KR101189096B1 - Method for Stabilization of Recombinant Protein Derived from Plant Cells - Google Patents

Abstract

The present invention relates to a method for stabilizing a recombinant protein induced in plant cell culture, and more particularly, to a method for stabilizing a recombinant protein by adding an ionic liquid during plant cell culture. According to the present invention, by using an ionic liquid in the culture of transformed plant cells, it is possible to provide stability that can prevent degradation and degeneration of the protein compared to the production of the existing recombinant protein, thereby providing a stable and high Productivity can be secured. In addition, since the ionic liquid used does not exhibit cytotoxicity, when the recombinant protein is mass-produced through the present invention in a transgenic plant cell culture, the production process can be realized at low cost and high efficiency.

Plant cell culture, transformation, recombinant protein, ionic liquid, protein stability

Description

Method for Stabilization of Recombinant Protein Derived from Plant Cells}

The present invention relates to a method for stabilizing a recombinant protein induced in plant cell culture, and more particularly, to a method for stabilizing a recombinant protein by adding an ionic liquid during plant cell culture.

Protein drugs refer to drugs produced by living organisms or tissues or cells derived from living organisms. It can be largely classified into antibodies, therapeutic proteins, cell therapies, gene therapies, and vaccines. The biopharmaceutical market is growing rapidly around therapeutic antibodies, and many therapeutic proteins have been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA). Since Humulin (recombinant human insulin, rHI) from Eli Lilly was first approved in 1982, more than 500 biologics have been approved until recently.

Recent trends have driven efforts to use plant cells or plants for the production of therapeutic high value-added proteins in protein products, driven by success in developed biotech countries. As a simplified experimental system, it can be used for breeding by cell fusion and genetic manipulation, so it is possible to introduce various biotechnological techniques and, unlike animals, has a total totipotency, so that a complete plant can be obtained from transformed cells. It is possible to produce foreign proteins by culturing the cells of transformed plants as well as in-flight cell culture. In particular, a suspension culture of transformed plant cells with the development of the conversion plant, the expression system becomes possible, through which the protein production was reached for the treatment with high economic efficiency. Production of foreign proteins can be achieved not only through transformed plant cell culture but also through culturing with animal cells and microorganisms. Plant cell culture, however, has distinct advantages over them.

The production of therapeutic proteins through animal cell culture is possible to produce high bioactive substances due to the complete post-translation modification, but the slow growth rate of the cells themselves, the difficulty of mass cultivation due to the weakness of the cell, and the high concentration due to serum use There are problems such as the price of the medium, high possibility of infection such as viruses harmful to the human body, difficulty in isolation and purification. In addition, the production of therapeutic proteins using microbial culture, despite the advantages of fast growth rate, high cell concentration, ease of operation and simple medium composition, and the establishment of background knowledge from many studies, The lack of post-translational modification makes it difficult to produce proteins with sufficient physiological activity. It also has difficulties such as solubility due to the formation of inclusion bodies in large amounts.

The research using plants as a means to overcome the problems of the production of useful protein through the culture of animal cells and microorganisms has emerged. Production of therapeutic proteins through plant cell culture is becoming increasingly important as an alternative means to solve the problems of existing animal cells or microbial culture. The production of therapeutic proteins through plant cell culture has the advantage of post-translational modification, easy to isolate and purify, less likely to infect viruses harmful to the human body, low production cost and easy production on a large scale.

In the present invention, when producing recombinant hCTLA4Ig protein using transformed rice suspension cells, a recombinant protein is produced by using an α-amylase promotor system that operates when the sugar is depleted in the medium (Terashima M. et al., Biochem Eng J 4: 31-36, 1999; Trexler MM. et al., Biotechnol Prog 21: 321-328,2005). In the production of recombinant protein, the cell growth stage and protein production stage are separated according to the use of the α-amylase promoter system, which is operated at the point of depletion of sugar. In the prior art, after performing the two-step culturing according to the present system, after the growth stage of the cells in the growth stage in which the sugar is present, it is exchanged with the medium from which the sugar is removed to produce the recombinant protein.

