Stable Expression of Adalimumab in Nicotiana tabacum

  • Tzvi Zvirin
  • Lena Magrisso
  • Amit Yaari
  • Oded Shoseyov
Original Paper


Production of monoclonal antibodies and pharmaceutical proteins in transgenic plants has been the focus of many research efforts for close to 30 years. Use of plants as bioreactors reduces large-scale production costs and minimizes risk for human pathogens contamination. Stable nuclear transformation of the plant genome offers a clear advantage in agricultural protein production platforms, limited only by the number of hectares that can be cultivated. We report here, for the first time, successful and stable expression of adalimumab in transgenic Nicotiana tabacum plants. The plant-derived adalimumab proved fully active and was shown to rescue L929 cells from the in vitro lethal effect of rhTNFα just as effectively as commercially available CHO-derived adalimumab (Humira). These results indicate that agricultural biopharming is an efficient alternative to mammalian cell-based expression platforms for the large-scale production of recombinant antibodies.


Plant-derived antibody Plant-made pharmaceuticals Adalimumab Humira TNF-α Nicotiana tabacum 


Plants have long been proposed as an attractive platform for the production of recombinant proteins. The most advanced biopharming application utilizes the plants as a bioreactor to generate antibodies required for medical use or industrial processes [1, 2, 3, 4, 5, 6, 7, 8]. The key advantages of plant expression systems, as compared to traditional approaches, such as bacterial or mammalian cells expression systems, include their low costs, particularly when considering large-scale production, and the limited risk of contamination by human pathogens [9]. Moreover, bacteria can neither produce full-size antibodies nor perform most of the important mammalian posttranslational modifications [4, 10, 11]. Plants, like other eukaryotes, can perform posttranslational modifications, including N-glycosylation, which is substantially similar to that found in mammalian cells [12]. In addition, disulfide bonds, which are required for folding and assembly of complex multimeric proteins such as mAbs [13] can be formed in proteins expressed in plants. Thus, molecular pharming presents an unprecedented opportunity to manufacture affordable modern medicines and to make them available on a global scale [14].

Today, the majority of therapeutic antibodies are generated in mammalian cell lines or by their transient expression in Nicotiana benthamiana. Stable nuclear transformation of Nicotiana tabacum has been established as an excellent alternative production platform, that can yield high levels of complex proteins [10, 15, 16], while providing marked advantages in upscaling processes, requiring conventional agricultural practices [17, 18]. More importantly, unlike transient expression systems, transgenic plant lines involving stable transformation require neither costly fermenters, nor vacuum infiltration equipment, nor sterile conditions [18]. Moreover, N. tabacum is a relatively easy-to-manipulate organism, with very high leaf biomass, compared with N. benthamiana.

Molecular pharming designs must determine the subcellular compartment and posttranslational modifications most appropriate for expression of the target proteins. To date, most antibodies produced in plants accumulate to higher levels when expressed in the apoplast or endoplasmic reticulum (ER), as compared to the cytosol. In addition, protein assembly and posttranslational modifications in the ER are important for the synthesis of full-size antibodies and Fab fragments [1].

Management of many autoimmune and inflammatory disorders is achieved via antibody-based neutralization of pro-inflammatory cytokines. Neutralization of TNF-α is the most common therapy for such conditions and has been reported to elicit clinically relevant immune response suppression [19]. Adalimumab is the first fully human therapeutic monoclonal antibody approved for clinical use by the FDA. It is a TNF-α-neutralizing mAb, generated by recombinant DNA techniques, and its structure is indistinguishable from its human counterpart. TNF-α is a potent pro-inflammatory cytokine expressed as a surface type II polypeptide on activated macrophages and lymphocytes, among other cell types. It exerts its biological functions, including cytotoxic activity and polyclonal B cell activation, in a cell-to-cell contact-dependent manner [20, 21]. The soluble form of TNF-α mediates its biological activities through type I and type II TNF receptors [21, 22].

