely scenario for launch of a new product. Our reasoning was that dedicated infrastructure could be built subsequently depending on market demand for the drug. As a result, we did not estimate capital equipment or total capital investment costs, and neglected depreciation, insurance, local taxes and factory expenses in the manufacturing operating cost analysis as these investments would have been made by the CMO. Our analysis assumed a 20% net profit margin/fee assessed by the CMO and this figure was added to the production cost of the product to arrive at the final total product cost. In addition to the techno economic analysis, an Environmental Health and Safety Assessment of the designed process was conducted using the method described by Biwer and Heinzle to evaluate the environmental, health and safety impact of Griffithsin manufacturing using the plant-based system, with the goal of assessing the sustainability of the process. The techno economic modeling for this study was performed using SuperPro Designer , Version 9.5 , a software tool for process simulation and flow sheet development that performs mass and energy balances, equipment sizing, batch scheduling/debottle necking, 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. SuperPro 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, chromatography, grinding or homogenization, and centrifugation. There are some specific unit operations and processes used in this study that are currently not included in SuperPro, such as indoor plant cultivation, transplantation, plant harvesting and screw press/disintegrator. Such unit operations were addressed through the “Generic Box” feature of the application. Unless otherwise noted, the maintenance costs of major equipment, unit operation-specific labor requirements and costs , pure components, stock mixtures, heat transfer agents, power and consumables used in the analysis were determined using the SuperPro built-in equipment cost model and default data banks. Additional case study specific design parameters were selected based on experimental data from journal articles, patent literature, the authors’ laboratories, 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.Process flow and unit operations were derived from published methods and unpublished results obtained by the authors and collaborators who have participated in the development and scale-up of the process described and in the development of Griffithsin products. On the basis of this information, the SuperPro software was used to select and size equipment for each of the unit operations to achieve the desired production target , simulate the operations by performing material and energy balances, and specify and schedule all operations taking place within each piece of equipment to calculate material inputs and outputs and process times. Costs for raw materials, utilities, consumables, labor, laboratory QA/QC, waste disposal and equipment maintenance were then used to determine annual operating costs, and per-unit mass or per-dose costs . The main case study model was based on an existing plant based manufacturing facility, operating in batch mode, and excluded new capital investments and other facility dependent costs, except for equipment maintenance costs, which were included.

For the downstream portion of the Griffithsin manufacturing process, an annual available operating time of 7,920 h for the facility was used with indoor-grown Nicotiana benthamiana plants. Operating time was based on Holtz et al. for a similar facility, which was designed with overlapping utility capacity and in which the largest single utility unit can be down for maintenance and/or repairs and the utility loads can be maintained with redundant equipment. Likewise, per Nandi et al. it was assumed that the plants would be grown continuously throughout the year . Land costs, upfront R&D, upfront royalties, and regulatory/certification costs were neglected in the model as these costs can vary widely. Griffithsin protein can be produced in plants in a number of ways. These include stable expression in recombinant plants; inducible expression in transgenic plants; transient expression induced directly by tobacco mosaic virus replicons; or via agrobacterial vectors introduced into the plants via vacuum assisted, or surfactant-assisted, infiltration . Relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility,nursery grow bag 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 plant-made pharmaceuticals . In our base-case analysis, we modeled expression of Griffithsin using TMV induction described in Fuqua et al. and results from 3 pilot-scale manufacturing runs because these batches provided the most extensive and complete data set; however, this process has been corroborated in 6 additional manufacturing runs at pilot-scale or larger.icotiana benthamiana host plants are generated from seed and propagated indoors under controlled environmental conditions until sufficient biomass is obtained for inoculation with the TMV vector carrying the Griffithsin gene. The process is summarized as follows. An N. benthamiana Master Seed Bank is generated from seeds obtained from the U.S. Department of Agriculture Repository. For bio-manufacturing, seeds from the TW- 16 line are obtained in bulk and stored securely. The Master Seed Bank is qualified for germination rate , freedom from disease, and genetic uniformity, and stored in sealed containers under temperature-controlled conditions . If the seed batch passes release tests, it becomes the Production Seed Batch and is used in the designated production run . Seedlings are allowed to grow for 21 days under controlled environmental conditions . At this stage, the seedlings are transplanted to accommodate their larger size and moved to another growth room to await inoculation, as described in the following sections.

The API extraction procedure modeled is per Holtz et al. except that a 1:1 ratio of biomass:buffer is used. Briefly, the aerial parts of the plants containing accumulated Griffithsin are mechanically inverted and cut with a mechanical cutter. The harvested biomass is collected in baskets for transport to the extraction suite, to initiate downstream processing. The harvested biomass fresh weight is determined to calculate the volume of extraction buffer to be added, typically at a rate of 1 kg biomass FW:1 L buffer mix . The pH is adjusted to 4.0 and the mixture is heated to 55◦C for 15 min to help precipitate major host plant proteins. The heated mixture is passively cooled and filtered to yield a crude extract. The crude extract is stirred overnight at 4◦C in the presence of bentonite and MgCl2. This procedure helps remove TMV coat protein , which at this step represents the largest protein impurity in the extract. The suspension is filtered to remove aggregated TMV CP, yielding a clarified and partially purified API-containing solution and is then sterile- filtered . In-process controls are applied throughout downstream processing unit operations to determine reagent volumes and assess yield and quality at key steps. To adequately meet the projected initial annual market demand for a rectal micro-bicidal formulation in the United States, approximately 6.67 million doses of Griffithsin API at 3 mg/dose would be needed. This translates into a production rate of 20 kg of purified Griffithsin API per year. The manufacturing facility to produce the required 20 kg of API per year was assumed to segregate production operations into two broad categories; namely, upstream production and downstream recovery and purification. To accommodate a large number of plants, the facility uses a vertical cultivation design with integrated irrigation and runoff collection system. Each rack is compatible with an integrated transportation infrastructure to move each tray to the next phase of the growth cycle. The upstream portion of the facility houses unit operations for N. benthamiana propagation, inoculation with TMV vector, and Griffithsin protein expression and accumulation. These processes begin with seeding and end when the biomass is taken to harvest. The downstream portion of the facility begins at harvest and continues through purification of the Griffithsin DS. Upstream processing is assumed compliant with good agricultural practices , whereas downstream processing is subject to FDA current good manufacturing practice . The general layout of the upstream growth rooms was adapted from Holtz et al. , and includes one germination chamber for seeds, one pre-inoculation room for biomass growth, and an isolated post-inoculation chamber where N. benthamiana inoculated with TMV expresses and accumulates Griffithsin. Plants are arrayed in equally sized trays under light-emitting diode light systems tuned to the optimized photosynthetic absorbance spectrum of N. benthamianaand are continuously illuminated. The plants are rooted in rock wool cubes held in the trays by polystyrene foam floats and perfused with a nutrient solution . Hydroponic irrigation is on a 12-h cycle and is accomplished via nutrient film technique . We modeled a hydroponic system because the nutrient solution is recycled; hence, water is conserved, and fertilizer runoff is reduced although not eliminated. The mass of nutrient solution taken up by the plants, the cost of the nutrient solution per liter, and the mass of residual nutrient solution that goes to the wastewater treatment system are shown in Supplementary Table 1 in Supplementary Material.