Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering.

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three-dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10-times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high-throughput screening to 150-mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.

From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants.

The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.

Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins.

Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins.

Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the same recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins.

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

BACKGROUND: Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10). RESULTS: We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5'-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. CONCLUSIONS: We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.

Locating Functionalized Gold Nanoparticles Using Electrical Impedance Tomography.

OBJECTIVE: An imaging device to locate functionalised nanoparticles, whereby therapeutic agents are transported from the site of administration specifically to diseased tissues, remains a challenge for pharmaceutical research. Here, we show a new method based on electrical impedance tomography (EIT) to provide images of the location of gold nanoparticles (GNPs) and the excitation of GNPs with radio frequencies (RF) to change impedance permitting an estimation of their location in cell models Methods: We have created an imaging system using quantum cluster GNPs as contrast agent, activated with RF fields to heat the functionalized GNPs, which causes a change in impedance in the surrounding region. This change is then identified with EIT. RESULTS: Images of impedance changes of around 80 ± 4% are obtained for a sample of citrate stabilized GNPs in a solution of phosphate-buffered saline. A second quantification was carried out using colorectal cancer cells incubated with culture media, and the internalization of GNPs into the colorectal cancer cells was undertaken to compare them with the EIT images. When the cells were incubated with functionalised GNPs, the change was more apparent, approximately 40 ± 2%. This change was reflected in the EIT image as the cell area was more clearly identifiable from the rest of the area. SIGNIFICANCE: EIT can be used as a new method to locate functionalized GNPs in human cells and help in the development of GNP-based drugs in humans to improve their efficacy in the future.

Cost-effective production of a vaginal protein microbicide to prevent HIV transmission.

A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Protein-based microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.

Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous beta1,4-galactosylated N-glycan profile.

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAb-associated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous beta1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human beta1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully beta1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.

Age-related changes in the response of rat adipocytes to insulin: evidence for a critical role for inositol phosphoglycans and cAMP.

Adipose tissue plays a pivotal role in ageing and longevity; many studies, both human and animal, have focussed on the effects of food limitation. Here we present a new model based on striking differences between two 'normal' inbred strains of albino Wistar rats the Charles River (CR) and Harlan Olac (HO) that have marked differences in age-related accumulation of fat and insulin-stimulated rates of glucose incorporation into lipid in the epididymal fat pads (EFP). The incorporation [U-(14)C]glucose into lipid by adipocytes showed that the CR group had a twofold higher basal rate of lipogenesis and a greater response to insulin in vitro, exceptionally, adipocytes from CR group maintained the high response to insulin to late adulthood while retaining the lower EFP weight/100 g body weight. Inositol phosphoglycan A-type (IPG-A), a putative insulin second messenger, was 3.5-fold higher and cAMP significantly lower per EFP in the CR versus HO groups. Plasma insulin levels were similar and plasma leptin higher in CR versus HO groups. The anomaly of a higher rate of lipogenesis and response to insulin and lower EFP weight in the CR group is interpreted as the resultant effect of a faster turnover of lipid and stimulating effect of leptin in raising fatty acid oxidation by muscle, potentially key to the lower accumulation of visceral fat. The metabolic profile of the CR strain provides a template that could be central to therapies that may lead to the lowering of both adipose and non-adipocyte lipid accumulation in humans in ageing.

Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody.

The use of vaginal microbicides containing human immunodeficiency virus (HIV)-neutralizing antibodies (nAbs) is a promising strategy to prevent HIV-1 infection. Although antibodies are predominantly manufactured using mammalian cells, elastin-like peptide (ELP) fusion technology improves the stability of recombinant, plant-produced proteins and facilitates their purification, making plants an alternative platform for antibody production. We generated transgenic tobacco plants accumulating four different formats of the anti-HIV-1 antibody 2G12 in the endoplasmic reticulum (ER), i.e. with ELP on either the light or heavy chain, on both, or on neither. Detailed analysis of affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters of all formats were identical to 2G12 lacking ELP produced in Chinese hamster ovary (CHO) cells. Importantly, protein purification from seeds by inverse transition cycling (ITC) did not affect the binding kinetics. Analysis of heavy chain N-glycans from leaf-derived antibodies showed that retrieval to the ER was efficient for all formats. In seeds, however, N-glycans on the naked antibody were extensively trimmed compared with those on the ELP fusion formats, and were localized to a different subcellular compartment. The in vitro HIV-neutralization properties of the tobacco-derived 2G12 were equivalent to or better than those of the CHO counterpart.

Production and localization of recombinant pharmaceuticals in transgenic seeds.

