Optimizing interleukin-6 and 8 expression, clarification and purification in plant cell packs and plants for application in advanced therapy medicinal products and cellular agriculture.

Healthcare and nutrition are facing a paradigm shift in light of advanced therapy medicinal products (ATMPs) and cellular agriculture options respectively. Both options heavily rely on some sort of animal cell culture, e.g. autologous stem cells. These cultures require various growth factors, such as interleukin-6 and 8 (IL-6/8), in a pure, safe and sustainable form that can be provided in a scalable manner. Plants seem well suited for this task because purification of small proteins can be readily achieved by membrane separation, human/animal pathogens do not replicate in plants and production can be scaled up using in-door farming or agricultural practices. Here, we illustrate this capacity by first optimizing the codon usage of IL-6/8 for translation in Nicotiana spp., as well as testing the effect of untranslated regions and product targeting to different sub-cellular compartments on expression in a high-throughput plant cell pack (PCP) assay. In the chloroplast, IL-6 accumulated up to 6.9±3.8 (SD, n=2) and 14.4±7.4 mg kg-1 (SD, n=5) were observed in case of IL-8. When transferring IL-8 expression into whole plants, accumulation was 12.3±1.5 mg kg-1 (SD, n=3). After extraction and clarification, IL-8 was purified using a two-stage process consisting of an ultrafiltration/diafiltration step with 100 kDa and 10 kDa cut off membranes followed by an IMAC polishing step. The purity, yield and recovery were 97.8%, 6.6 mg kg-1 and 38%, respectively. We evaluated the ability of the proposed purification process to remove endotoxins to ensure the compatibility of plant-made growth factors with cell culture.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Expression of Biofilm-Degrading Enzymes in Plants and Automated High-Throughput Activity Screening Using Experimental Bacillus subtilis Biofilms.

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg-1 Lg2 in extracts but 0.3 mg kg-1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

Precision analysis for the determination of steric mass action parameters using eight tobacco host cell proteins.

Plants are advantageous as biopharmaceutical manufacturing platforms because they allow the economical and scalable upstream production of proteins, including those requiring post-translational modifications, but do not support the replication of human viruses. However, downstream processing can be more labor-intensive compared to fermenter-based systems because the product is often mixed with abundant host cell proteins (HCPs). Modeling chromatographic separation can minimize the number of process development experiments and thus reduce costs. An important part of such modeling is the sorption isotherm, such as the steric mass action (SMA) model, which describes the multicomponent protein-salt equilibria established in ion-exchange systems. Here we purified ten HCPs, including 2-Cys-peroxiredoxin, from tobacco (Nicotiana tabacum and N. benthamiana). For eight of these HCPs, we obtained sufficient quantities to determine the SMA binding parameters (KSMA and ν) under different production-relevant conditions. We studied the parameters for 2-Cys-peroxiredoxin on Q-Sepharose HP in detail, revealing that pH, resin batch and buffer batch had little influence on KSMA and ν, with coefficients of variation (COVs) less than 0.05 and 0.21, respectively. In contrast, the anion-exchange resins SuperQ-650S, Q-Sepharose FF and QAE-550C led to COVs of 0.69 for KSMA and 0.05 for ν, despite using the same quaternary amine functional group as Q-Sepharose HP. Plant cultivation in summer vs winter resulted in COVs of 0.09 for KSMA and 0.02 for ν, revealing a small impact compared to COVs of 17.15 for KSMA and 0.20 for ν when plants were grown in different settings (climate-controlled phytotron vs greenhouse). We conclude that plant cultivation can substantially affect protein properties and the resulting SMA parameters. Accordingly, plant growth but also protein purification and characterization for chromatography model building should be tightly controlled and well documented.

Plant-made immunotoxin building blocks: A roadmap for producing therapeutic antibody-toxin fusions.

Molecular farming in plants is an emerging platform for the production of pharmaceutical proteins, and host species such as tobacco are now becoming competitive with commercially established production hosts based on bacteria and mammalian cell lines. The range of recombinant therapeutic proteins produced in plants includes replacement enzymes, vaccines and monoclonal antibodies (mAbs). But plants can also be used to manufacture toxins, such as the mistletoe lectin viscumin, providing an opportunity to express active antibody-toxin fusion proteins, so-called recombinant immunotoxins (RITs). Mammalian production systems are currently used to produce antibody-drug conjugates (ADCs), which require the separate expression and purification of each component followed by a complex and hazardous coupling procedure. In contrast, RITs made in plants are expressed in a single step and could therefore reduce production and purification costs. The costs can be reduced further if subcellular compartments that accumulate large quantities of the stable protein are identified and optimal plant growth conditions are selected. In this review, we first provide an overview of the current state of RIT production in plants before discussing the three key components of RITs in detail. The specificity-defining domain (often an antibody) binds cancer cells, including solid tumors and hematological malignancies. The toxin provides the means to kill target cells. Toxins from different species with different modes of action can be used for this purpose. Finally, the linker spaces the two other components to ensure they adopt a stable, functional conformation, and may also promote toxin release inside the cell. Given the diversity of these components, we extract broad principles that can be used as recommendations for the development of effective RITs. Future research should focus on such proteins to exploit the advantages of plants as efficient production platforms for targeted anti-cancer therapeutics.

