Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

Process Biochemistry 45 (2010) 67–74

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Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids Cheon-Ik Park a, Song-Jae Lee a, Seung-Hoon Kang a, Hahn-Sun Jung a, Dong-Il Kim b, Sang-Min Lim a,* a b

Boryung Central Research Institute, Boryung Pharmaceutical Co. Ltd., Ansan, Gyeong Gi-do 425-120, Republic of Korea Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 May 2009 Received in revised form 3 August 2009 Accepted 10 August 2009

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic Tlymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l1 at day 10. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Fed-batch culture hCTLA4Ig Transgenic plant cell culture RAmy3D promoter Oryza sativa

1. Introduction The use of transgenic plant cell cultures for the production of recombinant proteins, including plant-made pharmaceuticals (PMPs) and plant-made industrial proteins (PMIPs), is widespread in these days. Compared to mammalian cell cultures, plant cells are easy to cultivate and the culture media are inexpensive. Since the plant cells are higher eukaryotes, they can perform most of the post-translational modifications found in human cells. Plant cells are also intrinsically safe because they do not harbor any human pathogens, bacterial toxins and endotoxins [1,2]. In addition, plant systems as a source of human-derived proteins have several advantages, such as the potential of scale-up, low cost biomass production, secretion of foreign protein and the low cost for purification due to simple culture media. Because of these advantages, several mammalian proteins have been produced in transgenic plants or plant cell cultures, including human serum albumin [3], growth factors, hormones, cytokines [4–6], glucocerebrosidase [7] and monoclonal antibodies [8,9]. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is a recombinant fusion protein that contains the extracellular domain of hCTLA4 fused to the immunoglobulin IgG heavy chain [10]. The fusion protein is a 95 kDa homodimer and known to block the CD28-mediated co-stimulatory signal. It can

* Corresponding author. Tel.: +82 31 491 2271; fax: +82 31 491 5340. E-mail address: [email protected] (S.-M. Lim). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.08.004

inhibit immune responses in vitro and in vivo, and prolong graft survival [11–14]. We previously demonstrated that genetically engineered rice cells could express hCTLA4Ig at high level and secrete the protein into the medium using an inducible a-amylase (RAmy3D) promoter [15]. In this expression system, production of the recombinant protein is induced by two-stage culture composed of a cell growth stage, in which a sugar-rich medium is used, and a production stage, in which target recombinant proteins are produced under sugar-depleted conditions. The use of inducible RAmy3D promoter in transgenic rice cells showed high level expression of recombinant proteins, even though the growth rate is lower than those of the BY-2 and NT-1 tobacco cell lines [16–18]. The recombinant protein concentration obtained using this transgenic rice cell culture system is generally greater than 10 mg l1, which provides a promising starting point for process development considering the known level by tobacco cells previously [19,20]. One possible disadvantage of using the RAmy3D promoter is cell disruption, which can be caused by an imbalance in the osmotic pressure of the cells in a depleted sugar state as well as the absence of carbon sources for cell metabolism. Additionally, the medium exchange system frequently leads to contamination problems. Media change from the growth medium into sugar-free production medium is also difficult to perform in industrial largescale cultures. In previous studies, a low concentration of glucose (0.5 mM) was added to the induction medium to solve this problem. Although the addition of glucose has been shown to be effective at improving the a1-antitrypsin productivity [21], it may