    hCTLA4Ig is a fusion of CTLA-4 (CD152) and the Fc portion of human IgG, which is involved in immune responses by T cells. As a result, half-life in vivo was extended by forming dimeric hCTLA4Ig and effective purification by affinity chromatography was possible. An important T cell co-stimulatory signal for inducing immune responses is the binding between CD28 or CTLA-4 on the T cell surface and B7 receptors (CD80 and CD86) on the antigen presenting cells (APCs) surface. In this case, CTLA-4 plays a role in transmitting a signal that inhibits or reduces the activity of T cells, as opposed to CD28, and shows about 20-50 times higher affinity with the B7 receptor. HCTLA4Ig thus induces T cell specific immunosuppressive responses and acts as an immunosuppressive agent.

In December 2005, hCTLA4Ig from Bristol-Myers Squibb (BMS) is a drug used to treat rheumatoid arthritis, a type of autoimmune disease, approved by the US Food and Drug Administration (FDA) and sold under the name Orencia (abatacept) in 2008. Its sales are up to $ 440 billion. In the current rheumatoid arthritis market, the high-priced TNF-α inhibitors Amgen / Wyeth / Takeda's Enbrel and Abbott / Eisai's Humira are driving the market by increasing sales. It is also selling new drugs with new mechanisms such as BMS's Orencia, Biogen Idec / Genentech / Chugai / Zenyaku Industries' Rituxan and Roche's MabThera. As the demand for rheumatoid arthritis continues to grow, the market for these drugs is expected to expand gradually.

The target protein secreted out of the cell loses its stability by various mechanisms, and greatly affects productivity. Factors affecting the stability of these proteins include instability due to structural characteristics of the expressed protein secreted into the medium and instability due to aggregation. Specifically, physicochemical factors such as pH, temperature, shear stress, and the like the degradation factor for the desired protein by proteases (protease s) has had the greatest impact on productivity.

In the case of transformed rice cells using the α-amylase promoter and RAmy1A signal peptide, the expression of the target protein is strongly induced upon sugar depletion and secreted into the extracellular medium. However, the disadvantage of this system is that production is induced by sugar depletion, resulting in the synthesis of the desired protein in the absence of an adequate carbon source. These production conditions lead to decreased viability of cells and death, resulting in increased secretion of proteases, which are proteolytic agents into the medium, resulting in protein degradation, and degeneration of proteins due to physical and chemical stresses. Results in a decrease in stability and ultimately in productivity. Therefore, it is necessary to induce complete production with high activity through enhancement of the desired protein stability.

On the other hand, salts or salt compounds in which melting points are formed at a temperature of 100 ° C. or lower, unlike ionic salts or salt compounds composed of metal cations and nonmetal anions, such as salts, are usually melted at a high temperature of 800 ° C. or higher. In particular, the ionic liquid present as a liquid at room temperature is called a room temperature ionic liquid. This ionic liquid is composed of organic cations and anions, cations as dialkylimidazolium, alkylpyridinium, in this and the anion of quaternary ammonium, such as quaternary phosphonium is NO ₃ ^-, BF ₄ ^-, AlCl ^{_{4 -, Al 2 Cl 7 -}} , AcO -, PF 6 -, TfO - (trifluoromethanesufonate, CF 3 SO 3 -), Tf 2 N - (trifluoromethanesulfonylamide, (CF 3 SO 2) 2 N), CH 3 CH (OH and the like ^{_{-) CO 2 - (L-}} lacta te), SbF. Ionic liquids have excellent thermal stability due to ferroelectricity, non-combustibility, non-volatile, non-toxic, ionic conductivity, and can easily dissolve inorganic and organometallic compounds using high polarity and exist as liquids over a wide temperature range. Can be mentioned.

