Transgenic Res DOI 10.1007/s11248-015-9890-8

ORIGINAL PAPER

Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody Abel Herna´ndez-Vela´zquez . Alina Lo´pez-Quesada . Yanaysi Ceballo-Ca´mara . Gleysin Cabrera-Herrera . Kenia Tiel-Gonza´lez . Liliana Mirabal-Ortega . Marlene Pe´rez-Martı´nez . Rosabel Pe´rez-Castillo . Yamilka Rosabal-Aya´n . Osmani Ramos-Gonza´lez . Gil Enrı´quez-Obrego´n . Ann Depicker . Merardo Pujol-Ferrer

Received: 19 January 2015 / Accepted: 18 June 2015 Ó Springer International Publishing Switzerland 2015

Abstract The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to Electronic supplementary material The online version of this article (doi:10.1007/s11248-015-9890-8) contains supplementary material, which is available to authorized users. A. Herna´ndez-Vela´zquez (&)  A. Lo´pez-Quesada  Y. Ceballo-Ca´mara  K. Tiel-Gonza´lez  L. Mirabal-Ortega  M. Pe´rez-Martı´nez  R. Pe´rez-Castillo  Y. Rosabal-Aya´n  O. Ramos-Gonza´lez  G. Enrı´quez-Obrego´n  M. Pujol-Ferrer Plant Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), PO Box 6162, 10600 Havana, Havana, Cuba e-mail: [email protected] G. Cabrera-Herrera Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba A. Depicker Department of Plant Systems Biology, VIB, Plant-made Antibodies and Immunogens, Ghent, Belgium

produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5 mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented. Keywords Protein expression  Tobacco seeds  Phaseolin promoter  Monoclonal antibody  Hepatitis B  N-glycan

Introduction From the moment they were first developed (Ko¨hler and Milstein 1975), monoclonal antibodies (mAb) became one of the most versatile tools available for biological research in both industry and academia.

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Such has been the success of this technology, that the installed production capacities have never caught up with market demands for mAb-based biologicals, driving forward the search for more efficient and costeffective antibody production systems. Despite their disadvantages in terms of complexity and cost, mammalian cells are most commonly used to produce mAbs due to the exacting requirements these molecules impose on the production host, owing to their complex-type N-glycosyl moieties and the fact that they consist of four disulfide-linked polypeptides. One of the most promising solutions to this problem is the use of plants as heterologous hosts. Plants offer a number of significant advantages over mammalian cells, including significant cost savings and the fact that product contamination by adventitious animal pathogens is no longer a concern (Schillberg et al. 2003; Huang and Mcdonald 2012). The first report of mAb production in plants demonstrated that the synthesis and assembly of immunoglobulins could take place efficiently in tobacco leaves (Hiatt et al. 1989), and several other plant systems for expressing mAbs have been developed since for a variety of applications (Rodriguez et al. 2013; Zhang et al. 2014; He et al. 2014). Plants can be engineered to synthesize heterologous proteins in a specific organ, and seeds are the organ of choice whenever it is desirable to accumulate relatively large amounts of a heterologous protein. Seeds are designed for the synthesis and storage of proteins, which may account for 8–40 % of their weight. This endows them with a considerable advantage over green tissues and tubers, whose protein content is lower (De Jaeger et al. 2002; Shukla and Thommes 2010). Dehydration, another feature of seeds stemming from their role as a storage compartment, also reduces the levels of non-enzymatic hydrolysis and degradation of proteins (Fischer et al. 2009). It has been shown that antibodies, vaccine antigens and other heterologous proteins can accumulate to high levels in seeds, where they remain stable and functional for several years even upon storage at room temperature (Sto¨ger et al. 2002; Rhonda et al. 2011). Many different antibodies have been produced in seeds from a variety of species, including cereals and leguminosae. One early report, for instance, demonstrated the accumulation of a single-chain variable fragment (scFv) antibody in rice and wheat seeds at levels of 32 and 1.5 lg scFv/g, respectively (Sto¨ger et al. 2000),

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and another described the accumulation of a different scFv at 9 lg/g of pea seeds (Perrin et al. 2000). The anti-Human Immunodeficiency Virus mAb 2G12 was also produced in maize seed at levels of 75 lg of antibody per gram (Ramessar et al. 2008). Although several important biologicals with clinical applications are currently expressed in transgenic tobacco leaves (Gorantala et al. 2014; Shenoy et al. 2014), tobacco seed is not usually considered for use as a bioreactor despite documented yields of up to 1.1 tons of seed per ha (Giannelos et al. 2002). Tobacco seed has previously been studied as a production platform for biofuels (Andrianov et al. 2010) and as a protein source for livestock (Taheripour et al. 2010; Rossi et al. 2013). Since tobacco is not a food crop, regulatory issues concerning the production of recombinant proteins in this plant are far less contentious than for other seed crops such as corn, rice or soybean, and large-scale GMP-compatible production technologies for tobacco-derived biologicals are available from companies such as Fraunhofer IME, Planet Biotechnology and SAFC (Stoger et al. 2014). As is the case with many other plant species, the biochemical environment provided by tobacco seeds favors the accumulation of large amounts of protein under conditions of high stability with no substantial loss of activity, enabling manufacturers to decouple the processes of biomass production and protein purification. In 2003 a mouse IgG1 mAb (PHB-01) specific for the surface antigen of Hepatitis B Virus (HBsAg) was expressed in transgenic tobacco (N. tabacum cv. Havana 92) (Ramı´rez et al. 2003). These transgenic plants are currently used for the industrial production of PHB-01, which is employed for the immunoaffinity purification of HBsAg as part of the manufacturing process of a recombinant Hepatitis B vaccine (HeberBiovacÒ). Although the PHB-01 plantibody was the first plant-made reagent licensed for use in the production of a human vaccine, thus marking a milestone in the development of plant-made pharmaceuticals (PMP) (Pujol et al. 2005; Kaiser 2008), the expression level of this molecule in its plant host has never surpassed 60 mg PHB-01/kg of leaves (Ramı´rez et al. 2003), requiring a large scale, extensively optimized purification process (Valde´s et al. 2003a) and a well-engineered HBsAg immunoaffinity procedure (Valde´s et al. 2003b) for its use in a cost-effective manner. Additionally, analysis of the N-glycosylation

