KR101550677B1 - Highly efficient production methods of fusion proteins for antibody treatment using catechin - Google Patents

Abstract

The present invention relates to a highly efficient production method of therapeutic antibody fusion protein using catechin. A pAD11 expression vector expressing a TNFR-Fc fusion protein was prepared, and cell lines having high productivity were transformed into a cell line, , The content of misfolded protein and high molecular weight aggregate can be reduced to thereby produce TNFR-Fc fusion protein with high efficiency.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for efficiently producing an antibody fusion protein using catechin,

The present invention relates to a method for producing a therapeutic antibody fusion protein TNFR-Fc fusion protein with high efficiency using catechin.

Enbrel® (etanercept), developed by Immunex, is a TNF inhibitor, a Fc part of the TNF receptor and IgG (TNFR-Fc) fusion protein, and was approved by the US FDA in 1998. Enbrel is the world's first indications for rheumatoid arthritis (4 to 17 years old) in addition to the efficacy of rheumatoid arthritis treatment. In recent years, the approval of psoriasis indication has led to a steady increase in the market, have.

In order to produce TNFR-Fc fusion protein with high efficiency, various methods have been studied to induce an extension of culture period, an increase of living cell concentration and an increase of cell unit productivity. It is also important to produce the protein with high efficiency, but the quality of the produced protein is also important, and a lot of researches about it are being done as well. Glycosylation, misfolded protein, and high molecular weight aggregates can be a problem in terms of quality when producing TNFR-Fc fusion proteins, which are relatively complex substances. To solve these problems, cell culture conditions and methods using culture supplements have been studied. In the case of misfolded proteins and high molecular weight aggregates, the higher the ratio, the lower the productivity as a whole, the lower the quality, the effect on the efficacy, and side effects may appear.

Methods have been attempted to reduce misfolded proteins, high molecular weight aggregate ratios, during cell culture. One approach is to culture at low temperatures or use appropriate additives. Studies have shown that TNFR-Fc fusion proteins can reduce protein misfolding by culturing at low temperatures (U.S. Patent Publication No. 20080076155, Immunex corporation) There is also a study to reduce misfolding and aggregation of proteins by culturing methods using low temperature and low pH conditions, respectively (US Patent Publication No. 20080269132, Wyeth). In addition, studies have revealed that the addition of copper and glutamate separately or simultaneously at the time of culturing the TNFR-Fc fusion protein reduces the misfolding and aggregation of the protein, respectively (see US Patent Publication 20090068705, Wyeth). In addition, a method of reducing a high amount of aggregates such as misfolded proteins by using anti-aging compounds such as carnosine, which is an antioxidant, in cell culture has been disclosed (U.S. Patent Publication No. 20080274507, Wyeth). It can be seen that there has been an attempt to solve the misfolding or aggregation of proteins by changing the culture conditions or adding the additives during the culture. If only one method is used and the desired ratio is reached, the problem will be solved. However, if one of the methods has already been used and the desired misfolded protein or high proportion of high molecular weight aggregate is generated, There are also attempts to solve this problem.

