Extended space missions in a microgravity environment alter normal physiology in the human body. One critical issue in spaceflight is the loss of bone mineral density. In the process of bone homeostasis, specialized cells facilitate bone remodeling with coupled bone resorption and formation. The reduced mechanical loading in microgravity causes resorption to outpace formation, which results in loss of bone mass. Medications such as parathyroid hormone (PTH) [amino acids 1-34], a peptide fragment of naturally occurring human PTH, stimulate bone formation and may be able to restore bone mass in microgravity. However, this requires a daily subcutaneous injection for the duration of a mission, and in a limited-resource environment (LRE), this is impractical. An alternative to transporting an injectable medication is to instead build capacity to produce it during the mission, which we propose to do in transgenic plants. A whole plant can be thought of as a single-use organic bioreactor especially suited for spaceflight applications.

We are producing transgenic Lactuca sativa (lettuce) which expresses a fusion protein consisting of the PTH peptide linked with a fragment crystallizable (Fc) domain of human IgG1 via a flexible linker. The size increase by the addition of the Fc component and sequestration of the PTH-Fc within the plant endoplasmic reticulum is intended to increase bioavailability via oral delivery.

Developing a plant-based production system for use in LREs necessitates designing models to predict biomass production, which in turn requires the empirical analysis of basic plant growth characteristics. In Chapter 3, we discuss the characterization of plant growth, including biomass over time, water mass fraction in the plant, and seed production. We also describe the growth of a model plant in a vertical aeroponic growth system.

In Chapter 4, we discuss screening and selection strategies to verify the integration and expression of the PTH-Fc transgene. We present experimental results for a study of PTH-Fc expression over time and per leaf in transgenic lettuce. The expression level varied across different leaves and time points. It was found that the average PTH-Fc expression at a harvest time of 25 days after emergence was 68 mg/kg (fresh weight basis). We also show proof-of-concept expression in transgenic Nicotiana benthamiana.

While we suggest that stable expression of PTH-Fc in transgenic plants is the optimal solution to produce therapeutics in LREs for chronic disease states, transient expression, in which growing wild type plants are made to produce recombinant protein, is a valuable method for rapidly meeting health needs. We quantify transient expression of PTH-Fc in wild type Nicotiana benthamiana grown in a vertical aeroponic system in Chapter 5. It was found that plants grown in this system have expression levels comparable to plants grown in solid substrate in an environmentally controlled growth chamber. We show that the method used to induce expression in N. benthamiana is compatible with our reference cultivar of lettuce.

Modeling is a critical aspect of mission design, and our proposal to use plant biotechnology in space requires simulation of the growth system as part of a life support system, which includes the production of food and medicine. Chapter 6 concerns modeling of plant biomass growth using the NASA-sponsored Modified Energy Cascade model. We show an adaptation of the model for use in systems engineering and use cases for its output. The model is unable to account for the nitrogen required for plant growth and therefore must be extended. We discuss an approach to a nitrogen-dependent biomass growth model.

In Chapter 7, we discuss the purification of PTH-Fc from plant protein extracts via liquid chromatography. We show that purification methods that work well with N. benthamiana need to be modified for use with lettuce. Progress made toward purification of recombinant PTH-Fc from transgenic lettuce is discussed.

In Chapter 8, we review plant-based production of pharmaceuticals from a broader perspective. Production platforms and strategies are compared in the context of building a space medical foundry. Chapter 9 takes an even broader look at the use of biotechnology for long-duration space exploration surface missions. We discuss biotech-driven examples of production and recycling of food, pharmaceuticals, and materials and how they integrate with one another. In Chapter 10, the emerging field of space bioprocess engineering is given perspective. Metrics, technology deployment, and education are examined.

Finally, in Chapter 11, we discuss the challenges which lie ahead for spaceflight applications of plant biotechnology. We identify knowledge gaps and consider the difficulty of simulating microgravity conditions.