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patterns of tobacco leaf-derived PHB-01 has shown that despite the presence of a C-terminal KDEL endoplasmic reticulum (ER) retention signal, the antibody is modified both at the ER and, to a lesser extent, the Golgi apparatus (GA) (Triguero et al. 2005), although its performance on the immunoaffinity purification of HBsAg is undistinguishable from that of mouse-derived mAb (Ramı´rez et al. 2003). In the present work, therefore, we have set out to evaluate the accumulation of PHB-01 in tobacco seeds as an alternative production strategy for this molecule. PHB-01, including the KDEL tag, was expressed in tobacco (N. tabacum cv. BHmN) under seed-specific regulatory sequences. The integrity and activity of the resulting seed-derived mAb were studied, and N-glycan analysis was used to examine and compare the performance of KDEL as an ER anchor for PHB-01 in tobacco seed and leaves. Finally, an analysis of the productive potential and characteristics of both tobacco organs from the viewpoint of the PHB-01 manufacturing process was performed.

Materials and methods Plasmid constructs Regulatory sequences for storage proteins from Phaseolus vulgaris, provided by Prof. Ann Depicker, were used to express an anti-HBsAg mAb. Briefly, the heavy and light chain coding sequences from plantibody PHB-01, including a KDEL ER retention signal, were obtained from plasmid PDEHCLC (Ramı´rez et al. 2003) and cloned into plasmid pPhasG4 (De Jaeger et al. 2002) fully digested with EcoR I and partially digested with Nco I, yielding plasmids phasHB-HC and phasHB-LC. DNA fragments 3.0 and 2.3 kb long containing the phaseolin promoter together with the HC and LC coding sequences respectively from phasHB-HC and phasHB-LC were then cloned Xho I–Xba I into the Sal I and Xba I sites of patag50 -arc (De Jaeger et al. 2002) to obtain the binary vectors patagHB-HC and patagHB-LC (Online Resource 1). These contain the phaseolin promoter, the arcelin 50 (arc-5I) 50 -UTR, the 2S2 signal peptide, the HC or LC coding sequence inframe with the signal peptide and fused to an KDELcoding tag, and arcelin 30 flanking sequences.

Plant transformation, propagation and screening Vectors patagHB-LC and patagHB-HC were cointroduced into tobacco (Nicotiana tabacum, cv BHmN) using the Agrobacterium tumefaciens-mediated leaf disc method, as previously described (Horsch and Klee 1986). All the resulting kanamycin-resistant plants were self-pollinated to obtain T1 transgenic seeds. Total soluble proteins were extracted by adding freshly prepared extraction buffer (EB) (10 mM sodium phosphate, 0.15 M NaCl, 0.1 % (v/v) Tween 20, 0.1 % (v/v) b mercaptoethanol, pH 7.0) at a 1/10 (w/v) seed weight/buffer ratio, incubating the samples under gentle agitation for 1 h and then centrifuging them for 10 min at 12,0009g, analyzing the resulting supernatants by Western blotting with anti-mouse (HC ? LC) antibodies (Sigma-Aldrich, USA) or quantifying the amount of anti-HBsAg monoclonal antibody by ELISA. In the latter case, microtiter plates (Nunc Maxisorp, Life Technologies, Denmark) were coated by incubating them for 20 min at 50 °C with 5 lg/mL of recombinant HBsAg (CIGB, Cuba) in carbonate/bicarbonate buffer, pH 9.6. After washing the plates with PBS-0.05 % Tween 20 (washing buffer), the samples, the standard and the control were diluted in PBS-0.2 % BSA-0.005 % Tween 20, added to the wells of the plate and incubated again for 20 min at 50 °C. Subsequently, the wells were washed five times with washing buffer and incubated with 100 lL/well of a goat anti-mouse IgG horseradish peroxidase conjugate (Sigma-Aldrich, USA), in a 1:30,000 dilution for 20 min at 50 °C. Finally, the plates were washed again and developed by incubation for 15 min with o-phenylenediamine (OPD) as substrate in citrate buffer, pH 5.0 containing 0.015 % H2O2, stopping the reaction by adding 50 lL of 2 M H2SO4 and reading the plates immediately at a wavelength of 492 nm in a microELISA reader (Labsystem, Finland) (Leyva et al. 2007). The lines exhibiting the highest levels of expression from among all mAb-positive primary transformants were further selected and brought to a T3 generation. Purification of seed-produced PHB-01 (PHB-01seed) monoclonal antibody Fifty grams of seeds were powdered in a blender (40 pulses of 15 s each one) and mixed with 250 mL of