At present, ionic liquids having such properties are applied in a wide range of chemical fields such as catalysts, separations, and electrochemistry. For example, separation using ionic liquids, research on synthesis and application of ionic liquids as high activity catalysts containing organometallic compounds, application of ionic liquids to the development of optically active compound manufacturing technology, which is an advanced organic synthesis technology, The use of an ionic liquid as an immobilization catalyst has attracted attention for its high activity, reusability and environmental friendliness in the field of synthesis of compounds through transesterification of secondary alcohols. Such ionic liquids have been considered for use in the process of linking solvents, catalysts, separation media, nanochemistry, electrolytes, supercritical fluid-ionic liquids, etc. in homogeneous catalyst reactions, but are still used in biotechnology. Examples are extremely limited.

In this regard, the present inventors attempted to apply an ionic liquid to cell culture in terms of protein stabilization. Specifically, the present invention is intended to improve the conventional protein stability by applying to a recombinant plant cell culture system that produces a recombinant protein, and based on the research on the conventional ionic liquid applied to the stability problem of the recombinant protein cells It is intended to provide a method for stabilizing this in recombinant protein production through culture.

The present invention relates to a method for stabilizing a recombinant protein induced in plant cell culture, and more particularly, to a method for stabilizing a recombinant protein by adding an ionic liquid during plant cell culture.

The plant cell is preferably a genetically transformed plant cell, more preferably a plant cell transformed with an expression vector comprising a RAmy3D promoter. In the following examples, transgenic rice cells of transgenic plant cells were used as an example.

The ionic liquid is imidazolium having 1 to 4 carbon atoms of the alkyl group in which the cationic portion is substituted, and the anionic portion is methyl sulfate ([CH ₃ SO ₄ ]), ethyl sulfate ([CH ₃ CH ₂ SO). ₄ ]), trifluoromethanesulfonate ([CF ₃ SO ₃ ]), thiocyanate ([SCN]) and bis (trifluoromethylsulfonyl) imide ([(CF ₃ SO ₂ ) ₂ N] It may be any one selected from the group consisting of), more preferably the ionic liquid may be 1-butyl-2-methylimidazolium methyl sulfate.

The ionic liquid is preferably added in an amount of 0.0001 to 10% (v / v), and more preferably 0.001 to 1% (v / v).

The surfactant serves to reduce damage to plant cells by shear stress in the plant cell culture, and may be added together with the ionic liquid of the present invention. The type of the surfactant is not particularly limited, such as cationic surfactants, anionic surfactants, amphoteric surfactants, it is preferable to use Pluronic F-68 (PF-68) or Polyvinylpyrrolidone-10 (PVP-10). And, the addition content may be 0.001 to 10.0% (w / v), more preferred content may be 0.3% (w / v).

The recombinant protein is not particularly limited as long as it contains a disulfide bond as a protein induced in plant cell culture, but preferably may be a recombinant hCTLA4Ig protein.

According to the present invention, by using the ionic liquid in the culture of transformed plant cells, it is possible to provide stability that can prevent the degradation of the protein compared to the production of the existing recombinant protein, thereby providing a stable and high productivity of the recombinant protein It can be secured. In addition, since the ionic liquid used does not exhibit cytotoxicity, when the recombinant protein is mass-produced through the present invention in a transgenic plant cell culture, the production process can be realized at low cost and high efficiency.

Hereinafter, the present invention will be described in detail by examples. The following examples are only examples for describing the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Preparation of Transgenic Rice

The cell line was transformed into an expression vector transformed to produce recombinant hCTLA4Ig protein (see FIG. 7, Chonbuk National University, Boryeong Pharmaceutical Co., Ltd., Korea). Oryza sativa L. cv. It was used in suspension cells of Dongjin (Chonbuk National University, Boryeong Pharmaceutical, Korea). The transformed expression vector was introduced into the vector using a particle-bombardment method (Lee, SJ et al., Protein Expres. Purif. 51: 293-302, 2007). It was inserted and transformed, and the RAmy3D induction promoter is designed to operate in the absence of sucrose.

2. Transgenic Rice Cell Culture

Transgenic rice cell cultures were cultured in AA medium to which 3g / L transgenic rice cell line prepared in 1. was added 30g / L sucrose. The pH of the medium was adjusted to 5.8 and then dispensed into 500 mL Erlenmeyer flask to autoclave. Suspension culture was maintained at 120 rpm, 28 ℃, dark conditions and passaged at 9 days intervals.