Oxidative stress is an imbalance between biological systems that detoxify reactive oxygen species (ROS) and reactive intermediates. In case of a failure in the normal redox state of a cell, it shows a toxic effect, which damages all elements of cells such as protein, lipid and DNA. Antioxidants are substances that inhibit this oxidation. As shown in many studies, if an antioxidant is added to a cell culture, the cell will stop the stress caused by the active oxygen, so that the survival of the cell will be further maintained. . N-acetylcysteine (NAC), flavonoids (quercetin, catechin), alpha-tocopherol (alpha-tocopherol), and the like were added to an oxidative model of endometrial cells (RL95) Tocopherol has been used to increase the survival rate of oxidized cells with antioxidant effects of NAC and quercetin (Journal of Reproductive Immunology 75 (2007) 1-10, S. Estany). Alpha-tocopherol, a representative antioxidant, has also been shown to protect cells against oxidative stress in other studies (Toxicology 202 (2004) 227-235, Kalender, S.). Also, by using Q-media supplemented with Coenzyme-Q10 (coenzyme-Q10), which is known as a strong antioxidant in the respiratory chain, in a fed batch culture of YB2 / 0 cell line, (Cytotechnology 63 (2011) 163-170, Konno Y), which has been shown to increase production rates and to increase production even when this medium is applied to Chinese hamster ovary (CHO) and NS0 cell lines. (1999) 85-91, Chang KH), pyrrolidine dithiocarbamate (Biotechnol. Bioeng. 65 (1999) 298-305, Mastrangelo; Free Radic. Oncogene 17 (1998) 2515-2524, Higuchi, M.), ascorbic acid (J. Immunol. 148 (1992) 1817-1821, Ramakrishman , N; Toxicol. Appl. Pharmacol. 146 (1997) 270-280, Blankenship, LJ) were not cytotoxic and inhibited apoptosis and were also economical. As shown in various studies, it was confirmed that the antioxidant enhances cell viability and protein production rate in cell culture. However, there has been no research on the effect of antioxidants on the reduction of misfolded proteins or high molecular weight aggregates in proteins produced by cell culture.

Catechin belongs to the flavonoid group flavan-3-ols (flavan-3-ols) and is a kind of polyphenols. Catechin compounds include EGCG (-) - epigallocatechin-3-gallate, EGC [(-) - epigallocatechin], ECG [epicatechin-3-gallate) (1992) 334-350, Graham, HN; J. Cancer Res. 89 (1998) 254-261, Nakachi, K. J. Pharmacol. 606 (2009) 172-179, Zhang, Y.). Similar studies have shown the potential for cervical cancer suppression based on the use of catechin hydrate in human cervical cancer cells (Food and Chemical Toxicology 49 (2011) 3281-3286, Amal A. Al- Hazzani). In addition, there have been studies to confirm that cell growth of WI-38 human diploid fibroblasts is increased by experiments using polyphenols and catechins having radical scavenging activity ( J Nutr Sci Vitaminol 45 (1999) 263-273, Okada M). However, there is no known effect on misfolded proteins or high molecular weight aggregates.

Therefore, the inventors of the present invention studied a method for producing TNFR-Fc fusion protein with high efficiency. A pAD11 expression vector expressing a TNFR-Fc fusion protein was prepared and transformed into a cell line to select a highly efficient cell line , And that the content of misfolded proteins and high molecular weight aggregates decreased by catechin concentration by the addition of catechin, thereby completing the present invention.

It is an object of the present invention to provide a method for efficiently producing a TNFR-Fc fusion protein in which the content of misfolded protein and high molecular weight aggregate is reduced by adding catechin.

The present invention provides a method for producing a high efficiency TNFR-Fc fusion protein comprising the steps of:

1) preparing an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein;

2) transforming the expression vector of step 1) into a cell line;

3) adding catechin to the transformed cell line of step 2); And

4) separating the TNFR-Fc fusion protein from the cell culture medium of step 3).

The present invention provides a method for high-throughput production of a TNFR-Fc fusion protein with reduced misfolded protein and polymer aggregate ratios comprising the steps of:

1) preparing an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein represented by SEQ ID NO: 1;

2) transforming the expression vector of step 1) into a cell line;

3) culturing the transformed cell line of step 2) by adding catechin at a concentration of 50 to 400 uM once to four times;

4) adding CuCl ₂ to the culture medium of step 3); And

5) isolating the TNFR-Fc fusion protein from the cell line culture of step 4).

The high efficiency production method of TNFR-Fc fusion protein using the catechin of the present invention significantly reduces the content of misfolded protein and high molecular weight aggregate, thereby increasing TNFR-Fc fusion protein with high efficiency Can be usefully used as a production method for producing the polylactic acid.

1 is a diagram showing the structure of an expression vector expressing a TNFR-Fc fusion protein.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing a high efficiency TNFR-Fc fusion protein comprising the steps of:

1) preparing an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein;

2) transforming the expression vector of step 1) into a cell line;

3) adding catechin to the transformed cell line of step 2); And

4) separating the TNFR-Fc fusion protein from the cell culture medium of step 3).