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N-hexane in order to extract and remove fatty seed constituents, centrifuging the resulting hexane/macerated seed mixture for 5 min at 50009g and discarding the supernatant. This process was repeated three times, and the fine powder yielded by the procedure was dried under vacuum. The dried powder was then completely resuspended into 500 mL of ice-cold EB and incubated for 1 h at 4 °C with gentle agitation, after which the soluble fraction was obtained by centrifuging the sample for 30 min at 12,0009g. The extraction procedure was repeated afterwards with 250 mL of EB on the insoluble fraction, all the while keeping the soluble fraction from the previous extraction on ice. The supernatants were then pooled and filtered through a 0.45 lm filter, and the filtered pool was next loaded onto a column packed with 20 mL of Prosep-A matrix (Millipore, USA). The IgG fraction was eluted with 100 mM citric acid, pH 3.0 and instantly neutralized with 500 mM Tris pH 8.0, dialyzing the resulting sample (25 mL) against 3 L of phosphate buffer pH 7.0 for 16 h at 4 °C. The integrity of PHB-01seed was assessed by SEC-HPLC and SDSPAGE. A preparative TSK G3000 PW (55 9 600 mm, TosoH Biosep, Japan) HPLC-gel filtration column was used to determine the purity of the antibody and to isolate the full-size IgG fraction, employing phosphate buffer pH 7.0 as mobile phase, at a volumetric flow rate of 5 mL/min and measuring absorbance at 226 nm. Ten milliliters of the sample dissolved in phosphate buffered saline were directly applied onto the system, isolating three fractions that were concentrated with 10 kDa cut-off devices (Millipore, USA) and analyzed by immunoblotting to detect the antibody chains. Three separate runs were performed to assess the efficacy of the purification process. Protein concentration assays used the bicinchoninic acid (BCA) method, and antibody concentrations were determined by ELISA as described above. Isolation and purification of N-glycans Two-hundred micrograms of PHB-01seed were digested with 10 lg pepsin in 1 mL of 0.01 M HCl (pH 2.0) for 48 h at 37 °C, inactivating the enzyme at the end of this period by neutralizing the reaction mixture with 3.2 % NH4OH and heating it at 100 °C for 5 min. The glycopeptides were then de-glycosylated with PNGase A (Roche Diagnostics GmbH,

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Mannheim, Germany) at pH 5.0 for 37 °C during 24 h, using an enzyme/substrate ratio of 50 lU PNGase A per 200 lg of sample in a 10 ll reaction volume and purifying the resulting N-glycans with a GlycoClean H cartridge as described by the manufacturer (Glyko, Novato, CA, USA). The samples were next dried in a centrifugal vacuum evaporator without heating, and the dried N-glycans were labeled with 2-aminobenzamide (2AB) as described (Bigge et al. 1995). Briefly, 3 nmol of glycans were dissolved in 5 lL of 2AB solution (0.35 M 2AB in dimethyl sulfoxide/glacial acetic acid (3:7, v/v) containing 1.0 M sodium cyanoborohydride) and incubated at 60 °C for 2 h. The reaction mixture was then applied at the end of 3 MM chromatography strips (10 cm) and ran with acetonitrile for 1 h. After the run, the sample spot was cut off under ultraviolet light and the labeled N-glycans were eluted from the paper by washing four times with 500 lL of water, filtering the eluate with a plastic syringe fitted with a 13 mm 0.45 lm low protein binding PTFE membrane filter (Millipore, USA). Finally, the 2AB derivatives were dried in a centrifugal evaporator under vacuum without heating before submitting them to HPLC analysis. N-glycan analysis The mixture of 2AB oligosaccharide derivatives was analyzed by normal phase high performance liquid chromatography (NP-HPLC) on a TSK-GEL Amida80 (250 9 4.6 mm, TosoH, Japan) column and by electrospray ionization-mass spectrometry (ESI–MS). The oligosaccharides were separated by NP-HPLC as described (Triguero et al. 2011), monitoring fluorescence at kex = 330 nm and kem = 420 nm (Guile et al. 1996). The retention times of the N-glycans were converted to glucose units (GU) by comparison to a mixture of glucose oligomers with polymerization levels varying from 1 to 15, from a standard dextran ladder. For preliminary structure assignment, the experimental GU values were compared to reported values in GlycoBase, published by the Dublin-Oxford Glycobiology Laboratory (http:// glycobase.ucd.i.e/cgi-bin/profile_upload.cgi). For ESI–MS analysis in positive mode, the 2ABderivatized oligosaccharide mix was loaded into goldcoated borosilicate capillary needles (Micromass, UK) and introduced into the nanospray ionization source of a hybrid mass spectrometer with orthogonal geometry