A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies.

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity. Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution. We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems.

Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg-1 biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2-200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.

Plants as sources of natural and recombinant anti-cancer agents.

Herbal remedies were the first medicines used by humans due to the many pharmacologically active secondary metabolites produced by plants. Some of these metabolites inhibit cell division and can therefore be used for the treatment of cancer, e.g. the mitostatic drug paclitaxel (Taxol). The ability of plants to produce medicines targeting cancer has expanded due to the advent of genetic engineering, particularly in recent years because of the development of gene editing systems such as the CRISPR/Cas9 platform. These technologies allow the introduction of genetic modifications that facilitate the accumulation of native pharmaceutically-active substances, and even the production heterologous recombinant proteins, including human antibodies, lectins and vaccine candidates. Here we discuss the anti-cancer agents that are produced by plants naturally or following genetic modification, and the potential of these products to supply modern healthcare systems. Special emphasis will be put on proteinaceous anti-cancer agents, which can exhibit an improved selectivity and reduced side effects compared to small molecule-based drugs.

Determination of the thermal properties of leaves by non-invasive contact­free laser probing.

The thermal properties of materials provide valuable data for quality monitoring and the rational design of process steps where heating is required. Here we report a rapid, simple and reliable technique that determines the most important thermal properties of leaves, i.e. the specific heat capacity (cp) and thermal conductivity (λ). Such data are useful when leaves are heated during processing, e.g. for the precipitation of host cell proteins during the extraction of high-value products such as recombinant proteins produced by molecular farming. The cp of tobacco (Nicotiana tabacum) and Nicotiana benthamiana leaves was determined by infrared measurement of the temperature increase caused by a near-infrared laser pulse of defined length and intensity. We used the sample temperature profiles to calculate λ based on exponential fits of the temperature decline, taking convective heat transfer and thermal radiation into account. We found that the average cp was 3661 ± 323 J kg(-1) K(-1) (n=19) for tobacco and 2253 ± 285 J kg(-1) K(-1) (n=25) for N. benthamiana, whereas the average λ was 0.49 ± 0.13 (n=19) for tobacco and 0.41 ± 0.20 (n=25) Jm(-1) s(-1)K(-1) for N. benthamiana. These values are similar to those established for other plant species by photothermal imaging and other methods. The cp and λ values of leaves can be determined easily using our non-invasive method, which is therefore suitable for the in-line or at-line monitoring of plants, e.g. during the highly regulated production of biopharmaceutical proteins.

Controlling the interplay between Agrobacterium tumefaciens and plants during the transient expression of proteins.

In May 2012, the first plant-derived biopharmaceutical protein received full regulatory approval for therapeutic use in humans. Although plant-based expression systems have many advantages, they can suffer from low expression levels and, depending on the species, the presence of potentially toxic secondary metabolites. Transient expression mediated by Agrobacterium tumefaciens can be used to increase product yields but may also increase the concentration of secondary metabolites generated by plant defense responses. We have recently investigated the sequence of defense responses triggered by A. tumefaciens in tobacco plants and considered how these can be modulated by the transient expression of type III effectors from Pseudomonas syringae. Here we discuss the limitations of this approach, potential solutions and additional issues concerning transient expression in plants that should be investigated in greater detail.

The impact of Pseudomonas syringae type III effectors on transient protein expression in tobacco.

The production of recombinant proteins in plants is often achieved by transient expression, e.g. following the injection or vacuum infiltration of Agrobacterium tumefaciens into tobacco leaves. We investigated the associated plant defence responses, revealing that callose deposition is triggered by T-DNA transfer and that subsets of secondary metabolites accumulate in response to mechanical wounding or the presence of bacteria. We also tested the ability of five co-expressed type III effector proteins from Pseudomonas syringae to modulate these defence responses and increase the yield of two model proteins, the fluorescent marker DsRed and monoclonal antibody 2G12. HopF2 and AvrRpt2 induced necrotic lesions 5 days post-injection (dpi) even at low doses (OD600 nm  = 0.0078), and increased the concentration of certain secondary metabolites. HopAO1 significantly reduced the number of callose deposits at 2 dpi compared to cells expressing DsRed and 2G12 alone, whereas HopI1 reduced the concentration of several secondary metabolites at 5 dpi compared to cells expressing DsRed and 2G12 alone. Co-expression with HopAO1, AvrPtoB or HopI1 increased the concentrations of DsRed and 2G12 increased by ~6% but this was not a significant change. In contrast, HopF2 and AvrRpt2 significantly reduced the concentrations of DsRed and 2G12 by 34% and 22%, respectively. Our results show that type III effector proteins can modulate plant defence responses and secondary metabolite profiles but that transient co-expression is not sufficient to increase the yields of target recombinant proteins in tobacco.