3. Production of Recombinant hCTLA4Ig Protein through Transgenic Rice Cell Culture

Since the production of recombinant hCTLA4Ig protein in transgenic rice cells is performed by the α-amylase promoter expressed at the time of sugar depletion, the recombinant hCTLA4Ig protein is converted to a production medium without sucrose from the basic AA growth medium containing sucrose. Induced production. Protein-induced transgenic rice cell culture was also maintained in the same conditions as the cell culture of 2 .

4. Application of Cultured Ionic Liquids to the Phase

When producing recombinant hCTLA4Ig protein using the existing AA medium, since the expression of the α-amylase promoter is activated by sugar depletion, sugar was removed from the AA base medium and used as an induction medium, that is, a production medium. Transformed rice cells secrete proteins into the medium during the production of recombinant hCTLA4Ig protein.The cells are cultured in a production medium and recovered from the production point of the recombinant hCTLA4Ig protein. The degree of stabilization of the hCTLA4Ig protein was measured.

The ionic liquid used is 1-butyl-2-methylimidazolium methylsulfate ([Bmin] [MS]), and 100 mM Tris-HCl (pH 8.1), 1 mM EDTA, 3 mM reduced glutathione for use. (GSH) and 0.3 mM oxidized glutathione (GSSG) were used in a buffer. Dilute the ionic liquid prepared by the simple dilution method 10 times and 100 times by concentration to 1% (v / v) in the culture solution recovered after the culture. After the sampling (sampling) at 4 hours intervals at room temperature, the recombinant hCTLA4Ig protein was quantified to confirm the stability of the recombinant hCTLA4Ig protein in the culture medium through the ionic liquid, and the results are shown in FIG. 1.

According to Figure 1, the amount of recombinant hCTLA4Ig protein in the culture medium recovered after incubation in the existing AA medium is confirmed to be degraded due to physical factors such as the effects of protease or other temperature in the culture medium over time On the other hand, it can be seen that the amount of recombinant hCTLA4Ig protein is maintained in all cases when the ionic liquid is added by concentration.

5. Quantification of Recombinant hCTLA4Ig Protein

Quantification of the recombinant hCTLA4Ig protein secreted into the medium and the recombinant hCTLA4Ig protein after treatment with the ionic liquid was measured using an enzyme-linked immunosorbent assay (ELISA) assay. In order to perform Sandwich ELISA, goat anti-human IgG (Fc) (KPL) was used as a first antibody, and peroxidase-labeled goat anti-human IgG (g) (KPL) was used as a second antibody. ) Was used. The substrate was used as an ABTS peroxidase substrate (KPL), and the color development was measured by absorbance at 405 nm. ImmunoPure human IgG (Pierce) was used as a standard. The composition and detailed analysis method of the buffer used for the analysis are shown in the following [Table 1] and [Table 2].

Table 1 Composition of ELISA buffer

Buffer Composition Coating buffer 1.59 g / L Na ₂ CO ₃
2.93 g / L NaHCO ₃
pH 9.6 PBS 8.0 g / L NaCl
0.2 g / L KH ₂ PO ₄
1.15 g / L Na ₂ HPO ₄
0.2 g / L KCl
pH 7.4 Blocking solution         2% FBS in PBS Washing buffer (PBST)         0.05% tween20 in PBS Assay buffer         2% FBS in PBST

[Table 2] ELISA assay method

Step
Reagent
Dilution Loading volume
(μL / well)
Reaction time
Coating 1st antibody: Goat anti-human IgG (Fc) (KPL) 1: 1000
(Ab: coating buffer)
100 Overnight (4 ℃) or
2 h (37 ° C) Blocking Blocking solution 200 2 h
(room temp.) Washing 3 times with washing buffer (PBST) Standard /
sample loading
Standard: Human IgG (Pierce) 1 / 2-fold series dilution
100
1 h (37 ° C) Washing 4 times with washing buffer (PBST)
Detection 2nd antibody: Peroxidase-labled goat anti-human IgG (g) (KPL)
1: 5000
(Ab: assay buffer)
100
1 h (37 ° C) Washing 5 times with washing buffer (PBST)
Color development
Substrate:
ABTS peroxidase (KPL)
100 Until O.D of the first standard well reach to 1.0 Reading Read with microplate reader at 405nm