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(QTOF-2TM, Micromass, UK) at cone and capillary voltages of 35 and 900 V, respectively. The m/z values measured in the resulting mass spectra corresponded in all cases to the structures identified from GU values. N-glycan immunoblotting assays Isolated light or heavy chains were separated by 12.5 % SDS-PAGE under reducing conditions (Laemmli 1970), followed by electrotransfer onto a nitrocellulose membrane for 1 h at room temperature at 100 V in transfer buffer [50 mM Tris, 380 mM glycine, 0.01 % (w/v) SDS and 20 % ethanol (v/v)]. The membranes were blocked for 1 h at 37 °C with 5 % skimmed milk powder (w/v) in PBS and then washed three times in 0.05 % Tween-20 in PBS (10 min each). Later, the membrane was incubated for 1.5 h at 37 °C with anti-b1,2-Xyl and anti-a1,3-Fuc antibodies (diluted 1:100 and 1:500, respectively, in PBS) and then washed three times in 0.05 % Tween20 in PBS (10 min each). The blot was subsequently incubated at room temperature for 1 h with a goat antirabbit IgG—alkaline phosphatase conjugate (1:5000 dilution in PBS) and washed in 0.05 % Tween-20 in PBS as above. The immunostaining was developed with 0.1 mg/mL nitroblue tetrazolium and 0.06 mg/ mL 5-bromo-4-chloro-3-indolyl phosphate. Immunodetection with Con A-HRP (Sigma-Aldrich, USA) was carried out according to the manufacturer’s instructions. Immunoaffinity purification of Hepatitis B surface antigen with PHB-01seed Purified PHB-01seed was diluted in coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) at a ratio of 3.5 mg of antibody/mL of activated Sepharose CL-4B (Amersham Biosciences) and mixed for 2 h at 25 °C. The matrix was then blocked for 30 min at 25 °C with 0.2 M glycine, pH 8, and washed with acetate buffer (0.1 M sodium acetate, 0.5 M NaCl, pH 4). A sample from an intermediate step of the HBsAg production process (non-bound fraction from the negative ion exchange) just prior to the immunoaffinity step, provided by the Production Department of CIGB at Havana, Cuba, was used as starting material to evaluate the performance of PHB-01seed-coupled Sepharose. The procedures were performed as

previously described (Ramı´rez et al. 2003). Briefly, the column containing PHB-01seed-coupled Sepharose was equilibrated with 20 mM Tris–HCl, 3 mM EDTA, 1 M NaCl buffer, pH 7, and the equivalent of 1 mg of recombinant HBsAg/ml of immunoadsorbent was loaded at a lineal flow of 5.1 cm/h, equivalent to a residence time of approximately 1 h. The antigen was eluted with a similar buffer containing 3 M KSCN under a similar flow/residence time, analyzing the resulting fractions by SDS-PAGE in 12.5 % acrylamide gels and Western blotting with an anti-HBsAg monoclonal antibody-horseradish peroxidase conjugate. The column was regenerated by sequential washes with 20 mM Tris–HCl, 0.1 M sodium acetate, 0.5 M NaCl, pH 4, and 20 mM Tris–HCl, 0.5 M NaCl, pH 8.5.

Results Cloning and plant transformation With the aim of evaluating the convenience of tobacco seed as a target organ for antibody expression and storage, Nicotiana tabacum c.v. BHmN was chosen as host. The coding sequences of the light and heavy chains (LC and HC, respectively) of a monoclonal antibody against the Hepatitis B Virus surface antigen (HBsAg) (Agraz et al. 1994) were cloned under control of the phaseolin promoter and the 30 and 50 UTR of arcelin (De Jaeger et al. 2002). Both chains were cloned as in-frame fusions to the 2S2 signal peptide at their N-terminus and to a KDEL endoplasmic reticulum retrieval motif at their C-terminus (Online Resource 1). N. tabacum wild explants were co-transformed with both vectors, yielding transgenic plants expressing either HC or LC as well as HC ? LC co-transformants. mAb expression and purification One hundred and nine putative transgenic plants were obtained. Total soluble proteins were extracted from dry seed samples of these transformants, and the amounts of anti-HBsAg antibody were quantified by ELISA (Leyva et al. 2007) in order to generate homozygous lines from the plants exhibiting the highest expression levels. Maximum mAb accumulation levels ranged between 0.4 and 1.1 mg of mAb per

