Under the terms imposed by the DOJ, the companies are not allowed to exchange information about manufacturing cost of goods or sales prices of their drugs, and the duration of the collaboration is limited to the current pandemic. Yet another approach is a government-led strategy in which government bodies define a time-critical national security need that can only be addressed by sequestering critical technology controlled by the private sector. In the United States, for example, the Defense Production Act was first implemented in 1950 but has been reauthorized more than 50 times since then . Similar national security directives exist in Canada and the EU. In the United States, the Defense Production Act gives the executive branch substantial powers, allowing the president, largely through executive order, to direct private companies to prioritize orders from the federal government. The president is also empowered to “allocate materials, services, and facilities” for national defense purposes. The Defense Production Act has been implemented during the COVID-19 crisis to accelerate manufacturing and the provision of medical devices and personal protective equipment, as well as drug intermediates. Therefore, a two-tiered mechanism exists to create FTO and secure critical supplies: the first and more preferable involving cooperative licensing/cross-licensing agreements and manufacturing alliances, and alternatively , a second mechanism involving legislative directives.

Many companies have modified their production processes to manufacture urgently-required products in response to COVID- 19,square plastic plant pot including distillers and perfume makers switching to sanitizing gels, textiles companies making medical gowns and face masks, and electronics companies making respirators.27 Although this involves some challenges, such as production safety and quality requirements, it is far easier than the production of APIs, where the strict regulations discussed earlier in this article must be followed. The development of a mammalian cell line achieving titers in the 5 g L−1 range often takes 10–12 months or at least 5–6 months during a pandemic . These titers can often be achieved for mAbs due to the similar properties of different mAb products and the standardized DSP unit operations , but the titers of other biologics are often lower due to product toxicity or the need for bespoke purification strategies. Even if developmental obstacles are overcome, pharmaceutical companies may not be able to switch rapidly to new products because existing capacity is devoted to the manufacture of other important bio-pharmaceuticals. The capacity of mammalian cell culture facilities currently exceeds market demand by ~30% . Furthermore, contract manufacturing organizations , which can respond most quickly to a demand for new products due to their flexible business model, control only ~19% of that capacity. From our experience, this CMO capacity is often booked in advance for several months if not years, and little is available for short-term campaigns. Furthermore, even if capacity is available, the staff and consumables must be available too. Finally, there is a substantial imbalance in the global distribution of mammalian cell culture capacity, favoring North America and Europe.

This concentration is risky from a global response perspective because these regions were the most severely affected during the early and middle stages of the COVID-19 pandemic, and it is, therefore, possible that this capacity would become unusable following the outbreak of a more destructive virus. Patents covering several technologies related to transient expression in plants will end during or shortly after 2020, facilitating the broader commercial adoption of the technology. This could accelerate the development of new PMP products in a pandemic situation . However, PMP production capacity is currently limited. There are less than five large scale PMP facilities in operation, and we estimate that these facilities could manufacture ~2,200 kg of product per year, assuming a combined annual biomass output of ~1,100 tons as well as similar recombinant protein production and DSP losses as for mammalian cells. Therefore, plant-based production certainly does currently not meet the anticipated demand for pandemic countermeasures. We have estimated a global demand of 500–5,200 tons per year for mAbs, depending on the dose, but only ~259 tons per year can be produced by using the current global capacity provided by mammalian cell bioreactors and plant-based systems currently represent less than 1% of the global production capacity of mammalian cell bioreactors. Furthermore, the number of plant molecular farming companies decreased from 37 to 23 between 2005 and 2020, including many large industry players that would be most able to fund further technology development . Nevertheless, the current plant molecular farming landscape has three advantages in terms of a global first-line response compared to mammalian cells. First, almost two thirds of global production capacity is held by CMOs or hybrid companies , which can make their facilities available for production campaigns on short notice, as shown by their rapid response to COVID-19 allowing most to produce initial product batches by March 2020.