Plants represent a set of richly diverse and genetically tractable sources for high-value natural and recombinant products that can be used to support human health and industry. This work discusses the production of high-value recombinant products in plants, which is formally termed as plant molecular pharming (PMP), through the lens of the manufacturing techno-economics and applies these insights to the experimental development of plant-based pharmaceutical production technology. PMP must overcome significant challenges as a set of alternative manufacturing platforms. Of primary importance, particular emphasis is required to contextual PMP results in comparison to those of more traditional platforms (e.g., mammalian cell culture, bacterial fermentation) and in terms of economic impact. To encourage the proliferation of such critical context for the field of PMP, we describe a general method for techno-economic analysis of plant-based manufacturing in Chapter 3. We employ this techno-economic method to explore several different opportunities for plant-based manufacturing. In Chapter 4, we collaborate with industry partners to simulate the production of antimicrobial proteins in leafy green plants to respond a food safety-driven demand to protect against high-risk pathogens in a cost-sensitive and antibiotic resistance-concerned product landscape. In Chapter 5, we simulate scale-up of lab-scale results to evaluate the commercial potential of metabolically-regulated semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures. In Chapter 6, we approach a main barrier facing outdoor field-grown plant-based manufacturing, namely variation in plant quality driving process variation, by introducing uncertainty quantification to techno-economic simulation of large-market natural and biotechnology products in an outdoor field-grown plant system. After having evaluated manufacturing in several different established markets and environments for PMP, we use these general insights to consider and introduce the potential of PMP for supporting human health in the upcoming and uniquely resource-limited environments of deep-space exploration in Chapter 7. After identifying pharmaceutical purification as a bottleneck in the realization of a robust pharmaceutical foundry for space exploration, we evaluate the current state of terrestrial pharmaceutical purification costs in the space-relevant costing framework of equivalent system mass in Chapter 8. Lessons learned around the constraints of pharmaceutical purification in limited-resource environments, including deep-space exploration and rural terrestrial environments, motivated the experimental development of a plant virus-based immunosorbent nanoparticle (VIN) technology as a simple and bioregenerable reagent for the purification of therapeutic monoclonal antibody and Fc-fusion protein drugs using affinity sedimentation or, when coupled with magnetic particles, affinity magnetic separation in Chapter 9. We then further explore VINs as functional elements within larger system arrangements to exploit novel advantages of the virus-based nanomaterial structure and overcome limitations of the existing methodologies. In Chapter 10, we present entrapment and utility of VINs in silica sol-gel matrices by pore confinement, representing a novel system configuration for virus-based nanomaterials (VBNs) in general. In Chapter 11, we present a similarly novel process integration of capture, concentration, and diafiltration steps of pharmaceutical purification by circulation of VINs within a tangential flow filtration retentate loop. Having developed VINs for limited-resource secondary purification, in Chapter 12 we investigate a novel design for a 3D-printed hand-powered centrifuge that is capable of initial clarification steps of pharmaceutical purification. Lastly, in Chapter 13 we conclude and summarize a series of promising yet incomplete research investigations including the purification of VINs with the emerging technology of monolithic chromatography, an exploration of selective pressures to maintain VIN genetic stability over consecutive infection-based production cycles, development of a method for antibody-mediated capture of arbitrary proteins using VINs, important considerations for novel VIN design at the level of the three major structural components, and a novel strategy for the early-stage dynamics of plant cultivation on Mars.

Increases in global meat demands cannot be sustainably met with current methods of livestock farming, which has a substantial impact on greenhouse gas emissions, land use, and water consumption. Cultivated meat is a rapidly advancing technology that produces meat products by proliferating and differentiating animal stem cells in large bioreactors, avoiding conventional live-animal farming. While many companies are working in this area, there is a lack of existing infrastructure and experience at commercial scale, resulting in many technical bottlenecks such as scale-up of cell fermentation and media availability and costs. In this study, we evaluate theoretical cultivated beef production facilities with the goal of envisioning an industry with multiple facilities to produce in total 100,000,000 kg fresh weight (FW) of unstructured cultured beef per year or ~0.14% of the annual global beef production. Using the computer-aided process design software, SuperPro Designer®, facilities are modelled to create a comprehensive techno-economic analysis (TEA) to highlight improvements that can lower the cost of such a production system and allow cultivated meat products to be competitive. Three facility scenarios are presented with different sized production reactors; 42,000 L total volume stirred tank bioreactor (STR) with a base case cost of goods sold (COGS) of $31.5/kg, 210,000 L total volume STR with a COGS of $21.9/kg, and 260,000 L total volume airlift reactor (ALR) with a COGS of $14.0/kg. The SuperPro models that correspond to these three production reactor sizes are titled “40K STR 8-13-22”, “210K STR 8-13-22”, and “260K ALR 8-13-22”. This study outlines how advances in scaled up bioreactors and decreased media costs are necessary for commercialization of cultured beef products.

A large-scale facility was designed to analyze the techno-economics of current resveratrol manufacturing procedures to provide information for domestic production using a plant-based approach. The present model utilizes the plant Japanese knotweed as a source of resveratrol for production. All manufacturing steps and conditions were tuned to match manufacturing processes listed in publicly available literature and patents on resveratrol production. The model is designed to produce 100 metric tons of resveratrol at 98% purity. This simulated biomanufacturing facility operates for 330 days a year, 24 hours per day, performing 1,295 batches yielding 77.3 kg resveratrol per batch. This simulated manufacturing facility was designed in SuperPro Designer® allowing for initialization of bioprocessing conditions and pricing of materials and equipment utilized in the model. The analysis conducted here provided key insight into the capital expenditures (CAPEX), operating expenditures (OPEX), and Cost of Goods Sold (COGS) expected for such a process. The results from the base case model are as follows: CAPEX of $44.7 million, OPEX of $15.0 million per year, and a COGS of $150/kg resveratrol. A sensitivity and scenario analysis were conducted to assess the relationship between certain operating conditions to both the CAPEX and COGS. The implementation of additional equipment for an ethanol recovery unit was shown to reduce OPEX to about $14.1 million and COGS to $141.4//kg but increase CAPEX to $51.5 million. The simulated facility was assessed for its efficiency, and it was determined that the model has a process mass intensity (PMI) of 529 kg/kg resveratrol. The environment impact factor (EI) of the facility was also analyzed. Here, the EIs were determined to be 26 and 31.6 kg/kg resveratrol for input and outputs, respectively, suggesting a very safe and environmentally-friendly process.