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g dry seeds for the first generation, but increased to 6.5 mg PHB-01seed per g of seed in the homozygotic T3 generation (Table 1). Plantibody expression in the T1 transgenic tobacco seeds was also investigated by Western blotting with specific anti-mouse (HC ? LC) antibodies. Online Resource 2 shows bands from proteins extracts of T1 transgenic seeds whose relative migration matches that expected for the HC and LC under reducing conditions. No detectable protein bands were present in extracts from negative control plants. Degradation products are visible for mAb-positive plants as a protein band of 30 kDa together with other bands of molecular weights smaller than 25 kDa. The seed plantibody was purified using Protein A– Sepharose chromatography from a pool of heterozygous T2 transgenic A91 line, analyzing the resultant preparation by SDS-PAGE under reducing and nonreducing conditions. As shown in Fig. 1a lane 3, protein species whose relative migrations correspond to the expected molecular weight of the antibody chains are visible in the eluate, although additional bands of approximately 30 kDa are visible under reducing conditions. The Protein A-Sepharose PHB-01seed eluate had a purity of approximately 80 %, which was insufficient for further characterization. It was decided, therefore, to include an additional polishing step using gel exclusion chromatography. The protein fraction eluted from the Prosep protein A matrix was loaded onto a

preparative TSK G3000 PW column, collecting three fractions labeled as F1, F2 and F3 with retention times of 132–148, 150–165 and 172–182 min (Fig. 1b). Samples from each fraction were concentrated and analyzed by immunoblotting with specific anti-mouse (HC ? LC) antibodies under reducing and non-reducing conditions, identifying the full antibody in fraction F1 and antibody fragments in fractions F2 and F3 (Fig. 1c). The purity of the PHB-01seed antibody preparation after TSK G3000 PW chromatography and its subsequent concentration was higher than 90 % thanks to the removal of the main contaminants from the Protein A eluate, although the yield of plantibody diminished. Three independents purification batches were assayed with similar results, confirming the robustness of this procedure (Online Resource 3). PHB-01seed has a higher ratio of complex-type versus high-mannose type N-glycans than PHB-01leaf In order to study the intracellular localization of PHB01seed and the efficacy of the included KDEL signal on ER retention, its N-glycans were characterized by Western blotting with anti-a 1,3-Fuc- and anti-b 1,2Xyl-specific polyclonal antibodies and lectin concanavalin A-horseradish peroxidase (Con A-HRP) (Fig. 2), including PHB-01 purified from leaves (PHB-01leaf) and its mouse ascites counterpart (CB.Hep-1) in the same blot as controls. In the case of Con A (Fig. 2c), which binds strongly to high-

Table 1 Level of expression of PHB-01 in transgenic tobacco seeds Transgenic T1 lines

mg PHB-01/g seed (mean ± SD)

Transgenic T2 lines

mg PHB-01/g seed (range)a

Transgenic T3 lines

mg PHB-01/g seed (range)a

A10

0.532 ± 0.024

A35

0.853 ± 0.041

A50

0.648 ± 0.040

nd

nd

A68

0.602 ± 0.019

nd

nd

A83

0.810 ± 0.072

A91

1.109 ± 0.137

A35/4b

nd

nd

0.72–0.98

nd

nd A91/20

b

0.86–2.25

nd b

A91/20/25

3.21–6.53

seed

was quantified by an enzyme-linked immunosorbent assay (ELISA). The results are the average of three determinations PHB-01 for each plant. Two (A35, A91) out of the 109 primary transformants were further selected, evaluating individually more than 50 plants in order to select the best expressing lines in T2 seeds (A35/4 from A35, A91/20 from A91). More than 50 individual plants from the latter produced T3 seed, and A91/20/25 was selected as the line producing the largest amounts of PHB-01 mAb SD standard deviation nd not determined a

PHB-01seed expression range of the more than 50 plant individuals from best producer plants at T2 and T3 generation

b

PHB-01seed best producer plant in T2 and T3 generations

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Fig. 1 Purification of PHB-01 from tobacco seeds. a Coomassie blue-stained SDS-PAGE of PHB-01seed purified by Protein A chromatography and size exclusion high performance liquid chromatography (SEC-HPLC) on a TSK G3000 PW column. Lane 1 Crude seed extracts loaded onto the affinity column; lane 2 Unbound fraction; lane 3 Eluted fraction containing heavy and light chains besides other additional proteins; lane 4 F1 fraction from SEC-HPLC; lane 5 Purified PHB-01leaf control; lane 6 Broad Range Protein Marker (Biorad, USA). b SEC-HPLC chromatogram obtained from 10 mL

mannose-type N-glycans and weakly to short complex-type biantennary N-glycans, there were strong positive signals for PHB-01leaf, containing mainly high-mannose structures (Triguero et al. 2005), also strong but less intense signals for PHB-01seed, and a clear although relatively weak signal for CB.Hep-1, which contains predominantly complex-type N-glycans. In contrast, the anti-b 1,2-Xyl and anti-a 1,3-Fuc sera did not bind CB.Hep-1 and produced only very faint bands, if at all, with PHB-01leaf, while yielding very strong signals with PHB-01seed. This evidences that a large proportion of PHB-01seed molecules is modified with a 1,3-Fuc and b 1,2-Xyl groups (Fig. 2d, e), indicating that the frequency of appearance of complex-type N-glycans in the seed plantibody is higher than in the leaf plantibody or, in other words, that the fraction of PHB-01seed transiting through the Golgi is higher due to a lower efficiency of KDEL as ER retention signal in this particular case. In order to glean additional structural information regarding the N-glycans of PHB-01seed, they were isolated by trypsin treatment of the plantibody followed by PNGase A digestion, splitting the resulting free N-glycans into two samples, one of which was