In contrast, only ~20% of fermentation facilities are operated by CMOs . Second, despite the small number of plant molecular farming facilities, they are distributed around the globe with sites in the United States, Canada, United Kingdom, Germany, Japan, Korea, and South Africa, with more planned or under construction in Brazil and China . Finally, transient expression in plants is much faster than any other eukaryotic system with a comparable production scale, moving from gene to product within 20 days and allowing the production of up to 7,000 kg biomass per batch with product accumulation of up to 2 g kg−1 . Even if the time required for protein production in mammalian cells can be reduced to 6 months as recently proposed , Medicago has shown that transient expression in plants can achieve the same goals in less than 3 months . Therefore, the production of vaccines, therapeutics, and diagnostics in plants has the potential to function as a first line of defense against pandemics. Given the limited number and size of plant molecular farming facilities, we believe that the substantial investments currently being allocated to the building of bio-pharmaceutical production capacity should be shared with PMP production sites, allowing this technology to be developed as another strategy to improve our response to future pandemics.Myoblast, myocytes, and fibroblasts are cells of greatest interest for the field of cellular agriculture. For texture and taste, adipocytes may be used and grown either separately or co-cultured with muscle cells. The choice of animal will also have an effect on the final product and production process because cells from different animals will have different growth characteristics, morphology, and product qualities. The majority of these cell lines are adherent,25 liter square pot meaning they require a suitable substrate to grow. Ideally, cells may be grown in suspension culture , bringing cellular agriculture in line with typical pharmaceutical practice such as CHO cells. Micro-carriers may also be used to increase the surface area of the total surface. Proliferating many cells is not the only consideration in cellular agriculture. Stem cells differentiate into more complex tissue structures depending on time and environmental conditions, which is critical in forming final products that consumers are willing to purchase. For example, C2C12 immortalized murine skeletal muscle cells differentiate into myotubes at high density and when exposed to DMEM + 2% horse serum . However, because cell differentiation often precludes further proliferation, cells must be periodically pass aged to provide more physical space for growth. This is typically done by detaching the cells from the substrate using trypsin enzyme and physically placing the cells onto additional surface area. Fundamental techniques in cell culture can be found in and a general overview of mammalian cell culture for bio-production uses can be found in . Figure 1.1b shows a high level overview of the cellular agriculture process. Throughout this entire process, media is used to support cells by providing them with nutrients, signal molecules, and an environment for growth. We are focused on reducing the cost of the media while supporting cell proliferation. This is because the media has been identified as the largest contribution to cost . The main considerations for the design of cell culture media in cellular agriculture are the media must be inexpensive, it must be free of animal products, and it must support long-term proliferation of relevant cell lines and final differentiation into relevant products. The most basic part of a cell culture medium is the basal component, which supplies the amino acids, carbon sources, vitamins, salts, and other fundamental building blocks to cell growth.

The optimal pH of cell culture media is around 7.2 – 7.4 which is achieved through buffering with the sodium bicarbonate – CO2 or organic buffers like HEPES. Temperature should be maintained at around 37◦C at high humidity to prevent evaporation of media. Osmolarity around 260 – 320 mOsm/kg is maintained by the concentration of inorganic ions salts such as NaCl as well as hormones and other buffers. Inorganic salts also supply potassium, sodium, and calcium to regulate cell membrane potential which is critical for nutrient transport and signalling. Trace metals such as iron, zinc, copper, and selenium are also found in basal media for a variety of tasks like enzyme function. Vitamins, particularly B and C, are found in many basal formulations to increase cell growth because they cannot be made by the cells themselves. Nitrogen sources, such as essential and non-essential amino acids, are the building blocks of proteins so are critical to cell growth and survival.It is also unstable in water so is typically supplemented into media as L-alanyl-L-glutamine dipeptide . Carbon sources, primarily glucose and pyruvate, are essential as they are linked to metabolism through glycolysis and the pentose-phosphate pathway. Fatty acids like lipoic and linoleic acid act as energy storage, precursor molecules, and structural elements of membranes and are sometimes supplied through a basal medium like Ham’s F12. Having a sufficient concentration of all of these components is required for proliferating mammalian cells across multiple passages as per above. Having a robust basal media is a necessary but not sufficient condition for long-term cell proliferation and differentiation. Serum is a critical aspect of cell culture because it provides a mix of proteins, amino acids, vitamins, minerals, buffers and shear protectors . Serum stimulates proliferation and differentiation, transport, attachment to and spreading across substrates, and detoxification. Serum has large lot-to-lot variability, zoonotic viruses and contamination , as well as the ethical issues associated with collecting serum from animals. Therefore, while it often simplifies cell growth and differentiation, it is critical to remove serum as per point . Supplementation with growth factors like FGF2, TGFβ1, TNFα, IGF1, or HGF is a common way to induce growth of mammalian muscle cells without the use of serum. Transferrin, another protein found in serum, fulfills a transport role for iron into the cell membrane . PDGF and EGF are polypeptide growth factors that initiate cell proliferation. Such components enhance cell growth but are expensive and comprise the vast majority of the cost of theoretical cellular agriculture processes. Much work has been done on developing serum-free media. The E8 / B8 medium for human induced pluripotent stem cells is based on Dulbecco’s Modified Eagle Medium / F12 supplemented with insulin, transferrin, FGF2, TGFβ1, ascorbic acid, and sodium selenite. Beefy-9 by is similar to E8 but with additional albumin optimized for primary bovine satellite cells. The approach we will take in this dissertation is to use prior knowledge of biological processes to construct a list of potential media components, and use design-of-experiments methods to optimize component concentrations based on cell proliferation. This will be particularly useful for cellular agriculture because by developing and using these statistical tools, as we will see in the next section, DOEs will help develop media quickly and efficiently. One of the most difficult aspects of this work is measuring the quality of media. Viable cells must be counted after a period of time over which the scientist believes the medium will have an effect, which changes depending on cell type, media components, cell density, ECM, pH, temperature, osmolarity, and reactor configuration. If cells grow by adhering to a substrate, then subculturing / passaging may play a role on the health of a cell population, so discounting this effect may have deleterious effects on media design quality. Counting using traditional methods like a hemocytometer or more advanced automatic cell counters using trypan blue exclusion are labor-intensive and prone to error.