The therapeutic antibody fusion protein of the present invention is preferably fused with the Fc portion of the TNF receptor and IgG, and is most preferably composed of the nucleotide sequence of SEQ ID NO: 1 of the present invention, but is not limited thereto. In addition, the TNF-Fc fusion protein can be used for treatment of rheumatoid arthritis, pediatric rheumatoid arthritis, psoriasis and the like, but is not limited thereto.

In addition, the fusion protein generally has all or substantially all of the target peptide linked at the N- or C-terminus to all or a portion of the second polypeptide or protein, as well as a target residue, for example, a water soluble receptor fragment Or immunoglobulin chains, Fc fragments such as IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, have. Thus, the fusion protein may comprise the extracellular domain of the receptor and may be fused to a human immunoglobulin Fc chain (e. G., Human IgG1 or IgG4, or a mutant form thereof), but is not limited thereto.

In addition, the receptor is a transmembrane glycoprotein that functions by recognizing an extracellular cytotoxic ligand, often with a protein kinase domain other than the ligand cognitive domain. The protein kinase domain phosphorylates a molecule in a target cell upon binding to a ligand, thereby initiating a cell transduction pathway and causing development or metabolic change in the cell. In addition, the tumor necrosis factor inhibitor comprises a water soluble TNF receptor and comprises a water soluble TNF receptor fused to any part of the immunoglobulin protein, including the Fc region of the immunoglobulin. The TNF inhibitor of the present invention is preferably a water soluble form of TNF receptor I and TNF receptor II, more preferably a water soluble TNF binding protein.

According to the preferred embodiment of the present invention, the gene fragment encoding the therapeutic antibody fusion protein of step 1) is most preferably synthesized by the TOP gene technology. However, if it is a method widely known in the art, Can be used.

In the above production method, the expression vector of step 1) is preferably prepared as a backbone of the RcCMV vector, and the neomycin resistant site cleavage in the RcCMV vector and the 5 'and 3' cleavage of the CMV promoter It is more preferable to add DHFR (dihydrofolate reductase) gene and gIVS, but not always limited thereto.

In the above production method, the cell line of step 2) is most preferably a CHO cell line lacking the DHFR gene, but it is not limited thereto. According to a preferred embodiment of the present invention, Thymine-based transfection methods have been used, but other transfection methods can be used as long as they are well known in the art.

In the above production method, the transformed cell line of step 3) was obtained by selecting a primary cell line by measuring the concentration of TNFR-Fc fusion protein by ELISA and MTT method, and selecting the cell line by the above method as a DHFR inhibitor, methotrexate The secondary cell line was selected by a method of confirming the productivity of cells according to the concentration of the cell line. However, the present invention is not limited thereto. In addition, it is most preferable that the second screened cell line is used for culturing the final screened cell line by repeating the first screening method twice. However, the present invention is not limited thereto.

In the above production method, the addition of catechin in step 3) is most preferably added to the producer during the cell culture process, but the number of addition is preferably 1 to 4 times according to the preferred embodiment of the present invention But is not limited thereto. In addition, it is most preferable that the catechin is added within 0 to 3 days after the culture, and the amount of the catechin is preferably added at a concentration of 50 to 400 uM at one time, and it is more preferable that the catechin is added at a concentration of 300 uM or less Preferably, it is most preferably added at a concentration of 150 uM.

In the production method described above, the culturing method of step 3) is not particularly limited, and a method in which the protein production is performed in a manner such that the medium is supplemented at least once in an apparatus such as a bioreactor including a cell culture medium a batch culture in which a protein is produced in a cell culture medium originally supplied without supplementing the culture medium or a perfusion culture in which a cell culture medium is periodically or continuously added and harvested, Any cell culture method known in the art may be used.