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result in the inhibition of RAmy3D promoter used in this expression system. However, it may be possible to increase the a1-antitrypsin productivity in batch cultures by using induction medium containing an alternative carbon source, such as pyruvic acid, glyoxylic acid or lactic acid [22]. In this study, a fed-batch culture method was developed for the efficient production of a recombinant fusion protein, hCTLA4Ig, in transgenic rice cell suspension cultures. We attempted to develop a new feeding method using concentrated amino acids to enhance productivity without exchanging the medium for two-stage culture. It was found that the added amino acids solved the problems associated with the use of the inducible RAmy3D promoter for the expression of the target protein without any risk of contamination due to media exchange. 2. Materials and methods 2.1. Cell line and cultures The transgenic rice (Oryza sativa L.) cell line expressing hCTLA4Ig was established by Boryung Pharmaceutical Co. Ltd. (Ansan, Korea) as previously reported [15]. Transgenic rice cells were maintained in 500-ml Erlenmyer flasks with 140 ml of fresh medium on a shaking incubator at 110 rpm at 28 8C under dark conditions. To maintain the cells in shake flasks, 60 ml of the culture was transferred into fresh medium every 10 days. The amino acid (AA) medium, containing 2,4-dichlorophenoxyacetic acid (2 mg l1), kinetin (0.02 mg l1), and 3% sucrose (AAS(+)), was used for the rice cell cultures, and 50 mg l1 hygromycin B was added to provide selection pressure [16]. 2.2. Bioreactor operation Rice cell cultures were performed in a 7-l bioreactor (KoBiotech Co., Korea) modified for plant cell suspensions by adding a four-bladed hollow-paddle impeller and a disk type gas sparger to decrease the shear stress caused by increased aeration. The working volume was 3.2 l and the initial agitation and aeration rate were 80 rpm and 0.2 vvm, respectively. AA medium (2.3 l) was sterilized by autoclaving within the bioreactor at 121 8C for 25 min. After the sterilization, 300 ml of 10 concentrated amino acids solution filtered through a 0.22-mm filter (Millipore, USA) were infused into the bioreactor together with the cells. The bioreactor was then inoculated with 20% (v/v) of the suspension of cells that had been cultured in flasks for 7 days with the aid of a tube welder (Wave Biotech, Switzerland), which was used to make sterile connections between the tubing of the inoculation bottle and the bioreactor vessel. The culture temperature was maintained at 28 8C and the dissolved oxygen (DO) level was controlled by regulating the aeration rate and agitation speed during the culture period. Samples of 15–20 ml were collected aseptically through the sampling port at an interval of 1–3 days. A modified 15-l bioreactor (Applikon Co., Netherlands) with the same hollowpaddle impeller and disk type gas sparger was also used. The sterilization conditions, inoculation concentrations, and culture conditions were similar to those of the 7-l bioreactor except the regulation of dissolved oxygen (DO) level by using pure oxygen. To maintain the DO level at greater than 30%, pure oxygen was mixed with air and supplied intermittently into the bioreactor at a rate between 10 and 100 ml min1. 2.3. Induction of hCTLA4Ig expression To induce hCTLA4Ig expression in the batch culture during two-stage operation mode, a peristaltic pump was used to exchange the culture medium in the bioreactor with AA medium without any sugar (AAS() medium) sterilized by filtering through a 0.22-mm filter. A mesh port with a pore retention size of 20 mm was positioned at the bottom of the bioreactor to filter the medium while retaining the cells inside. For induction of hCTLA4Ig expression in the fed-batch culture during one-stage operation mode, 10 amino acids in soluble inorganic salts media were added into the bioreactor (Fig. 1). Composition of 10 amino acids was Larginine 1.3 mM, L-aspartic acid 2.0 mM, glycine 1.0 mM and L-glutamine 6.0 mM. 2.4. Measurements of cell mass Cell suspension samples were placed in a graduated cylinder and the total volume as well as the volume of the settled cells was recorded after 10 min. The medium was then centrifuged at 20,000  g for 5 min, after which the supernatant was frozen at 70 8C for later analysis. To determine the weight of the fresh cells, the cell suspension was filtered through Whatman No. 1 filter paper under vacuum and then washed two times with distilled water to remove residual sugar from the surface of the cells. The cells were then transferred to a pre-weighed dish and the cell mass was measured. The dry cell weight was estimated after drying at 60 8C for 2 days.