Adalimumab is a tetramer composed of two light kappa chains and two heavy immunoglobulin G1 (IgG1) chains, each of the latter containing one N-glycosylation site. The drug was first discovered by means of the “phage display” technique and was named D2E7. Currently, adalimumab is produced by cell culture using Chinese hamster ovary (CHO) cells [23].

This study aimed to achieve stable transgenic adalimumab expression in N. tabacum plants and to demonstrate that plant-derived mAb is fully functional, having similar bioactivity as its CHO-derived counterpart.


Accumulation of Adalimumab in Nicotiana tabacum cv. Samsun Plants

To identify the promoter/terminator set and signal peptide most efficient for stable transformation, adalimumab was first transiently expressed in N. tabacum cv. Samsun plants. Signal peptides directing protein accumulation to the vacuole (Vac) and apoplast (Cel1) [24] were tested. In addition, the CaMV 35s promoter with the NOS terminator (35s) and the RuBisCO promoter and terminator (Rb) were each tested with both signal peptides. To this end, 7-week-old N. tabacum cv. Samsun plants were syringe-infiltrated with Agrobacterium tumefaciens (A. tumefaciens) clones transformed with provectors containing the heavy chain (HC)- and light chain (LC)-coding sequences of adalimumab. Adalimumab assembly was examined 5 days post inoculation (d.p.i), by Western immunoblotting. Adalimumab expressed under either 35s with the Vac signal peptide, or with the Rb-Vac combination, yielded low mAb expression (Figs. 1, 2). Significantly higher levels of adalimumab accumulated when expressed by either the 35s-Cel1 or the Rb-Cel1 construct, with the RuBisCO promoter and terminator set providing superior expression as compared to the 35s promoter and NOS terminator set (Fig. 1).
Fig. 1

Transgenic Nicotiana tabacum cv. Samsun plant lines expressing adalimumab under different signal peptide and promoter/terminator sets. Bands correspond to the heavy (55 kDa) and light (25 kDa) chains. Lanes 1–4: 35S-Vac; lane 5: Protein ladder; lanes 6–9: 35S-Cel1; lane 10–11: Rb-Cell; lane 12: Positive control, 10 ng commercial adalimumab

Fig. 2

Transgenic Nicotiana tabacum cv. Samsun plant lines expressing adalimumab under RuBisCO promoter/terminator with the vacuolar signal peptide. Bands correspond to the heavy (55 kDa) and light (25 kDa) chains. Lane 1: WT; lane 2–3: positive control, 10–20 ng commercial adalimumab, respectively; lane 4: protein ladder; lanes 5–15: Rb-Vac plants

Stable Apoplastic Adalimumab Expression

Two vectors were constructed for the purpose of stable adalimumab expression in the apoplast: one for RuBisCO promoter-driven overexpression of the adalimumab heavy chain, tagged with the apoplast signal peptide, and the other for RuBisCO promoter-driven overexpression of the adalimumab light chain, tagged with the apoplast signal peptide. A. tumefaciens-mediated co-transformation of the two T vector into sterile N. tabacum cv. Samsun leaf tissue was performed.

Specific bands corresponding to the heavy and light adalimumab chains were detected at 55 and 25 kDa, respectively, indicating successful antibody expression in the apoplast. Figure 3 shows several of the best adalimumab-expressing N. tabacum plants (plant numbers 18, 19 and 20). The screening was performed with goat anti-human heavy and light chain IgG (Fig. 3b) and confirmed with goat anti-human kappa light chain IgG (Fig. 3a). Quantitative ELISA showed protein concentration ranging from 1 to 8.5 mg/kg fresh leaf (data not shown).
Fig. 3

Adalimumab stable expression in Nicotiana tabacum plants. Shown are the screening results of the best-expressing plant candidates. a Western blotting with goat anti-human (light chain) IgG, showing the mAb light chain at ~ 25 kDa. b Western blotting with goat anti-human (H + L chain) IgG, showing the mAb heavy and light chains at ~ 55 and ~ 25 kDa, respectively. Commercially available adalimumab served as a positive control