Also, the cell culture medium means a solution or substance containing nutrients to maintain the cell survival under conditions that allow the cells to grow to produce the desired protein. The essential amino acids, nonessential amino acids, Vitamins, energy sources, lipids and trace elements, and the like, all of which are provided at the time when the production of the target protein is initiated or after the initiation of cell culture. The cell culture medium used for culturing the therapeutic protein containing the antibody is well known to those skilled in the field of cell culture and the cell culture medium normally provided at the growing stage, . The cell culture medium according to the present invention may contain, in addition to catechin, additional components required for the purpose of increasing protein activity, increasing cell specific productivity, increasing cell viability, increasing integrated viable cell density, and decreasing the accumulation of high molecular weight aggregates .

Also, in the cell culture, the cells not only one is most preferred that the inoculation with 5 × 10 ⁶ cells / ml Thus, also not limited to one or desirable to add an additional CuCl ₂ to, CuCl ₂ added to the can According to a preferred embodiment of the present invention, it is most preferable to add at 10 uM on the third day after the incubation, but the present invention is not limited thereto.

In a specific embodiment of the present invention, we constructed a pAD11-pro expression vector that expresses the TNFR-Fc fusion protein with the RcCMV vector as a framework (see Figure 1).

The expression vector was transfected into a CHO cell line to select primary and secondary cell lines, and finally the cell line with the highest productivity was selected and a cell line adapted to the suspension culture was obtained.

Using the obtained cell lines, it was confirmed that as the amount of catechin was increased, the area of misfolded proteins and high molecular weight aggregates of the TNFR-Fc fusion protein decreased (see Table 1).

In addition, it was confirmed that the ratio of misfolded protein and high molecular weight aggregates was decreased regardless of the time point at which catechin was added (see Table 2).

In addition, it was confirmed that the formation rate of misfolded proteins and high molecular weight aggregates was suppressed when catechin was added even in a bioreactor culture process of large capacity culture (see Table 3).

Therefore, the production method of the therapeutic antibody fusion protein with the catechin of the present invention can be effectively used for the high-efficiency production method of the TNFR-Fc fusion protein by confirming that the formation rate of the misfolded protein and the high molecular weight aggregate is inhibited Do.

The present invention provides a highly efficient method of producing a TNFR-Fc fusion protein with reduced misfolded protein and polymer aggregate ratios comprising the steps of:

1) preparing an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein represented by SEQ ID NO: 1;

2) transforming the expression vector of step 1) into a cell line;

3) culturing the transformed cell line of step 2) by adding catechin at a concentration of 50 to 400 uM once to four times;

4) adding CuCl ₂ to the culture medium of step 3); And

5) isolating the TNFR-Fc fusion protein from the cell line culture of step 4).

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples illustrate the present invention in detail, and the present invention is not limited by the following examples.

< Example  1> TNFR - Fc  Preparation of fusion protein expressing cells

<1-1> Preparation of Expression Vector

An expression vector expressing the TNFR-Fc fusion protein was prepared by vector pAD11 expressing the TNFR-Fc fusion protein using the RcCMV (Invitrogen, USA) vector as a backbone.

Specifically, the neomycin resistant region of the RcCMV vector was removed by XhoI enzyme, gIVS was added to the 3 'end of the CMV promoter, and the mouse DHFR (dihydrofolate reductase) gene (Pubmed, NM 010049) Was added to the 5 ' end of the promoter. In addition, codon-optimized nucleic acid molecules encoding the amino acid sequence of TNFR-Fc were synthesized by TOP gene technology (www.topgenetech.com). In order to insert the TNFR-Fc gene (SEQ ID NO: 1) into the pAD11 expression vector constructed above, EcoRI at the 5 'end and XbaI restriction site at the 3' end were generated. Using these enzymes, the pAD11 expression vector Subcloning, which was named pAD11-pro.

As a result, as shown in Fig. 1, it contains the CMV promoter, the poly A sequence derived from the gestational growth hormone, the gIVS (globin intervening sequence) derived from the rabbit beta globin and other factors, and TNFR-Fc fusion PAD11-pro expression vector expressing the protein was prepared (Fig. 1).