(15 mg) of the Protein A affinity chromatography eluate. Three fractions (F1, F2, F3) were collected and concentrated for subsequent analysis. c Immunodetection, using anti-mouse (HC ? LC) antibodies, of the heavy and light chains (55 and 25 kDa respectively), the full-size antibody (150 kDa) or antibody fragments (\25 kDa) in SEC-HPLC fractions under reducing or non-reducing conditions; lane 1 Fraction F1 from SEC-HPLC; lane 2 Fraction F2 from SEC-HPLC; lane 3 Fraction F3 from SEC-HPLC

Fig. 2 Immunodetection of glycans on an anti-HBsAg monoclonal antibody purified from mouse ascites, leaves and tobacco seeds. Lane 1 Mouse ascites; lane 2 Tobacco leaves; lane 3 Tobacco seeds. a Coomassie blue stained SDS-PAGE; b immunodetection of heavy (HC) and light chain (HC) with a goat anti-mouse IgG-horseradish peroxidase conjugate (SigmaAldrich, USA); c detection with a Concanavalin A-horseradish peroxidase conjugate (Sigma-Aldrich, USA); d immunodetection with an anti-a 1,3-Fuc antibody-horseradish peroxidase conjugate (CIGB-Sancti Spiritus, Cuba); e immunodetection with an anti-b 1,2-Xyl antibody-horseradish peroxidase conjugate (CIGB-Sancti Spiritus, Cuba)

labeled with 2AB by reductive amination reaction (Triguero et al. 2005) for analysis by HPLC while the other was submitted to ESI–MS. The derivatized 2AB N-glycans were resolved by HPLC following previously described procedures (Bigge et al. 1995) yielding a profile that evidences

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that the N-glycan population of PHB-01seed contains both oligomannoside and complex-type structures (Fig. 3 and Online Resource 4). M5 to M9 structures were identified within the PHB-01seed profile, with M7 high-mannose-type N-glycan as the most abundant oligosaccharide structure, as also happens in PHB01leaf (Triguero et al. 2005). However, in PHB-01seed oligomannoside structures account for 56.2 % of its N-glycans, whereas this proportion increases to 80.7 % in PHB-01leaf (Online Resource 4). Thus, complex-type N-glycans account for 41.5 % of the total in PHB-01seed, with xylosylated/fucolylated and fucosylated N-glycans representing 22.2 and 5.2 %, respectively. Finally, 2.3 % of the oligosaccharides structures detected in the profile could not be identified (Online Resource 4). The underivatized PHB-01seed N-glycan sample was also analyzed by ESI–MS in positive mode (Online Resource 5). The m/z of the signals observed in the resulting spectrum corresponds to those expected for the oligosaccharide structures that were previously assigned on the basis of GU values. A peak with an m/z corresponding to the F(3)A1 structure was not detected, probably due to its low abundance within the total N-glycan population (Online Resources 4 and 5).

Immunoaffinity purification of HBsAg with PHB-01seed

Fig. 3 Amide 80-high-performance liquid chromatography (HPLC) profile of N-glycans from PHB-01seed released enzymatically by N-glycosidase A (PNGase A) treatment. The GU values of all peaks were interpolated from a standard dextran ladder, doing preliminary structural assignments by comparison

to the reported values in GlycoBase from the Dublin-Oxford Glycobiology Laboratory (http://glycobase.ucd.i.e/cgi-bin/ profile_upload.cgi). GlcNAc: N-acetylglucosamine, Man: mannose, Fuc: fucose, Xyl: xylose

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In order to assess the performance of PHB-01seed in the purification of HBsAg by immunoaffinity chromatography, a small-scale test run was done using PHB01seed coupled to activated Sepharose CL-4B. As shown in Fig. 4a, HBsAg eluted from the affinity gel prepared with PHB-01seed exhibited an electrophoretic pattern very similar to that of HBsAg eluted from the industrial affinity gel prepared with ascites-derived antibody, and also was recognized by an anti-HBsAg monoclonal antibody in a Western blot assay (Fig. 4b).

Discussion Seeds are often considered as the ideal organ for expressing heterologous proteins in plants (Sto¨ger et al. 2005). Promoters that drive gene expression only when placed in the regulatory context of seed tissues (i.e. seed-specific promoters) are usually employed for this purpose. Among the seed-specific promoters isolated from dicotyledonous plants, those derived from legume species appear to offer the greatest

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Fig. 4 Analysis by SDS-PAGE and Western blotting of fractions collected during the purification of recombinant Hepatitis B surface antigen (rHBsAg) using a PHB-01seedSepharose CL-4B affinity resin. a Coomassie blue-stained SDSPAGE. Lane 1 Starting sample (intermediate fraction from the rHBsAg purification process, just previous to the immunoaffinity chromatography); lane 2 unbound protein fraction; lane 3 rHBsAg eluate; lane 4 rHBsAg purified using a Sepharose CL-