Purification and Characterization of the Plant-Expressed Adalimumab

Protein elution was carried out under acidic conditions. The mAb elution peaked between 75 and 78 min (Fig. 4a). Eluents were collected in three fractions, which were then analyzed on an SDS–PAGE (Fig. 4b). Most of the mAb (~ 90%) was detected in the middle fraction (Fig. 4b, lane 3). Protein concentration was measured using the NanoDrop device, and serial dilutions (100, 250, 500, and 750 ng) were assessed by SDS–PAGE and compared to 1000 ng human IgG (Fig. 4c). Both mAb light and heavy chains were detected (Fig. 4b, c). LC–MS/MS sequencing of extracted bands showed that 98% of heavy chain sequence and 99.53% of light chain sequence were covered, with 100% identity to the template sequence for both chains (data provided in supplementary material).
Fig. 4

Purification of plant-derived adalimumab. a Hitrap protein A FPLC chromatography. The mAb was eluted under acidic conditions; elution peak was observed between 75 and 78 min (x-axis). b SDS–PAGE analysis of eluted samples. Sample eluted before (lane 2), from (lane 3) and after (lane 4) the mAb elution peak. The second of the three elution peak fractions (lane umber 3) contained most of the mAb. Bands observed at 25 and 55 kDa corresponding to mAb light and heavy chains, respectively. Lane 1 contains plant extract before mAb purification. c SDS–PAGE analysis of various dilutions of plant-derived adalimumab lane 1–100 ng, lane 2–250 ng, lane 3–500 ng and lane 4–750 ng, run next to 1000 ng human IgG lane 5, which served as a positive control

Plant-Derived Adalimumab Target-Binding Activity

To assess the specific target-binding activity of the purified adalimumab, antigen-specific ELISA was used. Microtiter 96-well plates were coated with rhTNF-α. HRP-labeled anti-human IgG was used for detection of bound CHO-derived adalimumab and plant-derived adalimumab (PDA) mAbs. Two standalone experiments were performed (biological replicates); each was performed in duplicate. The average standard error between the replicates was 0.03 for CHO-derived adalimumab and 0.01 for plant-derived adalimumab. The absorbance values for PDA and its CHO-derived counterpart were very similar confirming the efficient assembly of PDA with specific antigen-binding activity (Fig. 5).
Fig. 5

In vitro rhTNFα binding activity of plant-derived (square) versus CHO-derived (diamond) adalimumab. Serial twofold dilutions of plant-derived adalimumab (PDA) were compared with serial twofold dilutions of CHO-derived adalimumab for their ability to bind to rhTNF-α. mAb were incubated on rhTNF-α precoated microtiter plates for 1 h. at 37 °C. Detection was made with HRP-labeled anti-human IgG and TMB substrate solution. The reaction was terminated with H2SO4 and absorbance was measured at 450 nm. Two biological replicates where performed for rhTNFα binding activity assay each in duplicate. Each bar represents the standard error of the mean. N = 4

Adalimumab Neutralization Bioactivity

Functional bioactivity of plant-derived adalimumab was assessed and compared to that of CHO-derived adalimumab (Humira), by testing their ability to inhibit soluble TNF-α activity. To this end, serial dilutions of both mAbs were incubated with the lethal concentration 50 (LC50) (100 ng/ml) dose of recombinant human TNF-α (Fig. 6). L929 cells were then treated with the mAb and hrTNF-α mixture. TNF-α-mediated cytotoxicity in L929 fibroblasts was effectively neutralized by both plant-derived and CHO-derived adalimumab in a dose-dependent manner and L929 fibroblast cells viability was measured using MTT solution assay, five replicates were performed for both plant and CHO-derived adalimumab (Fig. 6). While at low doses, similar bioactivity was observed for the two antibodies, at the highest concentration tested (2 µg/ml), the PDA conferred a slightly toxic effect. This is possibly caused by soluble plant proteins that eluted with PDA during the protein A-based purification process.
Fig. 6