Fig. 1. Growth and hCTLA4Ig expression profiles of the rice suspension culture a two-stage operation (run A). Rice cells were cultured in a 7-l bioreactor at 28 8C and 80 rpm. The arrow indicates the time at which the media were exchanged with the sugar-free medium. (A) Dry cell weight and total sugar concentration. The run was induced by changing the medium into sugar-free medium while retaining the cells inside the bioreactor. (B) hCTLA4Ig concentration and culture pH. The concentration of hCTLA4Ig was determined by ELISA. The pH of the culture was not controlled during the run. 2.5. Sugar analysis Sucrose, glucose and fructose were analyzed using an HPLC (Hitachi, Japan) with a Sugar-Pak I column (Waters, USA) and a Guard-Pak guard column. The column was equilibrated with double distilled water containing 500 mg l1 of calcium disodium EDTA at a flow rate of 0.5 ml min1 while maintaining the column temperature at 80 8C. Samples that had been filtered with 0.45-mm syringe filters (Millipore, USA) were loaded onto the column and the sugars were then detected by a refractive index (RI) detector (Hitachi, Japan). The sugar concentration was then estimated using a standard curve generated using known concentrations of glucose, fructose, and sucrose, respectively. 2.6. Quantification of hCTLA4Ig The hCTLA4Ig expression level was measured by enzyme-linked immunosorbent assay (ELISA). The 96-well plates were coated with goat anti-human IgG (Fc) (1:1000; KPL Inc., USA), and then each well was loaded with sample or protein standard at concentrations ranged from 11 to 0.08 ng ml1, in eight 2-fold serial dilutions. Goat anti-human IgG (1:5000) was used as the detection antibody, followed by horseradish-peroxidase (HRP) and substrate 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, KPL Inc., USA). 2.7. SDS-PAGE and Western blotting Electrophoresis was performed using 10% Tris–glycine gels (Invitrogen, USA) and the gels were stained with Coomassie brilliant blue. The proteins were then electrotransferred onto polyvinylidene fluoride (PVDF) membrane (Invitrogen, USA) for Western blot analysis. Monoclonal goat anti-hCTLA4 antibody (R&D Systems, USA) or goat anti-human IgG (Fc) (KPL Inc., USA) was used as the primary

C.-I. Park et al. / Process Biochemistry 45 (2010) 67–74 antibody and alkaline phosphatase-conjugated rabbit anti-goat IgG (H+L) (KPL Inc., USA) was used as the secondary antibody, respectively. The immunoreactive hCTLA4Ig bands were then detected by a colorimetric method using the BCIP/NBT substrate system (KPL Inc., USA).

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USA) with a CarboPacTM PA1 column (Dionex, USA). The column was equilibrated with double distilled water containing 100 mM NaOH and 150 mM sodium acetate at a flow rate of 1.0 ml min1. N-glycans profiles were then estimated using a standard solution of fucose, galactose, xylose, mannose, N-acetylglucosamine, Dglucosamine, and N-acetylneuraminic acid, respectively.

2.8. Purification of hCTLA4Ig Rice-derived hCTLA4Ig (hCTLA4IgP) and CHO-derived hCTLA4Ig (hCTLA4IgM) were purified using the rProtein A column (GE healthcare, USA). Culture medium was mixed with binding buffer (20 mM sodium phosphate buffer, pH 7.0), and it was purified using the AKTA Prime FPLC system (GE healthcare, USA). The protein-bound rProtein A resin column was washed with 10 column volumes of binding buffer, and the bound hCTLA4Ig was eluted by 0.1 M glycine–HCl, pH 3.0. Fractions were collected and neutralized by the addition of 10 mM Tris–HCl buffer, pH 9.0. Fractions containing hCTLA4Ig were pooled and dialyzed using a Hiprep 26/10 desalting column (GE healthcare, USA). Purified hCTLA4IgP and hCTLA4IgM proteins were analyzed by SDS-PAGE and Western blot analysis. 2.9. N-terminal amino acid sequencing Peptide N-terminal sequencing was performed by Korea Basic Science Institute (Daejeon, Korea). The purified proteins were freeze-dried and directly sequenced on Procis 491 HT Protein Sequencer (Applied Biosystems, USA). 2.10. Monosaccharide composition analysis Monosaccharide composition was analyzed by Carbohydrate Bioproduct Research Center (Sejong University, Korea). N-glycans were released by acidic hydrolysis (100 8C, for 1 h) with 1 ml of 0.1N HCl and purified from 1 mg of hCTLA4Ig. After isolation of oligosaccharide chains, the samples were neutralized with 0.1N NH4OH. N-glycans were analyzed using HPAEC-PAD (high pH anion exchange chromatography with pulsed amperometric detector) system (Dionex,