4B affinity resin prepared with ascites-derived CB.Hep-1 antibody; lane 5 Broad Range Protein Marker (Biorad, USA). b Immunodetection of rHBsAg with an anti-HBsAg monoclonal antibody-horseradish peroxidase conjugate. Lane 1 rHBsAg purified using Sepharose CL-4B coupled to seed-derived antibody; lane 2 HBsAg purified using Sepharose CL-4B coupled to ascites-derived antibody

potential. The first seed-specific promoter reported in the literature, that of the phaseolin gene (SenguptaGopalan et al. 1985), is still widely used in the transgenic plant community, and has become the promoter of choice in many groups seeking to use transgenic seeds as bioreactors due to its strength and the tightness of its regulation. The levels of accumulation of heterologous proteins in seeds vary according to the complexity of the target molecule and specific host factors; Arabidopsis thaliana (De Jaeger et al. 2002; Van Droogenbroeck et al. 2007; Loos et al. 2011) and N. tabacum (Cheung et al. 2009; Morandini et al. 2011) are the most frequently employed host species. A single-chain variable fragment (scFv) of a murine antibody (G4) was expressed in A. thaliana at accumulation levels of up to 36 % of TSP (75 lg/ mg of seed) under homozygosis (De Jaeger et al. 2002), in what constitutes one of the highest levels of heterologous expression ever reported in plants. Four scFv-Fc from different human antibodies were also expressed in this species at levels ranging from 7 to 12.5 % of TSP (Van Droogenbroeck et al. 2007). Using the same expression system, two full-sized antibodies (HA78 against the Hepatitis A Virus and 2G12 against HIV) and their scFv-Fc derivatives reached accumulation levels of 10 lg/mg seed (Loos et al. 2010, 2011). The design used for these genetic constructs, however, is significantly less efficient in N.

tabacum. A comparison of the accumulation of a biopharmaceutical protein [the chimeric form (GAD67/65) of the 65 kDa human type I diabetes autoantigen, glutamic acid decarboxylase (GAD65)] in N. tabacum and A. thaliana revealed that product accumulation was ten times lower in the former (0.4 vs 4.5 mg per g of seed) at generation T3 (Morandini et al. 2011). The levels of accumulation achieved here for PHB-01 in N. tabacum at the heterozygotic T1 generation are within the same order of magnitude of those reported for the same species in that manuscript, although they increase to levels comparable to those of A. thaliana in higher generations, after screening and selecting the best mAb producer lines. It should be noted, though, that other results from our group [2.2 lg HBsAg/g of seed (Hernandez et al. 2013) and 3.3 mg Hemagglutinin Antigen/g of seed in T1 (article in preparation)] suggest that the complexity of the target protein is a major factor concerning the success of heterologous expression in this system. Immunoblotting of crude seed extracts (see Online Resource 2) was used to confirm expression of both the heavy and light chains in each transformant. The blots, however, also revealed the presence of degradation products in all cases at around 30 kDa together with additional bands below 25 kDa. This phenomenon has been previously observed for other seed plantibodies (Petruccelli et al. 2006; Rademacher et al. 2008; Ramessar et al. 2008; Shukla and Thommes

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2010), especially when using a C-terminal KDEL tag for ER retention (Loos et al. 2011). In leaves the KDEL signal retains efficiently a recombinant protein in the endoplasmic reticulum, whereas KDEL-tagged proteins expressed in seeds can also be directed to protein storage vacuoles (PSV) or the apoplast (Petruccelli et al. 2006). The content of complex-type N-glycans in PHB-01seed was about three times higher (See Online Resource 4) than that previously measured in PHB-01leaf (Triguero et al. 2005), implying a ratio of complex:oligomannoside structures in PHB01seed of approximately 1:1. This is suggestive of the same Golgi transit pattern previously described for the 14D9 tobacco seed plantibody (Petruccelli et al. 2006). Some seed-expressed antibodies bearing an ER retention signal have been found to still enter the secretory pathway and be routed, via ER and the Golgi apparatus, to vacuoles or the apoplast, undergoing specific N-glycan modifications along their passage. The relatively large fraction of complex-type oligosaccharides both in PHB-01seed and other plantibodies expressed in seed, therefore, evidences that in this system, the efficiency of KDEL as an ER retention signal is not high (Petruccelli et al. 2006; Floss et al. 2009). Taking into account that this antibody is used as an immunosorbent in the purification of HBsAg by immunoaffinity chromatography, and that N-glycosylation does not affect its ability to bind its cognate antigen or be coupled to activated Sepharose CL-4B (Ramı´rez et al. 2003; Valde´s et al. 2003b), it follows that the higher proportion of complex-type sugar groups in PHB-01seed does not limit the use of this technology for its manufacture, although it may certainly be inconvenient for other biologicals intended for systemic administration in humans, which can be rendered immunogenic or allergenic by the presence of foreign (i.e. non-mammalian) N-glycosylation patterns. Despite the many advantages of plants as bioreactors, purifying a recombinant protein from plant extracts remains a challenging task, as these preparations constitute very complex mixtures of large numbers of protein species. However, several recombinant proteins have been recently purified successfully from seeds using chromatographic and nonchromatographic methods, as reviewed recently (Wilken and Nikolov 2012). Since the purification process for PHB-01leaf uses protein A affinity