<1-2> Introduction and selection of cell lines

The pAD11-pro vector prepared in Example <1-1> was introduced into a CHO cell line (provided by Professor Lawrence Chasin of Columbia University) to select a cell line expressing the TNFR-Fc fusion protein.

Specifically, the CHO (DG44) cell line in which the DHFR gene was deleted is a MEM-alpha (DG44) cell line containing a fetal calf serum and HT [hypoxanthine and thymidine] on a 6- MEM-alpha) medium (Sigma, USA) at a concentration of 1 × 10 ⁵ cells / mL. After 48 hours of culture, it was confirmed whether the cells were evenly distributed when they were about 50%. Using the lipofectamine (Gibco, USA), pAD11-pro expression vector Were introduced into the cells. The expression vector was introduced into the cells. After 24 hours, the medium was changed to MEM-alpha medium in which the HT supplement was removed for primary selection, and the cell line into which the expression vector was introduced was added to a 96- Lt; / RTI &gt; cells, and cultured for 3 to 4 days while exchanging the medium. Growth colonies began to form on HT-free medium from day 7 after culturing. After about 3 weeks of selection, the cells were subcultured and divided into two 96-well plates. One of them was colonies The concentration of TNFR-Fc fusion protein in the culture supernatant was measured by ELISA and the other was the MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide: Thiazolyl blue] analysis was performed. The above two results were calculated and clones having relatively high expression were firstly selected.

For the selection of the secondary cell line, the primary selected clones were treated with increasing concentrations of methotrexate (MTX, methotrexate) from 20 nM to 100 nM in MEM-alpha medium supplemented with 10% dFBS to obtain TNFR -Fc fusion protein were simultaneously amplified, and the clones were adapted to MTX, cultured for each MTX concentration, and the clones having high productivity were secondly screened.

<1-3> Cell culture

The culture conditions of the selected cells were established by the method of Example <1-2>.

Specifically, the second-selected clone cell line in Example <1-2> was thawed at 37 ° C in a thermostat and then cultured in MEM-alpha medium (containing 10% bovine serum and 50 nM MTX) in a T-175 flask Cells were prepared. If fully grown in a T-175 flask, the culture medium was removed from the vessel and washed with PBS, and 5 mL of 0.25% trypsin solution was added to allow the attached cells to drop. The separated cells were collected using MEM-Alpha medium, centrifuged, the supernatant was removed and the cells were resuspended in MEM-alpha medium (containing 10% bovine serum and 50 nM MTX) and then transferred to a T-175 flask Lt; / RTI &gt; The cells were subcultured three times, and the cultured cells were separated in a flask with trypsin solution and then suspended in 20 mL of serum-free medium (EX-CELL CHO DHFR-medium, Sigma, USA). The suspended cells were collected by centrifugation again, and were subjected to the same procedure twice to remove MEM-alpha remaining in the cells. Finally, the collected cells were suspended in a serum-free medium, adjusted to an initial cell concentration of 5 × 10 ⁵ cells / mL to make a solution containing 30 mL of cells, and then inoculated into a 125 mL suspension culture flask.

The suspension culture was carried out at 37 ° C in a 5% CO ₂ incubator with stirring at 120 rpm. The serum-free medium was exchanged every 3 days. When the cell concentration was 1 × 10 ⁶ cells / mL or more, the cell concentration was adjusted to 5 × 10 ⁵ cells / mL. In the above method, more than 90% of the cells were allowed to grow normally by over 30 passages, and the clone fully adapted to the suspension culture was obtained by repeating the first cell line screening method of Example <1-2> Were finally selected.

< Example  2> catechin ( catechin ) Misfolded  Protein and High molecular weight  Inhibition of aggregate formation

We confirmed that the addition of catechin affects the inhibition of TNFR-Fc fusion protein misfolded protein and high molecular weight aggregate formation.