3. Results 3.1. Growth characteristics of transgenic rice cells Two-stage batch culture in a 7-l bioreactor with medium exchange (run A), one-stage fed-batch culture with amino acid feeding in a 7-l bioreactor (run B), and one-stage fed-batch culture with amino acid feeding in a 15-l bioreactor (run C) were performed to compare two-stage culture with one-stage fedbatch culture. The initial dry cell weights (Xo) of runs A, B, and C were 6.27, 3.77 and 4.53 g l1, respectively. All the runs were repeated three times and the results were averaged. The growth periods before induction were 6, 7 and 7 days for runs A, B and C, respectively. For run A, induction was initiated by exchanging the culture medium in the bioreactor with sugar-free AAS() medium, whereas induction of runs B and C was initiated by adding concentrated amino acids to the bioreactor without exchanging the culture medium. The maximum specific growth rates and doubling times (tD) were 0.121 day1 (tD = 8.26 day), 0.143 day1 (tD = 7.33 day) and 0.158 day1 (tD = 6.34 day) for runs A, B and C. The maximum dry cell weights (Xmax) were 12.06, 10.19 and 13.65 g l1 for runs A, B, and C, respectively.

Fig. 2. Growth and hCTLA4Ig expression profiles of the rice suspension during the fed-batch culture (one-stage operation, run B). Rice cells were cultured in a 7-l bioreactor and fed-batch culture was performed by adding 10 amino acids at day 7 without medium change. The arrow indicates the time of adding the concentrated amino acids to the culture. (A) Dry cell weight by cultivation time; (B) sugar concentration; (C) hCTLA4Ig concentration; (D) pH.

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Fig. 3. Scale-up of fed-batch culture (one-stage operation, run C). Transgenic rice cells were cultured in a 15-l bioreactor under culture conditions similar to those of the 7-l bioreactor. The arrow indicates the time at which the concentrated amino acids were fed to the cultures. The run was induced by 10 amino acid feeding without exchanging the media. (A) Dry cell weight; (B) sugar concentration; (C) hCTLA4Ig concentration; (D) pH; (E) DO. The pH of the culture was not controlled and DO level was regulated by adding pure oxygen during the run.

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Table 1 Comparison of each run for hCTLA4Ig production.

texpr (day) Maximum hCTLA4Ig (mg l1) Specific content (mg gDCW1) Harvest volume (l) Working volumea (l) Volumetric productivityb (mg l1 day1) a b

Batch (7-l, run A)

Fed-batch (7-l, run B)

Fed-batch (15-l, run C)

Run 1

Run 1

Run 2

Run 3

Run 1

Run 2

Run 3

7 63.9 7.1 1.7 3.0 5.2

9 69.3 10.3 1.8 3.1 4.8

9 71.2 10.0 1.9 3.2 4.7

9 64.8 8.0 1.8 3.1 4.2

10 76.5 9.1 5.8 7.6 5.8

11 75.5 8.5 5.9 7.7 5.3

10 75.6 8.5 6.0 7.8 5.8

Working volume measured in the induction phase. Volumetric productivity = (hCTLA4Ig (mg l1)  harvest volume (l))/(working volume (l)  texpr (day)).

3.2. Effects of medium exchange on induction and cell growth

3.4. hCTLA4Ig productivity in three different types of cultures

For the large-scale production of hCTLA4Ig, transgenic rice cells were grown in a 7-l bioreactor. The AAS(+) medium was exchanged into AAS() medium after 6 days of cell growth to induce the production hCTLA4Ig, and the concentrations of sucrose, glucose and fructose in the media were found to be less than 1 g l1 after induction (Fig. 1). When the medium in the 7-l two-stage culture was exchanged, the dry cell weight (DCW) was 12.06 g l1 (97.2 mg l1 in fresh cell weight (FCW)). In addition, the sucrose concentration decreased from 30.2 to 0 g l1. Furthermore, concentrations of fructose and glucose remained below 1 g l1 after the media change, and they were completely consumed during the induction phase (Fig. 1A). Analysis of the sugars in the medium demonstrated that glucose and fructose were formed by the hydrolysis of sucrose. The time course changes of the hCTLA4Ig concentration and the pH are shown in Fig. 1B. A maximum hCTLA4Ig concentration (63.9 mg l1) was obtained at 7 days after induction. In addition, the pH of the medium decreased after induction for 2 days, but then began to increase sharply from 5.22 to 7.36 until the end of the culture.

