The EU follows both decentralized processes as well as centralized procedures covering all Member States


TEA models have been developed and are publicly available for a variety of plant-based bio-manufacturing facilities, including whole plant and plant cell bioreactor processes for production of mAbs , antiviral lectins , therapeutics , and antimicrobial peptides . These tools are particularly useful for the development of new processes because they can indicate which areas would benefit most from focused research and development efforts to increase throughput, reduce process mass intensity, and minimize overall production costs.The rapid production of protein-based countermeasures for SARS-CoV-2 will most likely, at least initially, require bio-manufacturing processes based on transient expression rather than stable transgenic lines. Options include the transient transfection of mammalian cells , baculovirus-infected insect cell expression systems , cell-free expression systems for in vitro transcription and translation , and transient expression in plants . The longer term production of these countermeasures may rely on mammalian or plant cell lines and/or transgenic plants, in which the expression cassette has been stably integrated into the host genome, but these will take months or even years to develop, optimize, and scale-up. Among the available transient expression systems, only plants can be scaled-up to meet the demand for COVID-19 countermeasures without the need for extensive supply chains and/or complex and expensive infrastructure,black plastic pots for plants thus ensuring low production costs . These manufacturing processes typically use Nicotiana benthamiana as the production host and each plant can be regarded as a biodegradable, single-use bioreactor .

The plants are grown either in greenhouses or indoors, either hydroponically or in a growth substrate, often in multiple layers to minimize the facility footprint, and under artificial lighting such as LEDs. In North America, large-scale commercial PMP facilities have been built in Bryan, TX , Owensboro, KY , Durham, NC , and Quebec, Canada . The plants are grown from seed until they reach 4–6 weeks of age before transient expression, which is typically achieved by infiltration using recombinant A. tumefaciens carrying the expression cassette or by the introduction of a viral expression vector such as tobacco mosaic virus , for example, the GENEWARE platform . For transient expression by infiltration with A. tumefaciens, the plants are turned upside down and the aerial portions are submerged in the bacterial suspension. A moderate vacuum is applied for a few minutes, and when it is released, the bacteria are drawn into the interstitial spaces within the leaves. The plants are removed from the suspension and moved to an incubation room/chamber for 5–7 days for recombinant protein production. A recent adaptation of this process replaces vacuum infiltration with the aerial application of the A. tumefaciens suspension mixed with a surfactant. The reduced surface tension of the carrier solution allows the bacteria to enter the stomata, achieving a similar effect to agroinfiltration . This agrospray strategy can be applied anywhere, thus removing the need for vacuum infiltrators and associated equipment . For transient expression using viral vectors, the viral suspension is mixed with an abrasive for application to the leaves using a pressurized spray, and the plants are incubated for 6–12 days as the recombinant protein is produced. Large scale production facilities have an inventory of plants at various stages of growth and they are processed in batches. Depending on the batch size , the vacuum infiltration throughput, and the target protein production kinetics, the infiltration/ incubation process time is 5–8 days.

The inoculation/incubation process is slightly longer at 6–13 days. The overall batch time from seeding to harvest is 33–55 days depending on the optimal plant age, transient expression method, and target protein production kinetics . Importantly, plant growth can be de-coupled from infiltration, so that the plants are kept at the ready for instant use, which reduces the effective first-reaction batch time from gene to product to ~10–15 days if a platform downstream process is available . The time between batches can be reduced even further to match the longest unit operation in the upstream or downstream process. The number of plants available under normal operational scenarios is limited to avoid expenditure, but more plants can be seeded and made available in the event of a pandemic emergency. This would allow various urgent manufacturing scenarios to be realized, for example, the provision of a vaccine candidate or other prophylactic to first-line response staff.The speed of transient expression in plants allows the rapid adaptation of a product even when the process has already reached manufacturing scale. For example, decisions about the nature of the recombinant protein product can be made as little as 2 weeks before harvest because the cultivation of bacteria takes less than 7 days and the post-infiltration incubation of plants takes ~5–7 days. By using large-scale cryo-stocks of ready-to-use A. tumefaciens, the decision can be delayed until the day of infiltration and thus 5–7 days before harvesting the biomass . This flexibility is desirable in an early pandemic scenario because the latest information on improved drug properties can be channeled directly into production, for example, to produce gram quantities of protein that are required for safety assessment, pre-clinical and clinical testing, or even compassionate use if the fatality rate of a disease is high .

