A concise guide to choosing suitable gene expression systems for recombinant protein production.

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems.

Killer to cure: Expression and production costs calculation of tobacco plant-made cancer-immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg- and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine-tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high- and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.

Seed- and leaf-based expression of FGF21-transferrin fusion proteins for oral delivery and treatment of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a global disease with no effective medication. The fibroblast growth factor 21 (FGF21) can reverse this liver dysfunction, but requires targeted delivery to the liver, which can be achieved via oral administration. Therefore, we fused FGF21 to transferrin (Tf) via a furin cleavage site (F), to promote uptake from the intestine into the portal vein, yielding FGF21-F-Tf, and established its production in both seeds and leaves of commercial Nicotiana tabacum cultivars, compared their expression profile and tested the bioavailability and bioactivity in feeding studies. Since biopharmaceuticals need to be produced in a contained environment, e.g., greenhouses in case of plants, the seed production was increased in this setting from 239 to 380 g m-2 a-1 seed mass with costs of 1.64 € g-1 by side branch induction, whereas leaves yielded 8,193 g m-2 a-1 leave mass at 0.19 € g-1. FGF21-F-Tf expression in transgenic seeds and leaves yielded 6.7 and 5.6 mg kg-1 intact fusion protein, but also 4.5 and 2.3 mg kg-1 additional Tf degradation products. Removing the furin site and introducing the liver-targeting peptide PLUS doubled accumulation of intact FGF21-transferrin fusion protein when transiently expressed in Nicotiana benthamiana from 0.8 to 1.6 mg kg-1, whereas truncation of transferrin (nTf338) and reversing the order of FGF21 and nTf338 increased the accumulation to 2.1 mg kg-1 and decreased the degradation products to 7% for nTf338-FGF21-PLUS. Application of partially purified nTf338-FGF21-PLUS to FGF21-/- mice by oral gavage proved its transfer from the intestine into the blood circulation and acutely affected hepatic mRNA expression. Hence, the medication of NASH via oral delivery of nTf338-FGF21-PLUS containing plants seems possible.

Strategies for Efficient and Sustainable Protein Extraction and Purification from Plant Tissues.

Recombinant proteins produced in whole plants are usually extracted and purified from leaf or seed tissues. Here we describe a workflow that is suitable for most soluble pharmaceutical proteins produced in leafy biomass. We highlight options to streamline the process, reducing the total process time and costs, and eliminating certain unit operations if the product allows. The overall process is a combination of batch-wise or continuous extraction followed by a series of solid-liquid separation steps, which can be supported by chemically defined additives, and concludes with a sequence of optional membrane-based and regular chromatographic purification operations.

Statistical Designs to Improve Downstream Processing.

The efficient extraction and purification of recombinant proteins from leaf and seed tissues is often a challenging task, involving multiple steps that must be optimized by identifying and accommodating complex parameter interactions. Conventional one-factor-at-a-time approaches fail to reveal these complex interactions and often result in sub-optimal processes with unnecessary costs. Here, we describe generic considerations to identify global optima for the extraction and purification of recombinant proteins from complex plant matrices. The corresponding experiments can help to streamline downstream processing by reducing the time, costs, and number of unit operations. The procedure involves the knowledge-based selection of factors for screening, the systematic design and analysis of experiments, and the iterative refinement of suitable conditions. The resulting descriptive models can be used to guide process scale-up and offer scientific justifications for process development decisions in negotiations with regulatory authorities.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

Cryopreservation of plant cell cultures - Diverse practices and protocols.

Plant cell cultures can be used as biotechnological platforms for the commercial production of small-molecule active ingredients and recombinant proteins, such as biopharmaceuticals. This requires the cryopreservation of well-characterized cell lines as master cell banks from which uniform working cell banks can be derived to ensure high batch-to-batch reproducibility during production campaigns. However, the cryopreservation of plant cells is challenging due to their low viability and poor regrowth after thawing. Three approaches have been developed: slow freezing, vitrification, and encapsulation-dehydration. Typically, the protocols are iteratively adapted to accommodate the properties of different plant cell lines, taking time and resources while achieving moderate success. Since standardized processes are a prerequisite for industrial applications, this review presents an in-depth analysis of the different procedures for cryopreservation of plant suspension cell cultures, highlighting relevant parameters for effective cryopreservation and the re-establishment of vigorous plant cell cultures within weeks. The protocol variants are grouped into modules that facilitate the directed improvement of each step and allow protocol evolution by module recombination. Ultimately, such improved cryopreservation protocols will form the basis of processes that comply with good manufacturing practice and attract major biopharmaceutical companies to the benefits of plant molecular farming.

Glyco-Engineering of Plant-Based Expression Systems.