Table 1 summarizes the productivity of hCTLA4Ig in each run. The volumetric productivity was dependent both on the functional hCTLA4Ig concentration at the time of harvest and on the duration of each expression phase (texpr). Based upon the harvest volume of 1.7, 1.8  0.1 and 5.9  0.1 l, the hCTLA4Ig productivities for run A, B and C were 5.2, 4.6  0.3 and 5.6  0.3 mg l1 day1, respectively. In

3.3. Effect of amino acid feeding Fed-batch culture without AAS() medium exchange was preformed by adding concentrated amino acids to maintain cell mass during the induction period. Ten times concentrated amino acids were added at day 7 of culture, at which time the DCW was 10.17  1.22 g l1 (106.52  8.44 g l1 in FCW) (Fig. 2A), and the sugar concentration was 14.86  6.01 g l1 (Fig. 2B). Within 2 days after the addition of amino acids, the remaining fructose and glucose was completely consumed. The changes of pH and hCTLA4Ig concentration are shown in Fig. 2C and 2D, respectively. hCTLA4Ig reached a maximum level of 69.3 mg l1 (run 1), 71.2 mg l1 (run 2), 64.8 mg l1 (run 3) at 14, 12, 9 days after feeding, respectively. In addition, pH decreased 2–3 days after feeding; however, it then began to increase sharply in proportion to the increased level of hCTLA4Ig. Amino acid feeding was also conducted in a 15-l bioreactor under the same conditions. At the time of amino acids feeding, the cell mass of the 15-l bioreactor was similar to that of the 7-l bioreactor, and the sugar was completely consumed within 7 days after the addition of amino acids (Fig. 3A and B). The maximum concentrations of hCTLA4Ig, obtained 17 days (run 1), 16 days (run 2), 17 (run 3) days after the start of feeding, were 76.5, 75.5, 75.6 mg l1, respectively (Fig. 3D). In addition, the pH of the medium in the 15-l bioreactor decreased until the 5–8th day after induction and it began to increase sharply with the simultaneous increase in hCTLA4Ig and DO levels (Fig. 3C and E). These results were similar to those in 7-l bioreactor.

Fig. 4. SDS-PAGE and Western blot analysis of extracellular hCTLA4Ig in a 7-l bioreactor. Standard and samples were combined with 5 SDS-PAGE denaturing sample buffer and boiled for 5 min, and then 10 ml per well was loaded onto a 10% Tris–glycine 10-well gel. (A) Coomassie blue stained gel with pre-stain protein marker, standard and samples. Lane 1, animal-derived hCTLA4Ig standard (150 mg l1); lane 2, growth 7 days before amino acid feeding (0 mg/L), The rest of the lanes show samples taken after induction by amino acids; lane 3, immediately after amino acid feeding (0 mg l1); lane 4, induced 1 day (0.10 mg l1); lane 5, induced 2 days (0.03 mg l1); lane 6, induced 3 days (10.55 mg l1); lane 7, induced 5 days (40.69 mg l1); lane 8, induced 7 days (56.03 mg l1); lane 9, induced 9 days (64.8 mg l1). (B) Western blot.

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Table 2 Comparison of N-terminal amino acid sequences for hCTLA4Ig.