Among the many plant-based production systems that have been developed for pharmaceutical proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume, and recombinant proteins are very stable in dry seeds allowing long-term storage and facilitating distribution before processing.To take full advantage of the natural ability of endosperm cells to store large amounts of protein in a protected subcellular environment, it is useful to target recombinant proteins to appropriate storage organelles. In this chapter, we describe the distinct types of protein storage organelles in the cereal endosperm and a protocol for the detection of recombinant proteins in these organelles by immunofluorescence and immunogold labelling.The use of food and feed crops for the production of pharmaceutical proteins such as edible vaccines implies the need for strict separation of the transgenic seeds from the food and feed chain. For improved traceability visual markers may be co-expressed with the gene of interest in engineered seeds. DsRed is one example for a fluorescent protein that can be detected with high sensitivity using low tech equipment. We therefore describe the generation of transgenic maize plants expressing DsRed in a constitutive manner, and we point out the advantages of using this marker during the process of transformation and selection of plant tissue and later during breeding of transgenic lines into elite germplasm.

The influence of elastic strain on the early stages of decomposition in Cu-1.7at% Fe.

The initial stage of decomposition of homogenized Cu-1.7at% Fe at 722K was investigated by means of field ion microscopy (FIM), atom probe tomography (APT) and computer-assisted field ion image tomography (cFIIT). The agglomeration of atoms depending on time could be investigated and the growth of precipitates with a diameter of few nanometers was observed during ongoing nucleation. For the cFIIT measurements, an improved reconstruction algorithm was developed. Employing cFIIT in combination with FIM images, alignments of precipitates mainly in 100 directions were found. Besides, a general experimental concept to evaluate strain-related effects on the position of the proximate precipitate will be introduced. These analyses of the APT and cFIIT data show tendencies of a preferred 100 directed configuration as well. This effect can be associated with the elastic anisotropy of the Cu matrix (f.c.c.) to accommodate the volume misfit of precipitates. In accordance with previous model calculations, a preferred nucleation in 100 directions with respect to existing clusters can be concluded. For such cluster arrangements strain energy reduction is largest for precipitates adapting their sizes. Thus, additional stabilization against coarsening can be inferred for 100 alignments which result in the observed effects.

Increased inositol phosphoglycan P-type in the second trimester in pregnant women with type 2 and gestational diabetes mellitus.

A progressive insulin resistant state develops throughout human pregnancy. Inositol phosphoglycan P-type (P-IPG), a second messenger of insulin, was reported to negatively correlate with the degree of insulin resistance in non-pregnant diabetic subjects. Urinary levels of P-IPG were assessed in insulin resistant states during pregnancy such as gestational diabetes mellitus (GDM, n=44) and type 2 diabetes mellitus (type 2 DM, n=25) and in 69 normal pregnant women. Urinary levels of P-IPG were higher in GDM than controls with a positive trend of release throughout normal pregnancy (P<0.01). P-IPG excretion was higher in diabetic (GDM and type 2 DM) than in healthy women in the second trimester (P<0.05). A higher P-IPG urinary excretion occurs during the second trimester in pregnant women with clinically evident insulin resistance with a positive association with poor glycemic control.

Ready-to-Use Stocks of Agrobacterium tumefaciens Can Simplify Process Development for the Production of Recombinant Proteins by Transient Expression in Plants.

Large-scale automated transient protein expression in plants requires the synchronization of cultivation and bacterial fermentation, especially if more than one bacterial strain. Therefore, a ready-to-use approach that decouples bacterial fermentation and infiltration is developed. It is found that bacterial cultures can easily be reconstituted in infiltration medium at a user-defined time, optical density, and quantity. This allows the process flow to be staggered, avoiding bottlenecks in process capacity and labor. Using the red fluorescent protein, DsRed, as a model product, the ready-to-use preparations achieved the same yields in infiltrated plant biomass as Agrobacterium tumefaciens derived from regular fermentations. It is possible to store the ready-to-use stocks at -20 °C and -80 °C for more than two months without loss of activity. Using a consolidated cost model for the current fermentation process, it is found that the ready-to-use strategy can reduce operational costs by 20-95% and investment costs by up to 75%, which would otherwise offset the economic advantages of plants over mammalian expression systems during upstream production. Furthermore, the staggered cultivation of plants and bacteria reduces the likelihood of batch failure and thus increases the robustness and flexibility of transient expression for the production of recombinant proteins in plants.

Exploring strengths and limits of urinary D-chiro inositol phosphoglycans (IPG-P) as a screening test for preeclampsia: A systematic review and meta-analysis.

