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.

Reducing water uptake into BY-2 cells by systematically optimizing the cultivation parameters increases product yields achieved by transient expression in plant cell packs.

Plant-based production systems are inexpensive and easy to handle, allowing them to complement existing platforms for the production of protein-based vaccines, therapeutics and diagnostic reagents. However, screening product candidates in whole plants requires a large facility footprint and is challenging due to natural variations in recombinant protein accumulation. In contrast, plant cell packs (PCPs) allow more than 1000 samples to be screened per day in microtiter plates. PCPs enable rapid development cycles based on transient expression in as little as 3 days, and yield milligram quantities of product for initial quality assessment and functional testing. However, this requires high-level expression in BY-2 cells and consistent cell quality across batches. We therefore used a statistical design of experiments (DoE) approach to systematically assess factors that contribute to consistent high yields of recombinant proteins in PCPs. Specifically, we tested the osmolality, pH, carbon source, light source, and additives during cell cultivation, as well as cell and PCP harvest times. The careful adjustment of these factors increased overall productivity by approximately fourfold. Remarkably all cultivation conditions leading to high productivities during transient expression in PCPs were associated with a reduced water uptake into the central vacuole. The universal presence of a vacuole in plant cells indicates that our results should be transferrable to other cells lines. Our findings therefore support the broad application of PCPs for screening and product analysis during the development of protein-based pharmaceuticals and reagents in plants.

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.

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.