Fig. 5. SDS-PAGE analysis of purified hCTLA4IgP and hCTLA4IgM with affinity chromatography. Standard and samples were combined with 5 SDS-PAGE reducing or non-reducing sample buffer. Non-reducing samples were boiled for 5 min, and then 10 ml per well was loaded onto a 10% Tris–glycine 10-well gel. Coomassie blue stained gel with pre-stain protein marker and samples. Lane 1, purified hCTLA4IgM (0.1 mg ml1) in reducing; lane 2, purified hCTLA4IgP (0.1 mg ml1) in reducing; lane 3, purified hCTLA4IgM (0.1 mg ml1) in nonreducing; lane 4, purified hCTLA4IgP (0.1 mg ml1) in non-reducing condition.

addition, the specific contents of hCTLA4Ig per DCW, which was calculated by using the cell mass at the time of harvest, were 7.1, 9.4  1.5 and 8.7  0.3 mg gDCW1 for runs A, B and C, respectively. 3.5. SDS-PAGE and Western blot hCTLA4Ig produced in the 7-l fed-batch culture of run 3 was confirmed using SDS-PAGE (Fig. 4A) and Western blot (Fig. 4B). SDS-PAGE and Western blot profiles of 7-l two-stage batch and 15-l fed-batch cultures were similar. Based on the results of the SDS-PAGE analysis, the thick bands shown in lanes 4–9 (approximately 50 kDa) were thought to be a-amylase because they did not react with anti-hCTLA4 antibody. In addition, Western blot analysis indicated that the level of hCTLA4Ig increased as the culture time increased, similar to ELISA results. Furthermore, the immunoreactive band was not detected during the growth period prior to the amino acids feeding. Immunoreactivity was confirmed 3 days after the feeding of amino (lane 6). 3.6. Structural analysis of purified hCTLA4Ig Culture media were harvested and purified by protein A affinity chromatography. Because hCTLA4Ig has an Fc fragment, we performed one-step purification using Streamline rProtein A system. As expected, the hCTLA4Ig in rice cell culture media could be purified effectively.

Major amino acid among the detected amino acids are underlined; *, non-defined cycle.

Purified hCTLA4IgP was analyzed by SDS-PAGE under reducing or non-reducing conditions. As shown in Fig. 5, the molecular weight of purified hCTLA4IgP (lanes 2 and 4) was approximately 50 kDa, which was slightly lower than that of hCTLA4IgM (lanes 1 and 3). N-terminal amino acid sequences of hCTLA4IgP and hCTLA4IgM were found to be identical (Table 2). Those sequences are the same as theoretical N-terminal amino acid sequence of hCTLA4Ig. In terms of monosaccharide composition, fucose, galactose, xylose and mannose of hCTLA4IgP were detected in the molar ratio of 35.42:3.07:25.31:3.98 (Table 3). However, N-acetylneuraminic acid (sialic acid) was not detected at all. Molar ratio of fucose, galactose, mannose and N-acetylneuraminic acid of hCTLA4IgM was 19.06:20.47:3.62:37.98, which is totally different from plant version of the same protein. Therefore, it was apparent that the differences in molecular size between hCTLA4IgP and hCTLA4IgM were originated from the differences in glycosylation patterns. 4. Discussion In bioreactors, 7-l and 15-l fed-batch cultures were performed with feeding concentrated amino acids without medium exchange. This method was proved to be an effective way for inducing the expression of target recombinant protein and maintaining cell mass in transgenic rice cell suspension cultures. In a study regarding a1-antitrypsin production in rice cell cultures (300-ml flask) with alternative carbon sources (mannitol, pyruvic acid, glyoxilic acid, glycerol, lactic acid), 2.3–3.4 times higher target protein production was observed [22]. Pyruvic acid provided the highest level of product formation. However, repeated use of the cells by exchanging pyruvic acid

Table 3 Monosaccharide composition of hCTLA4IgM and hCTLA4IgP. Monosaccharide

CHO-derived hCTLA4Ig (hCTLA4IgM)

Plant-derived hCTLA4Ig (hCTLA4IgP)

Glycoprotein (mg mg1)

Molar ratio (%)

Glycoprotein (mg mg1)

Molar ratio (%)

Fucose Galactose Xylose Mannose N-Acetylglucosamine D-Glucosamine N-Acetylneuraminic acid