Most secreted proteins in eukaryotes are glycosylated, and after a number of common biosynthesis steps the glycan structures mature in a species-dependent manner. Therefore, human therapeutic proteins produced in plants often carry plant-like rather than human-like glycans, which can affect protein stability, biological function, and immunogenicity. The glyco-engineering of plant-based expression systems began as a strategy to eliminate plant-like glycans and produce human proteins with authentic or at least compatible glycan structures. The precise replication of human glycans is challenging, owing to the absence of a pathway in plants for the synthesis of sialylated proteins and the necessary precursors, but this can now be achieved by the coordinated expression of multiple human enzymes. Although the research community has focused on the removal of plant glycans and their replacement with human counterparts, the presence of plant glycans on proteins can also provide benefits, such as boosting the immunogenicity of some vaccines, facilitating the interaction between therapeutic proteins and their receptors, and increasing the efficacy of antibody effector functions. Graphical Abstract Typical structures of native mammalian and plant glycans with symbols indicating sugar residues identified by their short form and single-letter codes. Both glycans contain fucose, albeit with different linkages.

Molecular farming - The slope of enlightenment.

Molecular farming can be defined as the use of plants to produce recombinant protein products. The technology is now >30 years old. The early promise of molecular farming was based on three perceived advantages: the low costs of growing plants, the immense scalability of agricultural production, and the inherent safety of plants as hosts for the production of pharmaceuticals. This resulted in a glut of research publications in which diverse proteins were expressed in equally diverse plant-based systems, and numerous companies were founded hoping to commercialize the new technology. There was a moderate degree of success for companies producing non-pharmaceutical proteins, but in the pharmaceutical sector the anticipation raised by promising early research was soon met by the cold hard reality of industrial pragmatism. Plants did not have a track record of success in pharmaceutical protein manufacturing, lacked a regulatory framework, and did not perform as well as established industry platforms. Negative attitudes towards genetically modified plants added to the mix. By the early 2000s, major industry players started to lose interest and pharmaceutical molecular farming fell from a peak of expectation into a trough of disillusionment, just as predicted by the Gartner hype cycle. But many of the pioneers of molecular farming have refocused their activities and have worked to address the limitations that hampered the first generation of technologies. The field has now consolidated around a smaller number of better-characterized platforms and has started to develop standardized methods and best practices, mirroring the evolution of more mature industry sectors. Likewise, attention has turned from proof-of-principle studies to realistic techno-economic modeling to capture significant niche markets, replicating the success of the industrial molecular farming sector. Here we argue that these recent developments signify that pharmaceutical molecular farming is now climbing the slope of enlightenment and will soon emerge as a mature technology.

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

Non-invasive Imaging and Modeling of Liver Regeneration After Partial Hepatectomy.

The liver has a unique regenerative capability upon injury or partial resection. The regeneration process comprises a complex interplay between parenchymal and non-parenchymal cells and is tightly regulated at different scales. Thus, we investigated liver regeneration using multi-scale methods by combining non-invasive imaging with immunohistochemical analyses. In this context, non-invasive imaging can provide quantitative data of processes involved in liver regeneration at organ and body scale. We quantitatively measured liver volume recovery after 70% partial hepatectomy (PHx) by micro computed tomography (µCT) and investigated changes in the density of CD68+ macrophages by fluorescence-mediated tomography (FMT) combined with µCT using a newly developed near-infrared fluorescent probe. In addition, angiogenesis and tissue-resident macrophages were analyzed by immunohistochemistry. Based on the results, a model describing liver regeneration and the interactions between different cell types was established. In vivo analysis of liver volume regeneration over 21 days after PHx by µCT imaging demonstrated that the liver volume rapidly increased after PHx reaching a maximum at day 14 and normalizing until day 21. An increase in CD68+ macrophage density in the liver was detected from day 4 to day 8 by combined FMT-µCT imaging, followed by a decline towards control levels between day 14 and day 21. Immunohistochemistry revealed the highest angiogenic activity at day 4 after PHx that continuously declined thereafter, whereas the density of tissue-resident CD169+ macrophages was not altered. The simulated time courses for volume recovery, angiogenesis and macrophage density reflect the experimental data describing liver regeneration after PHx at organ and tissue scale. In this context, our study highlights the importance of non-invasive imaging for acquiring quantitative organ scale data that enable modeling of liver regeneration.

Ready-to-Use Stocks of Agrobacterium tumefaciens Can Simplify Process Development for the Production of Recombinant Proteins by Transient Expression in Plants.

Large-scale automated transient protein expression in plants requires the synchronization of cultivation and bacterial fermentation, especially if more than one bacterial strain. Therefore, a ready-to-use approach that decouples bacterial fermentation and infiltration is developed. It is found that bacterial cultures can easily be reconstituted in infiltration medium at a user-defined time, optical density, and quantity. This allows the process flow to be staggered, avoiding bottlenecks in process capacity and labor. Using the red fluorescent protein, DsRed, as a model product, the ready-to-use preparations achieved the same yields in infiltrated plant biomass as Agrobacterium tumefaciens derived from regular fermentations. It is possible to store the ready-to-use stocks at -20 °C and -80 °C for more than two months without loss of activity. Using a consolidated cost model for the current fermentation process, it is found that the ready-to-use strategy can reduce operational costs by 20-95% and investment costs by up to 75%, which would otherwise offset the economic advantages of plants over mammalian expression systems during upstream production. Furthermore, the staggered cultivation of plants and bacteria reduces the likelihood of batch failure and thus increases the robustness and flexibility of transient expression for the production of recombinant proteins in plants.

Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin.

Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria.

The Correlation Between DsRed mRNA Levels and Transient DsRed Protein Expression in Plants Depends on Leaf Age and the 5' Untranslated Region.

The yield of recombinant proteins in plants determines their economic competitiveness as a production platform compared to microbes and mammalian cells. The promoter, untranslated regions (UTRs) and codon usage can all contribute to the yield, but potential interactions among these components have not been examined in detail. Here the effect of two promoters (35SS and nos) and four 5'UTRs on the spatiotemporal expression of DsRed mRNA and the accumulation of DsRed protein during transient expression in tobacco (Nicotiana tabacum) mediated by Agrobacterium tumefaciens is investigated. The authors found that the mRNA levels peaked 2-3 days post-infiltration (dpi), and rapidly declined thereafter, whereas DsRed protein was first detected after ≈3 days and concentrations continued to increase until at least 5 dpi. This temporal decoupling of mRNA and protein expression was strongest in the older leaves, which also produced the lowest DsRed yields. The accumulation of DsRed linearly correlated with mRNA levels in all but the youngest leaves, where more DsRed was synthesized per mRNA molecule. This was the case for both promoters, although the nos promoter had a higher protein/mRNA ratio than the 35SS promoter. Furthermore, the type of 5'UTR affected DsRed protein accumulation by 50% starting from similar levels of mRNA. The authors concluded that DsRed mRNA levels are not the limiting factor for DsRed protein expression in plants, but that translation-associated processes such as initiation, elongation, and release are bottlenecks that should be addressed in future studies.

Plant Molecular Farming - Integration and Exploitation of Side Streams to Achieve Sustainable Biomanufacturing.

Plants have unique advantages over other systems such as mammalian cells for the production of valuable small molecules and proteins. The benefits cited most often include safety due to the absence of replicating human pathogens, simplicity because sterility is not required during production, scalability due to the potential for open-field cultivation with transgenic plants, and the speed of transient expression potentially providing gram quantities of product in less than 4 weeks. Initially there were also significant drawbacks, such as the need to clarify feed streams with a high particle burden and the large quantities of host cell proteins, but efficient clarification is now readily achieved. Several additional advantages have also emerged reflecting the fact that plants are essentially biodegradable, single-use bioreactors. This article will focus on the exploitation of this concept for the production of biopharmaceutical proteins, thus improving overall process economics. Specifically, we will discuss the single-use properties of plants, the sustainability of the production platform, and the commercial potential of different biomass side streams. We find that incorporating these side streams through rational process integration has the potential to more than double the revenue that can currently be achieved using plant-based production systems.

A Combined Ultrafiltration/Diafiltration Step Facilitates the Purification of Cyanovirin-N From Transgenic Tobacco Extracts.

The production of biopharmaceutical proteins in plants offers many advantages over traditional expression platforms, including improved safety, greater scalability and lower upstream production costs. However, most products are retained within plant cells or the apoplastic space instead of being secreted into a liquid medium, so downstream processing necessarily involves tissue and cell disruption followed by the removal of abundant particles and host cell proteins (HCPs). We investigated whether ultrafiltration/diafiltration (UF/DF) can simplify the purification of the model recombinant protein cyanovirin-N (CVN), an ~ 11 kDa HIV-neutralizing lectin, from tobacco extracts prior to chromatography. We compared different membrane types and process conditions, and found that at pH 8.0 and 50 mS cm-1 an UF step using a 100 kDa regenerated cellulose membrane removed more than 80% of the ~ 0.75 mg mL-1 total soluble protein present in the clarified plant extract. We recovered ~ 70% of the CVN and the product purity increased ~ 3-fold in the permeate. The underlying effects of tobacco HCP retention during the UF/DF step were investigated by measuring the zeta potential and particle size distribution in the 2-10,000 nm range. Combined with a subsequent 10 kDa DF step, this approach simultaneously reduced the process volume, conditioned the process intermediate, and facilitated early, chromatography-free purification. Due to the generic, size-based nature of the method, it is likely to be compatible with most products smaller than ~50 kDa.

Production of Functional Anti-Ebola Antibodies in Pichia pastoris.

The 2013-2016 Ebola outbreak highlighted the limited treatment options and lack of rapid response strategies for emerging pathogen outbreaks. Here, we propose an efficient development cycle using glycoengineered Pichia pastoris to produce monoclonal antibody cocktails against pathogens. To enable rapid genetic engineering of P. pastoris, we introduced a genomic landing pad for reliable recombinase-mediated DNA integration. We then created strains expressing each of the three monoclonal antibodies that comprise the ZMapp cocktail, and demonstrated that the secreted antibodies bind to the Ebola virus glycoprotein by immunofluorescence assay. We anticipate that this approach could accelerate the production of therapeutics against future pathogen outbreaks.

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.