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chromatography as its main step (Ramı´rez et al. 2003; Valde´s et al. 2003a), we decided to evaluate this method for PHB-01seed. After preliminary experiments performed to select the most appropriate extraction and pre-treatment strategies (data not shown), it was decided to use dry grinding followed by low-shear mixing with an aqueous buffer for recombinant protein extraction, also including a membrane filtration step before the immunoaffinity process. Figure 1 and Online Resource 3 show that the purity of PBH-01seed purified by protein A chromatography according to the selected methodology was consistently higher than 75 %, and that its main contaminants are minor protein species migrating between the heavy and light chains and, in some cases, additional species at molecular weights of 10–15 kDa. The resulting preparation, therefore, is considerably less pure than equivalent PHB-01leaf preparations obtained from tobacco leaves following similar methodologies (90 %) (Valde´s et al. 2003a), and does not meet the purity requirements of many regulatory agencies. Purity was increased above 90 % after an additional gel filtration step on a TSK G3000 PW chromatography column, where three separate fractions were isolated, one (F1) containing the full-size antibody and the others (F2, F3) containing antibody fragments (Fig. 1b, c) extensively cross-linked by disulfide bonding, according to the electrophoretic behavior of fractions F2 and F3 under reducing and non-reducing conditions. It should be noted that the contaminating antibody fragments are relatively more abundant and exhibit a different pattern in Protein A-purified PBH-01seed compared to Protein A-purified PHB-01leaf (Valde´s et al. 2003a), indicating an enhanced susceptibility of this antibody to seed proteases. After gel filtration chromatography, process yields decreased to 50 %, still in the same order of magnitude as the yields reported for plantibody 2G12 produced in corn seed and purified using two chromatographic steps (none of them with Protein A immunosorbents) (Ramessar et al. 2008). Unintended proteolysis influences both the quality and yield of the purified final product and is therefore major issue in recombinant protein production, so it would be desirable to gain deeper knowledge on the proteolytic processes related to tobacco seed proteins in order to enable the rational design of strategies aimed at minimizing the deleterious effects of this phenomenon. The performance of PHB-01seed in the

Transgenic Res Table 2 Comparative analysis of the production of PHB-01 in tobacco leaves versus seeds

Biomass production batchc Time to harvest

d

Tobacco leavesa

Tobacco seedsb

400–600 kg leaves

6.5–7.0 kg seedg

6–8 weeks

18–20 weeks

Expression level Purification batch (L)

30 mg PHB-01/kg leaves 400 L (1/1, w/v)e

6.5 g PHB-01/kg seeds 70 L (1/10, w/v)e

Process yieldf

9g

24 g

a

Data according the Monoclonal Antibody Production Department at CIGB

b

Data according to our experience and assumptions

c

Production batches from 840 plants

d

Time in a greenhouse after 6 weeks in seedbed

e

Ratio biomass weight/extraction buffer volume: 1/1 for leaves, 1/10 for seeds

g

Assuming that each plant produces 8 g of seeds

f

Assuming that the purification process recovers 50 % of the starting amount of PHB-01

immunoaffinity purification of HBsAg was similar (Fig. 4) to that of PHB-01leaf (Ramı´rez et al. 2003; Valde´s et al. 2003b), although additional parameters, such as the degree of antibody leakage, remain to be determined in future experiments. In addition, the results presented here reinforce the conclusions from previous studies suggesting that the glycosylation patterns of antibody CH2 domains do not affect antigen recognition (Wright and Morrison 1997; Ramı´rez et al. 2003). Therefore, the higher proportion of complex-type N-glycans in PHB-01seed affect neither its ability to be coupled to activated CL-4B Sepharose nor its binding to HBsAg. Finally, a short analysis was made regarding the economic feasibility of manufacturing PHB-01seed compared to PHB-01leaf (Table 2), based on data from a leaf biomass production batch (840 plants). Harvesting seeds takes approximately three times longer than harvesting leaves, so only 2–2.5 seed biomass batches would be produced in a year per area unit compared to 6–8 batches in the case of leaves, in what constitutes the main disadvantage of seeds as a production platform. However, seed production can still be increased by applying breeding techniques associated to higher seed yield germplasm, not to mention the fact that the starting concentration of PHB-01 in seeds is higher than in leaves, enabling the preparation of smaller volume seed extracts that shorten the length of chromatographic processes and thus, the effect of undesired proteolysis. In addition, the stability of seeds makes it possible to accumulate several biomass batches before initiating a purification

process, thereby increasing antibody yield per chromatographic run. For example, 400 L of seed extract would contain 260 g of PHB-01 from 40 kg of seeds (1/10, w/v), which is more than ten times the amount of antibody in 400 L of leaf extract. This, together with the absence of the pigments and phenolic compounds typical of leaves, would considerably simplify downstream processing, prolong resin life, and reduce overall downstream processing costs. In summary, the results obtained in this work evidence that it is feasible to use tobacco seeds as an alternative production system for plantibody PHB-01. Further costs savings can be realized through the optimization of biomass production and downstream processing of PHB-01seed, which constitute the subject of ongoing work. Acknowledgments We thank the Department of Plant Systems Biology of Ghent University Belgium for supplying the signals for expression in seeds. In addition, we would like to extend our appreciation to the staff of the experimental area at CIGB for their help in cultivating the tobacco plants used in this study and to the Monoclonal Antibody Production Department at CIGB for their help with the immunoaffinity purification of HBsAg. No potential conflicts of interest are declared in this work.

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