0.00405 0.00435 – 0.00077 0.00273 0.00128 0.00807

19.06 20.47 – 3.62 12.85 6.02 37.98

0.00948 0.00082 0.00677 0.00107 0.00634 0.00228 –

35.42 3.07 25.31 3.98 23.70 8.52 –

Total carbohydrate content

0.02125

0.02675

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media without sugar every 48 h lowered the protein level due to the deterioration of cellular physiological conditions. These results indicated that the induced expression by changing media may be harmful to the cells in long-term cultivation. In this study, a newly developed fed-batch culture method was used with feeding 10 amino acids, instead of using two-stage culture with media change. This method resulted in an increase in the production of hCTLA4Ig up to 76.5 mg l1, which was 1.2 times higher than that in two-stage batch culture. These results may be especially helpful in scale-up because they do not need a media change process. One-step process also reduces the risk of contamination with easy induction in large-scale cultivation of transgenic rice cells for the production of biopharmaceutical proteins using RAmy3D promoter. Compared to the production of hCTLA4Ig in shake flasks (usually 30–39 mg l1), the expression level of hCTLA4Ig in bioreactors was approximately 1.5–1.8 times higher. This difference in expression levels may be related with cell viability. When the maximum cell growth was reached and the sugar was completely consumed, transgenic rice cells with inducible RAmy3D promoter began to produce hCTLA4Ig. However, cellular activity decreases and the cells rapidly enter the apoptotic state due to the depletion of sugar. In the bioreactors, however, supplementation with pure O2 may alleviate the decreased cell activity and help the maintenance of cell mass, which could lead to better hCTLA4Ig production. Depletion of sugar leads to cell lysis and death due to the decreased osmolality due to the lack of a carbon source. Therefore, it is important for sugar-depleted cells to maintain their viability during the induction period for the maximal production of target proteins [23]. Previous study also showed that the control of culture conditions and monitoring of cell viability, oxygen uptake rate, and pH during the growth and production phases were important for enhancing productivity in bioreactors [24]. This study also showed that the oxygen uptake rate was a sensitive measure of the physiological state and overall metabolic activity of the cultures. Taken together, these findings indicate that the maintenance of the dissolved oxygen levels at greater than 20% in 15-l bioreactor may account for the higher hCTLA4Ig concentration in our system. The decrease in hCTLA4Ig level after the maximum point was thought to be due to the degradation by several kinds of proteases that were secreted into the cultivation medium as a result of cell death [23]. Protease assay indicated that the total protease concentration in the bioreactor was 50–70 ng ml1 at 10–12 days after induction. Therefore, determination of an optimum recovery time is necessary to maximize productivity while minimizing protein degradation. Denatured SDS-PAGE analysis revealed that the size of hCTLA4IgP was approximately 50 kDa, which was smaller than that of hCTLA4IgM. Additionally, the diffused band patterns of the hCTLA4IgP and hCTLA4IgM were different. The differences in sizes and patterns may be originated from the differences in glycosylation pattern of animal and plant cells. Erythropoietin (EPO) produced in tobacco BY-2 culture was found to have lower molecular weight than that of EPO produced in CHO cells [5]. In addition, the molecular weight of recombinant EPO varies with animal host-cells, and it has been reported that this variation occurs by the differences in N-linked oligosaccharides [25]. Western blot analysis using anti-hCTLA4 antibody revealed that the molecular weight of hCTLA4IgM was slightly higher than that of hCTLA4IgP, whereas the deglycosylated forms of both proteins had identical molecular weights of 80 and 40 kDa in non-reducing and reducing conditions, respectively [26]. Therefore, the molecular weights of the deglycosylated forms were the same as that of theoretical value calculated from their amino acid contents. These