Bioactivity of CHO-derived adalimumab versus plant-derived adalimumab. L929 cells were incubated with a premixed CHO-derived (diamonds) or plant-derived (squares) adalimumab-rhTNFa mixture. Cell viability was determined using the MTT assay. Each bar represents the standard error of the mean. N = 5


Ongoing efforts to advance the production of mAbs in plants have shown that plant-produced mAbs are as functionally active as their mammalian counterparts produced using traditional and well-established mammalian cell culture techniques [25, 26]. In this study, we, for the first time, demonstrate the production of fully active adalimumab, a human anti-TNF-α mAb, in transgenic N. tabacum plants. Plant-derived adalimumab exhibited in vitro activity (Fig. 5) and neutralization bioactivity similar to those measured for commercially available Humira (Fig. 6). At high PDA concentrations (2 μg/ml), a slight toxic effect was observed, likely due to plant-derived protein contaminants eluting out with the mAb. Therefore, for future production, further purification steps should be included, such as an additional protein A chromatography purification or ion-exchange chromatography step to remove impurities.

This study provides further evidence of the utility of plants for stable expression of human therapeutic antibodies. Plants can serve as cost-effective bioreactors, with enormous scalability potential. Furthermore, plant-derived adalimumab is free of mammalian pathogen contaminants. The N. tobacco-expressed adalimumab exhibited a sequence identical to that produced in CHO cell lines. In addition, PDA seemingly folded into its fully active form, as was shown by its functional activities and binding. The last was confirmed on the one hand by PDA binding to hrTNF-α through its Fab region, and on the other hand to protein A through its FC domain. The described heterozygous plants produced up to 10 mg adalimumab per kg fresh weight (data not shown). Further optimization of the purification process and homozygote plant production are expected to increase yields further.

Several expression systems are available for the expression of foreign proteins in plants. Agrobacterium-mediated plant tissue transformation is the most traditional technique, producing genetically modified plants, stably expressing the desired protein. Thus far, stable expression is the most promising approach, as it significantly cuts costs by requiring traditional agriculture for most of the production process. In contrast, transient expression systems require costly bioreactors for the production of the transformation vector, vacuum infiltration equipment, and sterile conditions [18], and fail to exploit the advantages embodied in plant expression systems. Yet, although production costs of plant-based mAbs are two orders of magnitude lower than mammalian cell-based mAbs [14, 27], there is still an urgent need to address two major limitations of plant-derived therapeutic mAbs. In mammalian proteins, the N-glycosylation structure is a central determinant of antibody half-life in the circulation and of its ability to be recognized by Fc receptors [12]. Recent works have shown that mAbs modified with plant N-glycans exhibit a half-live and antigen-binding properties similar to those decorated with mammalian N-glycans [6, 12, 28]. Yet, immunogenic effects ascribed to the plant-specific core N-glycans β1,2 xylose and α1,3/4 fucose have been noted in laboratory mammals and in humans [29, 30]. Therefore, plants N-glycans may render plant-derived antibodies immunogenic in humans when delivered parenterally [12], especially with repeated immunotherapy [31]. Thus, future research on the plant-produced adalimumab will require implementation of strategies to removing β1,2 xylose and α1,3/4 fucose immunogenic residues without impacting the bi-antennary complex glycan structure.

In addition, in order to comply with the demand for plant-derived mAb, hundreds of thousands of tons of green biomass will have to be processed and purified. This will present an enormous bottleneck and will demand effective downstream process strategies.

In this work, we established that a therapeutic mAb (PDA) can be produced in a plant-based expression system. PDA showed identical primary structure and similar in vitro bioactivities to those of the clinically approved CHO-derived mAb. We therefore provided further evidence that agricultural biopharming is a practical alternative to mammalian cell systems for the production of therapeutic mAbs.


In the future, the production of biological drugs will shift rapidly toward plant-based expression system, where plant stable nuclear transformation play a key role in making modern medicines affordable for all.

Materials and Methods

Construct Cloning

Coding gene sequences of human monoclonal D2E7 adalimumab heavy chain (AH) and light chain (AL), were synthesized with desired flanking regions, and optimized for expression in tobacco plants by GenScript Biotech (Piscataway, NJ, USA).

