Transient Production of Recombinant Pharmaceutical Proteins in Plants: Evolution and Perspectives.

During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection.

Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean.

Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed.

Production of hybrid-IgG/IgA plantibodies with neutralizing activity against Shiga toxin 1.

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.

Plant-based production of recombinant Plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate.

Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and MSP119. Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1∶11.000 and 1∶39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using αPf38 antibodies demonstrated strong inhibition (≥60%) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by αPf38 antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.

Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures.

For the commercialization of plant-made pharmaceuticals (PMPs) using transgenic plant cell cultures, the establishment of a cell-banking system has been known to be an essential process. Plant cells are traditionally maintained by repeated subcultures. However, this method has several problems including genetic instability of transformed cell lines, time- and cost-consuming. In this study, long-term cryopreserved rice suspension cells were firstly investigated for the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). The cryopreserved cells for 5 years were regrowed to callus successfully and then suspended into the liquid medium. Consequently, the maximum cell mass and the hCTLA4Ig production were similar levels compared to those of the non-cryopreserved cells (control) even though hCTLA4Ig productivity was 1.7-fold higher than that of control. To further assess the level of improvements in hCTLA4Ig productivity in cryopreserved cells, hCTLA4Ig production profiles were statistically assessed between data of the cryopreserved cells for 5 years and annual data of non-cryopreserved cells maintained by subculture for 5 years. These results also indicate that hCTLA4Ig productivity in cryopreserved cells for 5 years was significantly increased (p-value: <0.001, 95% confidence interval) and it could be related to cell lysis resulting in release of hCTLA4Ig which was confirmed by the measurement of electrolyte leakage. In conclusion, we show that the long-term cryopreservation of transgenic rice cells was possible to support stable cell lines for the production of PMPs.

Protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of Nicotiana benthamiana.

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ~3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology.

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.

Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes.

Infectious diseases represent a continuously growing menace that has severe impact on health of the people worldwide, particularly in the developing countries. Therefore, novel prevention and treatment strategies are urgently needed to reduce the rate of these diseases in humans. For this reason, different options can be considered for the production of affordable vaccines. Plants have been proved as an alternative expression system for various compounds of biological importance. Particularly, plastid genetic engineering can be potentially used as a tool for cost-effective vaccine production. Antigenic proteins from different viruses and bacteria have been expressed in plastids. Initial immunological studies of chloroplast-derived vaccines have yielded promising results in animal models. However, because of certain limitations, these vaccines face many challenges on production and application level. Adaptations to the novel approaches are needed, which comprise codon usage and choice of proven expression cassettes for the optimal yield of expressed proteins, use of inducible systems, marker gene removal, selection of specific antigens with high immunogenicity and development of tissue culture systems for edible crops to prove the concept of low-cost edible vaccines. As various aspects of plant-based vaccines have been discussed in recent reviews, here we will focus on certain aspects of chloroplast transformation related to vaccine production against human diseases.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting ß-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Plant-produced trastuzumab inhibits the growth of HER2 positive cancer cells.

To study the agricultural production of biosimilar antibodies, trastuzumab (Herceptin) was expressed in Nicotiana benthamiana using the magnICON viral-based transient expression system. Immunoblot analyses of crude plant extracts revealed that trastuzumab accumulates within plants mostly in the fully assembled tetrameric form. Purification of trastuzumab from N. benthamiana was achieved using a scheme that combined ammonium sulfate precipitation with affinity chromatography. Following purification, the specificity of the plant-produced trastuzumab for the HER2 receptor was compared with Herceptin and confirmed by western immunoblot. Functional assays revealed that plant-produced trastuzumab and Herceptin have similar in vitro antiproliferative effects on breast cancer cells that overexpress HER2. Results confirm that plants may be developed as an alternative to traditional antibody expression systems for the production of therapeutic mAbs.

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration.

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 µg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15-20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 µg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).

Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

Cowpea mosaic virus-based systems for the expression of antigens and antibodies in plants.

This chapter describes the use of Cowpea mosaic virus-based vectors for the production of foreign proteins such as antigens and antibodies in plants. The systems include vectors based on both full-length and deleted versions of RNA-2. In both cases, the modified RNA-2 is replicated by coinoculation with RNA-1. The constructs based on full-length RNA-2 retain the ability to spread systemically throughout an inoculated plant and the infection can be passaged. The vector based on a deleted version of RNA-2 can stably incorporate larger inserts but lacks the ability to move systemically. However, it has the added advantage of biocontainment. In both cases, vector constructs modified to contain a foreign gene of interest can be delivered by agroinfiltration to obtain transient expression of the foreign protein. If required, the same constructs can also be used for stable nuclear transformation. Both types of vector have proved effective for the production in plants of a diverse range of proteins including antigens and antibodies.

Transient expression of antibodies in plants using syringe agroinfiltration.

The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with varying levels of complexity, including monoclonal antibodies.

Production of plantibodies in Nicotiana plants.

Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described.

Characterization of mechanical properties of transgenic tobacco roots expressing a recombinant monoclonal antibody against tooth decay.

In this article, we describe a new approach that allows the determination of the magnitude of force required to break single plant roots. Roots were taken from transgenic tobacco plants, expressing a secreted monoclonal antibody. They were divided into four key developmental stages. A novel micromanipulation technique was used to pull to breakage, single tobacco roots in buffer in order to determine their breaking force. A characteristic uniform step-wise increase in the force up to a peak force for breakage was observed. The mean breaking force and mean work done were 101mN and 97microJ per root respectively. However, there was a significant increase in breaking force from the youngest white roots to the oldest, dark red-brown roots. We speculate that this was due to increasing lignin deposition with root stage of development (shown by phloroglucinol staining). No significant differences between fresh root mass, original root length, or mean root diameter for any of the root categories were found, displaying their uniformity, which would be beneficial for bioprocessing. In addition, no significant difference in antibody yield from the different root categories was found. These data show that it is possible to characterise the force requirements for root breakage and should assist in the optimisation of recombinant protein extraction from these roots.