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results indicate that the structures of hCTLA4IgP and hCTLA4IgM are the same except for their glycan contents. Overall, the results of this study show that the fed-batch cultivation process using amino acid feeding may be useful for the scale-up of transgenic rice cell cultures in bioreactors because it is relatively easy and prevents contamination by removing troublesome medium exchange processes. Optimization of the oxygen uptake rate, osmotic control to maintain cell mass and the control of protein degradation by adding some stabilizing agents should be studied further to maximize the production of biopharmaceutical protein in transgenic rice cell suspension cultures. Acknowledgements This work was supported by a grant (A18-014) from the Next Generation New Technology Development Project of the Ministry of Commerce, Industry, and Energy of the Republic of Korea, and also by the Center for Advanced Bioseparation Technology, KOSEF. References [1] Doran PM. Foreign protein production in plant tissue cultures. Curr Opin Biotechnol 2000;11:199–204. [2] Hellwig S, Drossard J, Twyman RM, Fischer R. Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 2004;22:1415–22. [3] Sabharwal N, Icoz I, Saxena D, Stotzky G. Release of the recombinant proteins, human serum albumin, b-glucuronidase, glycoprotein B from human cytomegalovirus, and green flurescent protein, in root exudates from transgenic tobacco and their effects on microbes and enzymatic activities in soil. Plant Physiol Biochem 2007;45:464–9. [4] Bai JY, Zeng L, Hu YL, Li YF, Lin ZP, Shang SC, et al. Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plant. Biotechnol Lett 2007;29:2007–12. [5] Matsumoto S, Ikura K, Ueda M, Sasaki R. Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol 1995;27:1163–72. [6] Sardana R, Dudani AK, Tackaberry E, Alli Z, Porter S, Rowlandson K, et al. Biologically active human GM-CSF produced in the seeds of transgenic rice plants. Transgenic Res 2007;16:713–21. [7] Aviezer D, Almon-Brill E, Shaaltiel Y, Galili G, Chertkoff R, Hashmueli S, et al. Novel enzyme replacement therapy for Gaucher disease: on-going phase III clinical trial with recombinant human glucocerebrosidase expressed in plant cells. Mol Genet Metab 2008;93:S15. [8] Girard LS, Fabis MJ, Bastin M, Courtois D, Petiard V, Koprowski H. Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture. Biochem Biophys Res Commun 2006;345:602–7. [9] Hong S-Y, Kim T-G, Kwon TH, Jang Y-S, Yang M-S. Production of an anti-mouse MHC class II monoclonal antibody with biological activity in transgenic tobacco. Protein Exp Purif 2007;54:134–8. [10] Linsley PS, Brady W, Urnes M, Grosmaire LS, Damle NK, Ledbetter JA. CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med 1991;174:561–9. [11] Lui VCH, Tam PKH, Leung MYK, Lau JYB, Chan JKY, Chan VSF. Mammary glandspecific secretion of biologically active immunosuppressive agent cytotoxic-Tlymphocyte antigen 4 human immunoglobulin fusion protein (CTLA4Ig) in milk by transgenesis. J Immunol Meth 2003;277:171–83. [12] Levisetti MG, Padrid PA, Szot GL, Mittal N, Meehan SM, Wardrip CL, et al. Immunosuppressive effects of human CTLA4Ig in a non-human primate model of allogeneic pancreatic islet transplantation. J Immunol 1997; 159:5187–91. [13] Kirk AD, Harlan DM, Armstrong NN, Davis TA, Dong Y, Gray GS, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 1997;94:8789–94. [14] Kremer JM, Westhovens R, Leon M, Giorgio ED, Alten R, Steinfeld S, et al. Treatment of rheumatoid arthritis by selective inhibition of T-cell activation with fusion protein CTLA4Ig. N Engl J Med 2003;349:1907–15. [15] Lee S-J, Park C-I, Park M-Y, Jung H-S, Ryu W-S, Lim S-M, et al. Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Exp Purif 2007;51:293–302. [16] Terashima M, Ejiri Y, Kawamura M, Nakanishi S, Stoltz T, Chen L, et al. Production of functional human a1-antitrypsin by plant cell culture. Appl Microbiol Biotechnol 1999;52:516–23. [17] Huang J, Sutliff TD, Wu L, Nandi S, Benge K, Terashima M, et al. Expression and purification of functional human alpha-1-antitrypsin from cultured plants cells. Biotechnol Prog 2001;17:126–33. [18] Huang J, Wu L, Yalda D, Adkins Y, Kelleher SL, Crane M, et al. Expression of functional recombinant human lysozyme in transgenic rice cell culture. Transgenic Res 2002;11:229–39.

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