AH and AL were cloned into separate expression cassettes in the pUC18 plasmid, that were previously developed in our laboratory [32]. The pUC18 plasmid included a Chrysanthemum rbcS1 promoter, Chrysanthemum rbcS1 terminator, and either the vacuolar-targeting signal peptide (SP) (Rb-Vac) or the apoplast-targeting SP (RbCel1) (Fig. 7a). The complete expression cassettes (promoter, SP, coding region, and terminator) were cloned into the multiple cloning site of the pBINPLUS plant transformation vector, using the HindIII restriction enzyme (Fig. 7b).
Fig. 7

Adalimumab construct for expression in plants. a A modular pUC18 plasmid designed to generate four variants each of the heavy and light adalimumab chains: 1. CaMV35s promoter, Vac SP, heavy chain, NOS terminator; 2. CaMV35s promoter, Vac SP, light chain, NOS terminator; 3. CaMV35s promoter, Cel1 SP, heavy chain, NOS terminator; 4. CaMV35s promoter, Cel1 SP, light chain, NOS terminator; 5. RuBisCO promoter, Vac SP, heavy chain, RuBisCO terminator; 6. RuBisCO promoter, Vac SP, light chain, RuBisCO terminator; 7. RuBisCO promoter, Cel1 SP, Heavy Chain, RuBisCO terminator; 8. RuBisCO promoter, Cel1 SP, Light Chain, RuBisCO terminator; b The pBINPLUS binary vector. Each pUC18 cassette was cloned into pBINPLUS vector using the HindIII restriction enzyme for RuBisCO promoter/terminator cassettes and using EcoRI/SacI for CaMV35s promoter/NOS terminator cassettes

AH and AL were also cloned into a plasmid containing the CaMV 35S promoter, Agrobacterium nopaline synthase (NOS) terminator, and either the vacuolar-targeting SP (35sVac) or the apoplast-targeting SP (35sCel1) (Fig. 7a). The complete expression cassettes were cloned into the multiple cloning site of pBINPLUS vectors, using the EcoRI and SacI restriction enzymes (Fig. 7b).

Signal peptide sequences:
  1. 1.

    Vacuole SP sequence of barley thiol protease aleurain precursor (NCBI accession P05167 GI:113603): MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA.

  2. 2.

    Apoplast signal of Arabidopsis thaliana endo-1,4-β-glucanase (Cel-1, NCBI accession CAA67156.1 GI:2440033): RKSLIFPVILLAVLLFSPPIYSAGHDYRDALRKSSMA.


Transgenic Tobacco Plant Production

Tobacco plants were grown under sterile conditions for approximately 4–5 weeks. Agrobacterium tumefaciens bacteria were grown in 25 ml LB medium with 50 mg/ml kanamycin, for 48 h, on a shaker, at 28 °C, to a stationary stage. Starters were then centrifuged for 10 min, 4000×g, at room temperature. The supernatant was aspirated and the bacterial pellet was diluted with sterile liquid MS medium (4.4 g/L Murashige and Skoog medium including vitamins (Duchefa (cat#M0222.0050)) and 30 g/L sucrose (J.T.Baker (cat#4072-05), pH = 5.8) and grown to a final O.D600 of 0.5. Bacteria (10 ml) were then applied to tobacco leaf sections and incubated for 5 min. Inoculated explants were then transferred to Petri dishes containing solid MS medium (liquid MS medium with 0.7% plant agar (Duchefa (cat# P1001.1000)), supplemented with 0.8 ml/L indole-3-acetic acid (IAA) and 2 ml/L kinetin. Plates were incubated at 28 °C, in the dark, for 48 h, AND then explants were transferred to new Petri dishes containing selective MS medium (0.8 ml/L IAA, 2 ml/L kinetin, 400 mg/L carbenicillin and 100 mg/L kanamycin). Petri dishes were incubated at 25 °C, in the light, for 3 weeks; medium was exchanged every 10 days. During this period, shoots formed. The shoots were detached and transferred to rooting medium (MS medium with 100 mg/L kanamycin and 400 mg/L carbenicillin), under the same light conditions. Shoots that produced roots were transferred to soil pots in the greenhouse, equipped with an upper irrigation system, and grown to mature transgenic plants. Transgenic plants were grown in pots in the greenhouse in the controlled environment. Temperature was 25 °C under a natural photoperiod. The plants were watered in a frequency of three daily 5-min irrigation events. Liquid fertilizer (Shefer 7 – 3 – 7 + 3, ICL Fertilizers, Israel) was injected into the irrigation water.

