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.

Targeted genome editing of plants and plant cells for biomanufacturing.

Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as "plant molecular farming" (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose "chassis" for PMF.

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.

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.

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.

Product safety aspects of plant molecular farming.

Plant molecular farming (PMF) has been promoted since the 1990s as a rapid, cost-effective and (most of all) safe alternative to the cultivation of bacteria or animal cells for the production of biopharmaceutical proteins. Numerous plant species have been investigated for the production of a broad range of protein-based drug candidates. The inherent safety of these products is frequently highlighted as an advantage of PMF because plant viruses do not replicate in humans and vice versa. However, a more nuanced analysis of this principle is required when considering other pathogens because toxic compounds pose a risk even in the absence of replication. Similarly, it is necessary to assess the risks associated with the host system (e.g., the presence of toxic secondary metabolites) and the production approach (e.g., transient expression based on bacterial infiltration substantially increases the endotoxin load). This review considers the most relevant host systems in terms of their toxicity profile, including the presence of secondary metabolites, and the risks arising from the persistence of these substances after downstream processing and product purification. Similarly, we discuss a range of plant pathogens and disease vectors that can influence product safety, for example, due to the release of toxins. The ability of downstream unit operations to remove contaminants and process-related toxic impurities such as endotoxins is also addressed. This overview of plant-based production, focusing on product safety aspects, provides recommendations that will allow stakeholders to choose the most appropriate strategies for process development.

An overview of descriptors to capture protein properties - Tools and perspectives in the context of QSAR modeling.

Proteins are important ingredients in food and feed, they are the active components of many pharmaceutical products, and they are necessary, in the form of enzymes, for the success of many technical processes. However, production can be challenging, especially when using heterologous host cells such as bacteria to express and assemble recombinant mammalian proteins. The manufacturability of proteins can be hindered by low solubility, a tendency to aggregate, or inefficient purification. Tools such as in silico protein engineering and models that predict separation criteria can overcome these issues but usually require the complex shape and surface properties of proteins to be represented by a small number of quantitative numeric values known as descriptors, as similarly used to capture the features of small molecules. Here, we review the current status of protein descriptors, especially for application in quantitative structure activity relationship (QSAR) models. First, we describe the complexity of proteins and the properties that descriptors must accommodate. Then we introduce descriptors of shape and surface properties that quantify the global and local features of proteins. Finally, we highlight the current limitations of protein descriptors and propose strategies for the derivation of novel protein descriptors that are more informative.

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.

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.

Spherical nanoparticles can be used as non-penetrating tracers to determine the extra-particle void volume in packed-bed chromatography columns.

The optimization of downstream processing in silico can accelerate bioprocess development by limiting experiments to the most promising separation conditions that have been identified using chromatography models. Such models describe protein binding and mass transport in packed-bed columns and thus require precise knowledge about the columns and the resins they contain. One important set of properties is the resin porosities, often determined using combinations of penetrating and non-penetrating tracers. However, the former can be disproportionately small, providing data of limited practical relevance, and the latter can undergo unwanted interactions with the resin, interfering with porosity calculations. Here we characterize and minimize the interactions of three novel hard-sphere non-penetrating tracers with the model resin Q Sepharose HP under various conditions and determine the corresponding inter-particle porosities. We found that conductivities > 100 mS cm-1 were necessary to suppress tracer-resin interactions despite them sharing the same surface charge. We combined these data with those from proteins studied under non-binding conditions, which can be used as authentic penetrating tracers, to determine both the intra-particle and total porosities. Furthermore, we found that the inter-particle porosity was below the theoretical limit of dense sphere packing (25.95%) and provide experimental data showing that the discrepancy is caused by resin particle deformation during the packing of columns under pressure.

The use of predictive models to develop chromatography-based purification processes.

Chromatography is the workhorse of biopharmaceutical downstream processing because it can selectively enrich a target product while removing impurities from complex feed streams. This is achieved by exploiting differences in molecular properties, such as size, charge and hydrophobicity (alone or in different combinations). Accordingly, many parameters must be tested during process development in order to maximize product purity and recovery, including resin and ligand types, conductivity, pH, gradient profiles, and the sequence of separation operations. The number of possible experimental conditions quickly becomes unmanageable. Although the range of suitable conditions can be narrowed based on experience, the time and cost of the work remain high even when using high-throughput laboratory automation. In contrast, chromatography modeling using inexpensive, parallelized computer hardware can provide expert knowledge, predicting conditions that achieve high purity and efficient recovery. The prediction of suitable conditions in silico reduces the number of empirical tests required and provides in-depth process understanding, which is recommended by regulatory authorities. In this article, we discuss the benefits and specific challenges of chromatography modeling. We describe the experimental characterization of chromatography devices and settings prior to modeling, such as the determination of column porosity. We also consider the challenges that must be overcome when models are set up and calibrated, including the cross-validation and verification of data-driven and hybrid (combined data-driven and mechanistic) models. This review will therefore support researchers intending to establish a chromatography modeling workflow in their laboratory.

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.

On the verge of the market - Plant factories for the automated and standardized production of biopharmaceuticals.

The market for biopharmaceuticals is dominated by recombinant proteins and is driven mainly by the development of vaccines and antibodies. Manufacturing predominantly relies on fermentation-based production platforms, which have limited scalability and suffer from high upstream process costs. As an alternative, the production of recombinant proteins in whole plants (plant molecular farming) provides a scalable and cost efficient upstream process because each plant functions as a self-contained bioreactor, avoiding costs associated with single-use devices and cleaning-in-place. Despite many proof-of-concept studies and the approval of a few products as medical devices, the only approved pharmaceutical proteins manufactured in whole plants have been authorized under emergency protocols. The absence of approvals under standard clinical development pathways in part reflects the lack of standardized process equipment and unit operations, leading to industry inertia based on familiarity with fermenter systems. Here we discuss the upstream production steps of plant molecular farming by transient expression in intact plants, including seeding, plant cultivation, infiltration with Agrobacterium tumefaciens, post-infiltration incubation, and harvesting. We focus on cultivation techniques because they strongly affect the subsequent steps and overall process design. We compare the benefits and drawbacks of open field, greenhouse and vertical farm strategies in terms of upfront investment costs, batch reproducibility, and decoupling from environmental impacts. We consider process automation, monitoring and adaptive process design in the context of Industry 4.0, which can boost process efficiency and batch-to-batch uniformity to improve regulatory compliance. Finally, we discuss the costs-benefit aspects of the different cultivation systems.

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.

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.

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.

Very-large-scale production of antibodies in plants: The biologization of manufacturing.

Gene technology has facilitated the biologization of manufacturing, i.e. the use and production of complex biological molecules and systems at an industrial scale. Monoclonal antibodies (mAbs) are currently the major class of biopharmaceutical products, but they are typically used to treat specific diseases which individually have comparably low incidences. The therapeutic potential of mAbs could also be used for more prevalent diseases, but this would require a massive increase in production capacity that could not be met by traditional fermenter systems. Here we outline the potential of plants to be used for the very-large-scale (VLS) production of biopharmaceutical proteins such as mAbs. We discuss the potential market sizes and their corresponding production capacities. We then consider available process technologies and scale-down models and how these can be used to develop VLS processes. Finally, we discuss which adaptations will likely be required for VLS production, lessons learned from existing cell culture-based processes and the food industry, and practical requirements for the implementation of a VLS process.

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.