Specifically, the cell line selected in Example 1 was cultured in a serum-free medium supplemented with 4 mM glutamine and 50 nM methotrexate (MTX) in a shake flask at 37 ° C and 120 0.0 &gt; rpm. &lt; / RTI &gt; When the cell concentration of the shake culture reached an appropriate level, the culture medium was centrifuged at 1000 x g to remove the used culture medium, suspended in serum-free medium, and cultured in 5 shake flasks at a concentration of 5 x ¹⁰⁶ cells / mL Were inoculated and cultured. The cultivation was carried out in a shaker incubator at 30 ° C and 120 rpm in an oil-bed culture mode, catechin was added at a concentration difference of 0 to 300 μM on the third day of culturing, and 10 μM CuCl ₂ was further added on the third day of culturing . On the 15th day, the supernatant of the culture was subjected to hydrophobic interaction chromatography (HIC). Specifically, TSK butyl-NPR (0.46 × 3.5 cm) column was charged with 0.1 M sodium phosphate phosphate buffer, pH 7.0) at a flow rate of 1.0 ml / min for 50 min. The concentration gradient of ammonium sulfate was changed from 1.8 M to 0 M and eluted. In this case, the main peak to be separated is referred to as peak 2 (HIC peak 2), and the small peak after the peak is referred to as peak 3 (HIC peak 3). In the peak 2, the TNFR- In peak 3, misfolded proteins or high molecular weight aggregates were eluted and separated. And the% of the peak was calculated by the area of the peak 2 and the peak 3.

As a result, as shown in Table 1, it was confirmed that as the amount of catechin increases, the area of the misfolded protein and the high molecular weight aggregate (peak 3) of the TNFR-Fc fusion protein decreases, And decreased the content of high molecular weight aggregates (Table 1).

flask Catechin (uM) HIC peak 2 (%) HIC peak 3 (%) One 0 76.4 23.4 2 50 78.5 21.5 3 100 79.3 19.7 4 150 82.0 18.0 5 300 85.6 14.4

< Example  3> When the catechin was added Misfolded  Protein and High molecular weight  Change in aggregate ratio

The percentage of misfolded proteins and high molecular weight aggregates was determined at the time of catechin addition.

Specifically, serum-free medium in which glutamine and methotrexate were removed and 5 mM N-acetyl-D-mannosamine was added, The cell line was inoculated at a cell concentration of 5 × 10 ⁶ cells / ml and cultured at 30 ° C. with shaking at 100 rpm. 150 μM of catechin was added on day 0 and day 3 of the shaking culture, and on day 15, a supernatant sample was extracted and the ratio of the productivity, misfolded protein and high molecular weight aggregate was confirmed using HIC-HPLC. On the third day in the culture process, 10 uM CuCl ₂ was added to all the flasks.

As a result, it was confirmed that the ratio of about 5% misfolded proteins and high molecular weight aggregates was decreased in both of the flasks to which catechin was added on day 0 and day 3 as shown in Table 2, Fold decrease in the proportion of misfolded proteins and high molecular weight aggregates without any significant changes (Table 2).

flask When to add Catechin (uM) HIC peak 2 (%) HIC peak 3 (%) One - 0 73 27 2 0 150 78 22 3 3 150 78 22

< Example  4> Bioreactor ( bioreactor ) Effect of catechin addition on culture

Changes in misfolded protein and high molecular weight aggregate ratios depending on whether catechin was added or not were confirmed in the bioreactor.

Specifically, the cell line selected in Example 1 was shake-cultured in an environment of 37 ° C. When the cell line reached an appropriate concentration, the cells were transferred to a 5 L bioreactor and cultured at 37 ° C. in the environment. In the perfusion incubator, the selected cell line was continuously supplied with the medium, and at the same time, the same volume of the used medium was removed to prepare a high-concentration inoculating cell on the bioreactor. When the cells were grown at an appropriate concentration, cells were inoculated at a cell concentration of 5 × 10 ⁶ cells / ml in a bioreactor of 10 L working volume. Dissolved oxygen was maintained at 30 ℃, pH 7.0 ~ 7.2 and 50% for 21 days, and nutrient was added 5% over 6 times every 3 days from 3 to 18 days. Catechin and 10 uM CuCl ₂ were added on the third day, and the productivity, misfolded protein and high molecular weight aggregates of the cultured cells were sampled on the 21st day and analyzed by HIC-HPLC.