6. Recombinant protein stabilization method using ionic liquids in culture

The ionic liquid was transferred to a production medium without sucrose for application in transgenic plant cell culture to induce recombinant hCTLA4Ig protein production. Protein-induced cell culture was also maintained in the same conditions as the cell culture conditions of growth. Based on studies that confirmed the toxicity to cells when ionic liquids were added during cell culture (Lee, SM et al., Korean. J. Chem. Eng., 22: 4, 687-690, 2005) In plant cell culture, ionic liquid was added to confirm the toxicity on the cells and the role on the stability of the protein.

The ionic liquid added to the production medium was the same as the composition treated in the recovered culture, and was applied during incubation after sterilization with a 0.22 μm filter for application to the culture. After treating the ionic liquid prepared by the simple dilution method by concentration, the effect of each concentration was confirmed, and the stability effect according to the addition time was confirmed. In addition, we tried to observe the effect on the protein stability by treating the suspension cell culture with a combination of ionic liquids and surfactants known to reduce the damage of cells caused by shear stress.

In order to confirm the effect of ionic liquid on the stability of hCTLA4Ig, 1% of ionic liquid and PF-68, a surfactant, were added at the initial concentration of 0.3%. Based on the results of day 12, which showed the maximum required protein stabilizer to increase productivity during time-course observation, it was confirmed that the productivity of 1% ionic liquid added compared to the control was increased by 50%. It was confirmed that the productivity of the ionic liquid and 0.3% PF-68 added was improved by more than 80%, the results are shown in FIG. After confirming the effect of the ionic liquid, 0.01, 0.1, 0% of the ionic liquid was added to each concentration at the beginning of the culture in order to confirm the effect of the concentration of the ionic liquid. As a result of quantifying hCTLA4Ig by recovering the culture solution on the 8th day, when the ionic liquid of 0.01% was added as shown in FIG. 3, the productivity was increased by more than 270% compared to the control.

After confirming the optimum concentration of the ionic liquid, we tried to confirm the maximum effect through a combination experiment with the addition of a surfactant. The effect was confirmed after the addition of surfactants PVP10 and PF-68 0.3% to the addition of 0.01% ionic liquid showed the most enhanced productivity. 4 and 5 show the results of quantifying hCTLA4Ig by recovering the culture solution on the 8th day after the addition of the ionic liquid and the surfactant on the 5th day after the initial culture and the start of the culture. According to FIG. 4, when the ionic liquid and the surfactant were added at the beginning of the culture, the productivity increased by more than 150% compared to the control when 0.01% of the ionic liquid and 0.3% PF-68 were added. there was. According to FIG. 5, when 0.01% of the ionic liquid and 0.3% PF-68 were added on the 5th day after the start of the culture, the productivity was increased by 400% or more compared to the control. Through this, the production of therapeutic protein hCTLA4Ig through transgenic rice cell culture was confirmed to increase productivity through protein stabilization through the addition of ionic liquid, especially 0.01% ionic liquid and 0.3% PF-68. When added to the 5th day after the start of the culture it could be confirmed that it shows the most enhanced productivity effect.

7. The effect of ionic liquids on cells

To determine the effect of ionic liquids on cells by observing cell viability, dry cell weight, and pH in a time-course in culture with a combination of ionic liquids and surfactants It was.

Cell viability was measured by relative cell viability by TTC (2,3,5-triphenyltetrazolium chloride) reduction method. 1.6 ml of a 1.5% TTC solution (0.05 M potassium phosphate solution, pH 9.0) was added to the cells of 0.1 gFCW and reacted at 20 ° C. for 24 hours. Thereafter, the TTC solution was removed, and 1 mL of 95% ethanol was added thereto, followed by reaction at 60 ° C. for 30 minutes, and red formazan was extracted. After centrifugation at 15,000 rpm, the supernatant was measured for absorbance at 485 nm. As a blank, 95% ethanol was used.