Although infiltration is typically a discontinuous process requiring stainless-steel equipment due to the vacuum that must be applied to plants submerged in the bacterial suspension, most other steps in the production of PMPs can be designed for continuous operation, incorporating single-use equipment and thus complying with the proposed concept for bio-facilities of the future . Accordingly, continuous harvesting and extraction can be carried out using appropriate equipment such as screw presses , whereas continuous filtration and chromatography can take advantage of the same equipment successfully used with microbial and mammalian cell cultures . Therefore, plant-based production platforms can benefit from the same >4-fold increase in space-time yield that can be achieved by continuous processing with conventional cell-based systems . As a consequence, a larger amount of product can be delivered earlier, which can help to prevent the disease from spreading once a vaccine becomes available. In addition to conventional chromatography, several generic purification strategies have been developed to rapidly isolate products from crude plant extracts in a cost-effective manner . Due to their generic nature, these strategies typically require little optimization and can immediately be applied to products meeting the necessary requirements, which reduces the time needed to respond to a new disease. For example, purification by ultrafiltration/diafiltration is attractive for both small and large molecules because they can be separated from plant host cell proteins ,plant pot with drainage which are typically 100–450 kDa in size, under gentle conditions such as neutral pH to ensure efficient recovery . This technique can also be used for simultaneous volume reduction and optional buffer exchange, reducing the overall process time and ensuring compatibility with subsequent chromatography steps. HCP removal triggered by increasing the temperature and/ or reducing the pH is mostly limited to stable proteins such as antibodies, and especially, the former method may require extended product characterization to ensure the function of products, such as vaccine candidates, is not compromised . The fusion of purification tags to a protein product can be tempting to accelerate process development when time is pressing during an ongoing pandemic.

These tags can stabilize target proteins in planta while also facilitating purification by affinity chromatography or non-chromatographic methods such as aqueous two-phase systems . On the downside, such tags may trigger unwanted aggregation or immune responses that can reduce product activity or even safety . Some tags may be approved in certain circumstances , but their immunogenicity may depend on the context of the fusion protein. The substantial toolkit available for rapid plant biomass processing and the adaptation of even large-scale plant-based production processes to new protein products ensure that plants can be used to respond to pandemic diseases with at least an equivalent development time and, in most cases, a much shorter one than conventional cell-based platforms. Although genetic vaccines for SARS-CoV-2 have been produced quickly , they have never been manufactured at the scale needed to address a pandemic and their stability during transport and deployment to developing world regions remains to be shown.Regulatory oversight is a major and time-consuming component of any drug development program, and regulatory agencies have needed to revise internal and external procedures in order to adapt normal schedules for the rapid decision-making necessary during emergency situations. Just as important as rapid methods to express, prototype, optimize, produce, and scale new products are the streamlining of regulatory procedures to maximize the technical advantages offered by the speed and flexibility of plants and other high-performance manufacturing systems. Guidelines issued by regulatory agencies for the development of new products, or the repurposing of existing products for new indications, include criteria for product manufacturing and characterization, containment and mitigation of environmental risks, stage-wise safety determination, clinical demonstration of safety and efficacy, and various mechanisms for product licensure or approval to deploy the products and achieve the desired public health benefit. Regardless of which manufacturing platform is employed, the complexity of product development requires that continuous scrutiny is applied from preclinical research to drug approval and post-market surveillance, thus ensuring that the public does not incur an undue safety risk and that products ultimately reaching the market consistently conform to their label claims. These goals are common to regulatory agencies worldwide, and higher convergence exists in regions that have adopted the harmonization of standards as defined by the International Council for Harmonization ,2 in key product areas including quality, safety, and efficacy.Both the United States and the EU have stringent pharmaceutical product quality and clinical development requirements, as well as regulatory mechanisms to ensure product quality and public safety. Differences and similarities between regional systems have been discussed elsewhere and are only summarized here. Stated simply, the United States, EU, and other jurisdictions follow generally a two-stage regulatory process, comprising clinical research authorization and monitoring and result’s review and marketing approval. The first stage involves the initiation of clinical research via submission of an Investigational New Drug application in the United States or its analogous Clinical Trial Application in Europe. At the preclinicalclinical translational interphase of product development, a sponsor must formally inform a regulatory agency of its intention to develop a new product and the methods and endpoints it will use to assess clinical safety and preliminary pharmacologic activity . Because the EU is a collective of independent Member States, the CTA can be submitted to a country-specific regulatory agency that will oversee development of the new product. The regulatory systems of the EU and the United States both allow pre-submission consultation on the proposed development programs via discussions with regulatory agencies or expert national bodies. These are known as pre-IND meetings in the United States and Investigational Medicinal Product Dossier 3 discussions in the EU. These meetings serve to guide the structure of the clinical programs and can substantially reduce the risk of regulatory delays as the programs begin. PIND meetings are common albeit not required, whereas IMPD discussions are often necessary prior to CTA submission. At intermediate stages of clinical development , pauses for regulatory review must be added between clinical study phases. Such End of Phase review times may range from one to several months depending on the technology and disease indication. In advanced stages of product development after pivotal, placebo-controlled randomized Phase III studies are complete, drug approval requests that typically require extensive time for review and decision-making on the part of the regulatory agencies. In the United States, the Food and Drug Administration controls the centralized marketing approval/authorization/ licensing of a new product, a process that requires in-depth review and acceptance of a New Drug Application for chemical entities, or a Biologics License Application for biologics, the latter including PMP proteins. The Committee for Medicinal Products for Human Use , part of the European Medicines Agency , has responsibilities similar to those of the FDA and plays a key role in the provision of scientific advice, evaluation of medicines at the national level for conformance with harmonized positions across the EU, and the centralized approval of new products for market entry in all Member States.