Simulation and optimization of nutrient uptake and biomass formation using a multi-parameter Monod-type model of tobacco BY-2 cell suspension cultures in a stirred-tank bioreactor.

Introduction: Tobacco (Nicotiana tabacum) cv Bright Yellow-2 (BY-2) cell suspension cultures enable the rapid production of complex protein-based biopharmaceuticals but currently achieve low volumetric productivity due to slow biomass formation. The biomass yield can be improved with tailored media, which can be designed either by laborious trial-and-error experiments or systematic, rational design using mechanistic models, linking nutrient consumption and biomass formation. Methods: Here we developed an iterative experiment-modeling-optimization workflow to gradually refine such a model and its predictions, based on collected data concerning BY-2 cell macronutrient consumption (sucrose, ammonium, nitrate and phosphate) and biomass formation. Results and discussion: The biomass formation was well predicted by an unstructured segregated mechanistic Monod-type model as long as the nutrient concentrations did not approach zero (we omitted phosphate, which was completely depleted). Multi-criteria optimization for sucrose and biomass formation indicated the best tradeoff (in a Paretian sense) between maximum biomass yield and minimum process time by reducing the initial sucrose concentration, whereas the inoculation biomass could be increased to maximize the biomass yield or minimize the process time, which we confirmed in calibration experiments. The model became inaccurate at biomass densities > 8 g L-1 dry mass when sucrose was almost depleted. We compensated for this limitation by including glucose and fructose as sucrose hydrolysis products in the model. The remaining offset between the simulation and experimental data might be resolved by including intracellular pools of sucrose, ammonium, nitrate and phosphate. Overall, we demonstrated that iterative models can be used to systematically optimize conditions for bioreactor-based processes.

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.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia.

Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

Toxin type and domain sequence affect accumulation of recombinant immunotoxins.Transient expression in plant cell packs and intact plants correlates well.IC₅₀ values of toxicity correlate with the cell surface receptor concentration.

Risk assessment and bioburden evaluation of Agrobacterium tumefaciens-mediated transient protein expression in plants using the CaMV35S promoter.

Large-scale transient expression of recombinant proteins in plants is increasingly used and requires the multi-liter cultivation of Agrobacterium tumefaciens transformed with an expression vector, which is often cloned in Escherichia coli first. Depending on the promoter, unintentional activity can occur in both bacteria, which could pose a safety risk to the environment and operators if the protein is toxic. To assess the risk associated with transient expression, we first tested expression vectors containing the CaMV35S promoter known to be active in plants and bacteria, along with controls to measure the accumulation of the corresponding recombinant proteins. We found that, in both bacteria, even the stable model protein DsRed accumulated at levels near the detection limit of the sandwich ELISA (3.8 µg L-1). Higher levels were detected in short cultivations (< 12 h) but never exceeded 10 µg L-1. We determined the abundance of A. tumefaciens throughout the process, including infiltration. We detected few bacteria in the clarified extract and found none after blanching. Finally, we combined protein accumulation and bacterial abundance data with the known effects of toxic proteins to estimate critical exposures for operators. We found that unintended toxin production in bacteria is negligible. Furthermore, the intravenous uptake of multiple milliliters of fermentation broth or infiltration suspension would be required to reach acute toxicity even when handling the most toxic products (LD50 ~ 1 ng kg-1). The unintentional uptake of such quantities is unlikely and we therefore regard transient expression as safe in terms of the bacterial handling procedure.

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.

Simple plant-based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor-targeted drug delivery and bioimaging in cancer therapy.

Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g-1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5'-untranslated regions in PCPs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg-1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.

Liposome manufacturing under continuous flow conditions: towards a fully integrated set-up with in-line control of critical quality attributes.

Continuous flow manufacturing (CFM) has shown remarkable advantages in the industrial-scale production of drug-loaded nanomedicines, including mRNA-based COVID-19 vaccines. Thus far, CFM research in nanomedicine has mainly focused on the initial particle formation step, while post-formation production steps are hardly ever integrated. The opportunity to implement in-line quality control of critical quality attributes merits closer investigation. Here, we designed and tested a CFM setup for the manufacturing of liposomal nanomedicines that can potentially encompass all manufacturing steps in an end-to-end system. Our main aim was to elucidate the key composition and process parameters that affect the physicochemical characteristics of the liposomes. Total flow rate, lipid concentration and residence time of the liposomes in a high ethanol environment (i.e., above 20% v/v) emerged as critical parameters to tailor liposome size between 80 and 150 nm. After liposome formation, the pressure and the surface area of the filter in the ultrafiltration unit were critical parameters in the process of clearing the dispersion from residual ethanol. As a final step, we integrated in-line measurement of liposome size and residual ethanol content. Such in-line measurements allow for real-time monitoring and in-process adjustment of key composition and process parameters.

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.

Bayesian optimization using multiple directional objective functions allows the rapid inverse fitting of parameters for chromatography simulations.