To measure cell growth, fresh cell weight (FCW) and dry cell weight (DCW) were measured. Suspension cells incubated in 100-mL Erlenmeyer flasks were prepared using Whatman No. 1 was separated from the culture medium of the sample on the filter paper. After washing 2-3 times with the same amount of medium and distilled water, water was removed using a vacuum pump and the cells were transferred to a weighting dish to measure FCW. After FCW measurement, DCW was measured by drying in a dry oven at 60 ° C. for 48 hours until a constant weight was obtained.

In addition, the pH of the culture solution was measured using a pH meter (Thermo, Electron Co.). After the pH meter was turned on, the pH probe was washed with distilled water and then gently wiped with a soft tissue. After performing the calibration, the pH probe was added to the solution to be measured, waited until the value became stable, and the pH value was entered. Then, washed with distilled water and immersed in the storage solution and stored so as not to dry the probe.

The cell viability, dry cell weight, and pH were observed in time-course, and the results are shown in FIG. 6 (relative cell viability), FIG. 7 (dry cell weight), and FIG. 8 (pH). As shown. 6 to 8, it can be seen that the cell viability is reduced in a similar pattern to the control (control), thereby confirming that the addition of the ionic liquid is harmless to the cells.

As described above, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. The scope of the present invention is shown by the claims to be described later rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. Should be.

Figure 1 shows the change in the amount of recombinant hCTLA4Ig protein over time when the ionic liquid according to the present invention is added by concentration during the recombinant hCTLA4Ig protein production culture.

Figure 2 shows the amount of recombinant hCTLA4Ig protein when the ionic liquid and surfactant PF-68 according to the present invention are added together in the culture of recombinant hCTLA4Ig protein.

Figure 3 shows the amount of recombinant hCTLA4Ig protein according to the concentration of the ionic liquid according to the present invention added during the recombinant hCTLA4Ig protein production culture.

Figure 4 shows the amount of recombinant hCTLA4Ig protein when the ionic liquid and the surfactants PVP10 and PF-68 according to the present invention are added at the initial stage of the culture during the culture of the recombinant hCTLA4Ig protein.

Figure 5 shows the amount of recombinant hCTLA4Ig protein when the ionic liquid and the surfactants PVP10 and PF-68 according to the present invention were added 5 days after the start of the culture of the recombinant hCTLA4Ig protein.

Figure 6 shows the relative cell viability in time-course after the addition of the ionic liquid and surfactant (PF-68) according to the invention during the culture of the recombinant hCTLA4Ig protein at the beginning of the culture.

Figure 7 shows the change in dry cell weight in time-course after the addition of the ionic liquid and surfactant (PF-68) according to the invention during the culture of recombinant hCTLA4Ig protein at the start of the culture step.

Figure 8 shows the change in pH in time-course after the addition of the ionic liquid and surfactant (PF-68) according to the invention during the culture of the recombinant hCTLA4Ig protein at the beginning of the culture.

Claims (13)

Method of stabilizing recombinant protein by the addition of 1-butyl-2-methylimidazolium methyl sulfate in plant cell culture. The method of claim 1, The plant cell is characterized in that the transformed plant cell. 3. The method of claim 2, The transformed plant cell is characterized in that the transformed rice cells. 3. The method of claim 2, The transformed plant cell is characterized in that transformed with an expression vector comprising a RAmy3D promoter. delete delete delete The method of claim 1, Wherein said 1-butyl-2-methylimidazolium methylsulfate is added in an amount of 0.0001 to 10% (v / v). The method of claim 1, Wherein said 1-butyl-2-methylimidazolium methylsulfate is added with a surfactant. 10. The method of claim 9, The surfactant is any one of pluronic F-68 or polyvinylpyrrolidone-10. 10. The method of claim 9, The surfactant is added in an amount of 0.001 to 10.0% (w / v). The method of claim 1, The recombinant protein is a protein comprising a disulfide bond. 13. The method of claim 12, The protein comprising a disulfide bond is a recombinant hCTLA4Ig protein.

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