SDS–PAGE and Western Blot Analysis

SDS–PAGE analysis was performed on 12.5% SDS gels, using a “mini-protein gel system” (Hercules, CA, USA). Western blot analysis was performed as previously described [33]. Then, the Membrane was blocked with 4% skim milk for 0.5 h, at room temperature (RT). The membrane was incubated with a primary antibody (alkaline phosphatase-conjugated goat anti-human IgG heavy and light chain antibody or goat anti-human kappa light chain antibody) overnight at RT, washed three times with tris-buffered saline (TBS) Tween 20 (TBST), and then incubated with an alkaline phosphatase-conjugated secondary antibody, for 2 h, followed by three washes with TBST. Finally, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) substrates (Sigma) diluted in substrate buffer (100 mM Tris, 100 mM sodium chloride, and 5 mM MgCl2, pH 9.5. adjusted with HCl), were added.

Plant tissue Preparation for ELISA Assay

For sample preparation, pre-weighed Eppendorf tubes were prepared, and 100 μl grinding buffer (100 mM Tris–HCl pH 8, 25 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA, and 1 mM potassium metabisulfite (PMBS)) were added to each Eppendorf. Six leaf disks from each plant were sampled by clipping with the Eppendorf lid directly into pre-weighed Eppendorf and immediately placed on ice. Samples were taken from lower, middle, and upper plant sections. The samples were then weighed and ground for 30 s with a plastic pistol on mortar homogenizer, at 500 RPM. Soluble fraction was extracted by centrifugation for 15 min, at 12,000 × g, in 4 °C. Samples were then 500-fold-diluted in TBS.

Quantitative ELISA

Untreated 96-well plates were coated with 100 ng/ml rhTNF-α solution (Merck) and then thoroughly washed. A calibration curve was produced by twofold serial dilutions of commercially available adalimumab (AbbVie Inc. North Chicago, Illinois, USA) (0.097–100 ng/ml). PDA twofold serial dilutions were produced, to compare PDA activity to that of commercially available counterpart. Unknown samples were prepared as described previously, in plant tissue preparation for ELISA assay paragraph. Both calibration curve and unknown samples were prepared in duplicates. Samples were incubated in the wells for 1 h, at 37 °C. The plate was washed four times with TBST wash solution and then incubated with 1:50000 HRP-conjugated goat anti-human IgG (Merck Millipore), for 1 h, at 37 °C. The wells were then washed four times with TBST and incubated for 20 min with 100 µl TMB substrate solution (Thermo Scientific, Rockford, USA). The reaction was terminated with 100 µl H2SO4 0.5 N. Absorbance was measured in a microplate reader (BioTek Synergy 2 plate reader), at 450 nm. For each calibration curve sample, duplicate average O.D read was obtained, and concentration was calculated using the four-parameter logistic (4PL) regression model. The equation for this model is:
$$x = c\left( {\frac{a - d}{y - d} - 1} \right)^{{\frac{1}{b}}} ,$$
where x is the independent variable (O.D.) and y is the dependent variable (commercially available adalimumab concentration). a is a minimum asymptote (response value for 0 concentration) and d is maximum asymptote (response value for infinite standard concentration). c is the point of inflection (i.e., the point on the S-shaped curve halfway between a and d), b is Hill’s slope of the curve. a and d will always define the upper and lower asymptotes (horizontals) of the curve.