The modeling of chromatographic separations can speed up downstream process development, reducing the time to market and corresponding development costs for new products such as pharmaceuticals. However, calibrating such models by identifying suitable parameter values for mass transport and sorption is a major, time-consuming challenge that can hinder model development and improvement. We therefore designed a new approach based on Bayesian optimization (BayesOpt) and Gaussian processes that reduced the time required to compute relevant chromatography parameters by up to two orders of magnitude compared to a multistart gradient descent and a genetic algorithm. We compared the three approaches side by side to process several internal and external datasets for ion exchange chromatography (based on a steric mass action isotherm) and hydrophobic interaction chromatography (a modified version of a recently published five-parameter isotherm) as well as different input data types (gradient elution data alone vs gradient elution and breakthrough data). We found that BayesOpt computation was consistently faster than the other approaches when using either single-core or 12-cores computer processing units. The error of the BayesOpt parameter estimates was higher than that of the competing algorithms, but still two orders of magnitude less than the variability of our experimental data, indicating BayesOpts applicability for chromatography modeling. The low computational demand of BayesOpt will facilitate rapid model development and improvement even for large datasets (e.g., > 100 proteins) and increase its suitability for research laboratories or small and medium enterprises lacking access to dedicated mainframe computers.

The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope-labelled substrates to cultivate the tobacco-derived cell line BY-2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with 15 N and 2 H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost-efficient alternative for the production of isotope-labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems.

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.

Monitoring EPR Effect Dynamics during Nanotaxane Treatment with Theranostic Polymeric Micelles.

Cancer nanomedicines rely on the enhanced permeability and retention (EPR) effect for efficient target site accumulation. The EPR effect, however, is highly heterogeneous among different tumor types and cancer patients and its extent is expected to dynamically change during the course of nanochemotherapy. Here the authors set out to longitudinally study the dynamics of the EPR effect upon single- and double-dose nanotherapy with fluorophore-labeled and paclitaxel-loaded polymeric micelles. Using computed tomography-fluorescence molecular tomography imaging, it is shown that the extent of nanomedicine tumor accumulation is predictive for therapy outcome. It is also shown that the interindividual heterogeneity in EPR-based tumor accumulation significantly increases during treatment, especially for more efficient double-dose nanotaxane therapy. Furthermore, for double-dose micelle therapy, tumor accumulation significantly increased over time, from 7% injected dose per gram (ID g-1 ) upon the first administration to 15% ID g-1 upon the fifth administration, contributing to more efficient inhibition of tumor growth. These findings shed light on the dynamics of the EPR effect during nanomedicine treatment and they exemplify the importance of using imaging in nanomedicine treatment prediction and clinical translation.

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.

A combined pH and temperature precipitation step facilitates the purification of tobacco-derived recombinant proteins that are sensitive to extremes of either parameter.

Incubation at pH 4.0 or blanching at ∼65°C facilitates the purification of biopharmaceutical proteins from plants by precipitating most of the host cell proteins (HCPs) before chromatography. However, both methods are compatible only with pH or thermostable target proteins whereas many target proteins may irreversibly denature, e.g., at pH < 4.0. Here, we developed a combined pH/temperature treatment for clarified tobacco extracts and intact leaves. The latter were subjected to a blanching procedure, i.e., the submersion into a hot buffer. Using a design of experiments approach we identified conditions that remove ∼70% of HCPs at ∼55°C, using the thermosensitive antibody 2G12 and the pH-sensitive DsRed as model proteins. We found that pH and temperature exerted a combined effect during the precipitation of HCPs in the pH range 5.0-7.0 at 35°C-60°C. For clarified extracts, the temperature required to achieve a DsRed purity threshold of 20% total soluble protein (TSP) increased from 54°C to 63°C when the pH was increased from 6.4 to 7.3. The pH-stable antibody 2G12 was less responsive to the combined treatment, but the purity of 1% TSP was achieved at 35°C instead of 44°C when the pH was reduced from 6.3 to 5.8. When blanching intact leaves, product losses were not exacerbated at pH 4.0. Indeed, the highest DsRed purity (58% TSP) was achieved at this pH, combined with a temperature of 60°C and an incubation time of 30 min. In contrast, the highest 2G12 purity (0.7% TSP) was achieved at pH 5.1 and 40°C with an incubation time of 20 min. Our data suggest that a combined pH/temperature regime can avoid extreme values of either parameter; therefore, broadening the applicability of these simple purification techniques to other recombinant proteins.

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.

The Emergency Response Capacity of Plant-Based Biopharmaceutical Manufacturing-What It Is and What It Could Be.

Several epidemic and pandemic diseases have emerged over the last 20 years with increasing reach and severity. The current COVID-19 pandemic has affected most of the world's population, causing millions of infections, hundreds of thousands of deaths, and economic disruption on a vast scale. The increasing number of casualties underlines an urgent need for the rapid delivery of therapeutics, prophylactics such as vaccines, and diagnostic reagents. Here, we review the potential of molecular farming in plants from a manufacturing perspective, focusing on the speed, capacity, safety, and potential costs of transient expression systems. We highlight current limitations in terms of the regulatory framework, as well as future opportunities to establish plant molecular farming as a global, de-centralized emergency response platform for the rapid production of biopharmaceuticals. The implications of public health emergencies on process design and costs, regulatory approval, and production speed and scale compared to conventional manufacturing platforms based on mammalian cell culture are discussed as a forward-looking strategy for future pandemic responses.