The use of quantitative structure-activity relationship models to develop optimized processes for the removal of tobacco host cell proteins during biopharmaceutical production.

The production of recombinant pharmaceutical proteins in plants benefits from the low cost of upstream production and the greater scalability of plants compared to fermenter-based systems. Now that manufacturing processes that comply with current good manufacturing practices have been developed, plants can compete with established platforms on equal terms. However, the costs of downstream processing remain high, in part because of the dedicated process steps required to remove plant-specific process-related impurities. We therefore investigated whether the ideal strategy for the chromatographic removal of tobacco host cell proteins can be predicted by quantitative structure-activity relationship (QSAR) modeling to reduce the process development time and overall costs. We identified more than 100 tobacco proteins by mass spectrometry and their structures were reconstructed from X-ray crystallography, nuclear magnetic resonance spectroscopy and/or homology modeling data. The resulting three-dimensional models were used to calculate protein descriptors, and significant descriptors were selected based on recently-published retention data for model proteins to develop QSAR models for protein retention on anion, cation and mixed-mode resins. The predicted protein retention profiles were compared with experimental results using crude tobacco protein extracts. Because of the generic nature of the method, it can easily be transferred to other expression systems such as mammalian cells. The quality of the models and potential improvements are discussed.

Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5'UTR combination.

The promoter and 5'-untranslated region (5'UTR) play a key role in determining the efficiency of recombinant protein expression in plants. Comparative experiments are used to identify suitable elements but these are usually tested in transgenic plants or in transformed protoplasts/suspension cells, so their relevance in whole-plant transient expression systems is unclear given the greater heterogeneity in expression levels among different leaves. Furthermore, little is known about the impact of promoter/5'UTR interactions on protein accumulation. We therefore established a predictive model using a design of experiments (DoE) approach to compare the strong double-enhanced Cauliflower mosaic virus 35S promoter (CaMV 35SS) and the weaker Agrobacterium tumefaciens Ti-plasmid nos promoter in whole tobacco plants transiently expressing the fluorescent marker protein DsRed. The promoters were combined with one of three 5'UTRs (one of which was tested with and without an additional protein targeting motif) and the accumulation of DsRed was measured following different post-agroinfiltration incubation periods in all leaves and at different leaf positions. The model predictions were quantitative, allowing the rapid identification of promoter/5'UTR combinations stimulating the highest and quickest accumulation of the marker protein in all leaves. The model also suggested that increasing the incubation time from 5 to 8 days would reduce batch-to-batch variability in protein yields. We used the model to identify promoter/5'UTR pairs that resulted in the least spatiotemporal variation in expression levels. These ideal pairs are suitable for the simultaneous, balanced production of several proteins in whole plants by transient expression.

Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs.

The transient expression of recombinant biopharmaceutical proteins in plants can suffer inter-batch variation, which is considered a major drawback under the strict regulatory demands imposed by current good manufacturing practice (cGMP). However, we have achieved transient expression of the monoclonal antibody 2G12 and the fluorescent marker protein DsRed in tobacco leaves with ∼ 15% intra-batch coefficients of variation, which is within the range reported for transgenic plants. We developed models for the transient expression of both proteins that predicted quantitative expression levels based on five parameters: The OD(600 nm) of Agrobacterium tumefaciens (from 0.13 to 2.00), post-inoculation incubation temperature (15-30°C), plant age (harvest at 40 or 47 days after seeding), leaf age, and position within the leaf. The expression models were combined with a model of plant biomass distribution and extraction, generating a yield model for each target protein that could predict the amount of protein in specific leaf parts, individual leaves, groups of leaves, and whole plants. When the yield model was combined with a cost function for the production process, we were able to perform calculations to optimize process time, yield, or downstream costs. We illustrate this procedure by transferring the cost function from a production process using transgenic plants to a hypothetical process for the transient expression of 2G12. Our models allow the economic evaluation of new plant-based production processes and provide greater insight into the parameters that affect transient protein expression in plants.

Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG.

Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model. We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1g of protein per 1 kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50 g to 1 kg of processed leaf biomass.