As a result, as shown in Table 3, when the catechin was added in the bioreactor culture process of a large capacity culture, it was confirmed that the formation ratio of misfolded proteins and high molecular weight aggregates was suppressed (Table 3).

Bioreactor Catechin (uM) HIC peak 2 (%) HIC peak 3 (%) One 0 80.0 20.0 2 150 85.0 15.0

&Lt; 110 > Dong-A Pharmaceutical Co., Ltd. <120> Highly efficient production method of fusion proteins for          antibody treatment using catechin <130> 13P-01-24 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> TNFR-Fc <400> 1 atggctcccg tggccgtgtg ggccgctctg gccgtgggcc tggagctgtg ggccgctgcc 60 cacgccctgc ctgcccaggt ggcctttaca ccctatgctc ccgagcccgg cagcacctgc 120 agactgagag agtactacga ccagacagcc cagatgtgtt gcagcaagtg cagccccggc 180 cagcacgcca aggtgttctg caccaagacc agcgacaccg tgtgtgacag ctgcgaggac 240 agcacctaca cccagctgtg gaactgggtg cccgagtgcc tgagctgtgg cagcagatgc 300 agctccgacc aggtggagac ccaggcctgc accagagagc agaatagaat ctgcacctgc 360 agacctggct ggtactgtgc cctgagcaag caggagggct gcagactgtg tgcccctctg 420 agaaagtgca gacctggctt tggcgtggcc agacccggca ccgagaccag cgacgtggtg 480 tgcaaaccct gtgcccctgg caccttcagc aacaccacat ccagcaccga catctgcaga 540 ccccaccaga tctgcaacgt ggtggccatt cccggcaatg ccagcatgga tgccgtgtgc 600 accagcacca gccccaccag aagcatggcc cctggcgccg tgcacctgcc ccagcccgtg 660 agcaccagaa gccagcacac ccagcccaca cccgagccca gcacagcccc tagcaccagc 720 ttcctgctgc ccatgggccc cagccctcct gccgagggca gcacaggcga tgagcccaag 780 cggcggcccc 840 agcgtgttcc tgtttccccc taaacccaag gacaccctga tgatcagcag aacacccgag 900 gtgacctgcg tggtggtgga cgtgagccac gaggatcccg aggtgaagtt caactggtac 960 gtggatggcg tggaggtgca caatgccaag accaaaccca gagaggagca gtacaacagc 1020 acctacagag tggtgagcgt gctgacagtg ctgcaccagg actggctgaa tggcaaggag 1080 tacaagtgca aggtgagcaa caaggccctg cctgccccca tcgagaagac catcagcaag 1140 gccaagggcc agcccagaga gccccaggtg tacaccctgc ctcccagcag agaggagatg 1200 accaagaacc aggtgagcct gacctgcctg gtgaagggct tctatcccag cgacattgcc 1260 gtggagtggg agagcaacgg ccagcccgag aataactaca agaccacacc tcccgtgctg 1320 gacagcgatg gcagcttctt tctgtacagc aagctgaccg tggacaagag cagatggcag 1380 cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1440 aagagcctga gcctgagccc tggcaagtga 1470

Claims (9)

1) preparing an expression vector comprising a polynucleotide encoding a TNFR-Fc fusion protein consisting of the nucleotide sequence shown in SEQ ID NO: 1;
2) transforming the expression vector of step 1) into a cell line;
3) adding catechin to the transformed cell line of step 2) at a concentration of 50 to 400 uM; And
4) isolating the TNFR-Fc fusion protein from the cell line culture of step 3).
delete 2. The method according to claim 1, wherein the cell line of step 2) is a CHO cell line.
delete 2. The method according to claim 1, wherein the catechin of step 3) is added one to four times.
The method according to claim 1, wherein the culture of step 3) is cultured by addition of CuCl ₂ .
The method according to claim 1, wherein the culture in step 3) is a fed batch culture, a batch culture, or a perfusion culture.
delete delete

Images