Preeclampsia is a severe complication of human pregnancy as it leads to significant maternal and perinatal mortality and morbidity worldwide. A prompt recognition of women that develop this syndrome can improve clinical management, increase surveillance and, finally, improve outcomes. Different methods (based on history, ultrasound, serum and urinary biomarkers) were proposed a screening tests for this disease but their performance showed limited results. Urinary inositol phosphoglycans P-type (IPG-P) were shown to identify in advance most of the women who will develop preeclampsia in case-control and longitudinal studies, so we undertook a systematic review and meta-analysis of published studies. Seven studies met the entry criteria so were evaluated. All case-control studies showed excellent statistical performances in a quality statistical assessment. The meta-analysis considered three longitudinal, prospective studies that showed high sensitivity and specificity with ranges of 0.82- 0.99 and 0.90-1.00, respectively. Univariate measures of accuracy revealed a positive and negative likelihood ratio respectively of 3.61 (95% CI 1.56-5.67) and -2.35 (95% CI -3.79 to -0.91). By univariate approach, we found a pooled logarithm of diagnostic odds ratio of 6.15 (95% CI 2.64-9.67). A limitation of this analysis is that, although conducted in different settings (UK, Italy, France, South Africa, and Mauritius) and different clinical groups, they were based on a single academic group. According to our findings, IPG-P test showed very encouraging results as a rapid noninvasive screening test for preeclampsia. Further studies are needed to verify and to validate the reported findings.

Russell-Like Bodies in Plant Seeds Share Common Features With Prolamin Bodies and Occur Upon Recombinant Protein Production.

Although many recombinant proteins have been produced in seeds at high yields without adverse effects on the plant, endoplasmic reticulum (ER) stress and aberrant localization of endogenous or recombinant proteins have also been reported. The production of murine interleukin-10 (mIL-10) in Arabidopsis thaliana seeds resulted in the de novo formation of ER-derived structures containing a large fraction of the recombinant protein in an insoluble form. These bodies containing mIL-10 were morphologically similar to Russell bodies found in mammalian cells. We confirmed that the compartment containing mIL-10 was enclosed by ER membranes, and 3D electron microscopy revealed that these structures have a spheroidal shape. Another feature shared with Russell bodies is the continued viability of the cells that generate these organelles. To investigate similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds, we crossed plants containing ectopic ER-derived prolamin protein bodies with a line accumulating mIL-10 in Russell-like bodies. This resulted in seeds containing only one population of protein bodies in which mIL-10 inclusions formed a central core surrounded by the prolamin-containing matrix, suggesting that both types of protein aggregates are together removed from the secretory pathway by a common mechanism. We propose that, like mammalian cells, plant cells are able to form Russell-like bodies as a self-protection mechanism, when they are overloaded with a partially transport-incompetent protein, and we discuss the resulting challenges for recombinant protein production.

A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits.

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.

Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants.

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N-Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N-glycans and that the ELP fusions had no significant effect on N-glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.

Recombinant antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc N-glycans.

Antibody 2G12 is one of a small number of human immunoglobulin G (IgG) monoclonal antibodies exhibiting potent and broad human immunodeficiency virus-1 (HIV-1)-neutralizing activity in vitro, and the ability to prevent HIV-1 infection in animal models. It could be used to treat or prevent HIV-1 infection in humans, although to be effective it would need to be produced on a very large scale. We have therefore expressed this antibody in maize, which could facilitate inexpensive, large-scale production. The antibody was expressed in the endosperm, together with the fluorescent marker protein Discosoma red fluorescent protein (DsRed), which helps to identify antibody-expressing lines and trace transgenic offspring when bred into elite maize germplasm. To achieve accumulation in storage organelles derived from the endomembrane system, a KDEL signal was added to both antibody chains. Immunofluorescence and electron microscopy confirmed the accumulation of the antibody in zein bodies that bud from the endoplasmic reticulum. In agreement with this localization, N-glycans attached to the heavy chain were mostly devoid of Golgi-specific modifications, such as fucose and xylose. Surprisingly, most of the glycans were trimmed extensively, indicating that a significant endoglycanase activity was present in maize endosperm. The specific antigen-binding function of the purified antibody was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the maize-produced antibody were equivalent to or better than those of its Chinese hamster ovary cell-derived counterpart.

Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures.

We report the production of an important human therapeutic antibody in plant cell suspension cultures and the functional analysis of that antibody, including a comparison with the same antibody produced in CHO cells. We established transgenic tobacco BY2 suspension cell cultures expressing the human monoclonal antibody 2F5, which shows broadly neutralizing activity against HIV-1. The antibody was directed to the endoplasmic reticulum of the plant cells and was isolated by cell disruption, followed by protein A chromatography. The plant-derived antibody was shown to be largely intact by SDS-PAGE and immunoblot. Antigen binding activity was investigated by electrophoretic mobility shift assay and quantitatively determined by ELISA and Biacore biosensor technology. Ligand binding properties were analyzed using the ectodomain of human Fc gammaRI for kinetic analysis. The plant-derived antibody showed similar kinetic properties and 89% of the binding capacity of its CHO-derived counterpart, but was only 33% as efficient in HIV-1 neutralization assays. Our results show that plant suspension cultures can be used to produce human antibodies efficiently and that the analysis methods used in this study, including biosensor technology, provide useful functional data about antibody performance. This highlights important issues raised by the use of plant systems to produce human biologics.