The average concentration in each set of PDA samples was calculated from the calibration curve. Then, the PDA concentration in the sample was multiplied by the dilution factor. Ab yield in the transgenic plant was calculated by dividing PDA concentration in the sample by fresh tissue weight. The obtained result represents mg PDA per kg fresh leaf tissue.

Plant Tissue Preparation for mAb Purification

Leaves (1 kg) from PDA-expressing plants were screw-pressed with homogenizing buffer (100 mM Tris–HCl pH 8, 25 mM NaCl, 1 mM PMSF, 10 mM EDTA), (w/v 1:1). The soluble mAb fraction was obtained by centrifugation for 30 min, 10,000 × g at 4 °C and then incubated with 3% polyvinylpolypyrrolidone (PVPP), for 4 h, at 4 °C, with shaking, to remove polyphenols. Samples were then centrifuged three times at 10,000 × g for 30 min, at 4 °C, and then filtrated through 0.8- and 0.45-µm filters.

Antibody Purification

Soluble fraction obtained from plants expressing adalimumab was filtered through a 0.2-µm filter and passed through a HiTrap Protein A high-performance 5-ml column was prepacked with Protein A Sepharose, on an ÄKTA design chromatography system. Samples were diluted in 20 mM sodium phosphate buffer pH7.4, which was also used to wash the column. Elution was performed under acidic conditions, with 100 mM citric acid, pH 3. Fractions were collected and analyzed by SDS–PAGE. Protein concentration was measured using ELISA assay.

Protein Sequence Analysis

Samples were digested with trypsin and analyzed by liquid chromatography coupled to a LTQ mass spectrometer (Hybrid-2D-Linear Quadrupole Ion Trap—mass spectrometer, Thermo Scientific or Thermo Electron, Bremen, Germany) and identified using the Discoverer software, using the adalimumab D2E7 heavy and light chain sequences as reference (performed in The Smoler Proteomics Center, Department of Biology, Technion, Israel). Peptide contig was produced with BioEdit software CAP feature, peptides were assembled to a protein, and CLUSTALW protein alignment was performed in comparison with the adalimumab sequence (D2E7).

Neutralization Test

The adalimumab activity assay was performed by Harlan Biotech Ltd., Israel. Briefly, activity was tested by antibody-driven neutralization of TNFα-mediated cytotoxicity in L929 fibroblast cells. Two 96-well tissue culture plates were filled with 100 μl of a L929 cell suspension, prepared at a density of 3.5 × 105 cells/ml, and incubated overnight at 37 °C, 5% CO2, in a humidified incubator. After 12-h incubation, rhTNF-α and actinomycin D were added to a final concentration of 1 ng/ml rhTNF-α [LC50 (lethal concentration 50%) as determined in preliminary experiments (data not shown)] and 1 µg/ml actinomycin D, and incubated for 2 h at 37 °C, 5% CO2, in a humidified incubator. The first plate (experiment plate) was then treated with PDA (1.95–1000 ng/ml). The second plate (control plate) was treated with Humira (1.95–1000 ng/ml). MTT solution (tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well, to a final concentration of 0.5 mg/ml, and incubated for approximately 4 h, at 37 °C. MTT solution was then aspirated, and 100 µl isopropanol was added to each well for no less than 30 min. Absorbance was measured using a microplate spectrophotometer (Multiscan® FC; Thermo Scientific), with 570–650 nm wavelength filters.



We thank Dr. Meirav Blanka for her help in the development of the quantitative ELISA and Dr. Yehudit Posen for critically reviewing this manuscript. This study was supported by the Minerva Center for bio-hybrid complex system.

Supplementary material

12033_2018_75_MOESM1_ESM.docx (181 kb)
Supplementary material 1 (DOCX 181 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Tzvi Zvirin
    • 1
  • Lena Magrisso
    • 1
  • Amit Yaari
    • 1
  • Oded Shoseyov
    • 1
  1. 1.The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and EnvironmentThe Hebrew University of JerusalemRehovotIsrael

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