KR19990000214A - Cell line producing erythropoietin (EPO), its preparation method and method of producing EPO using the same - Google Patents

Abstract

The present invention relates to a cell line producing erythropoietin (hereinafter abbreviated as 'EPO') and a method for producing EPO using the same.

More specifically, the present invention provides efficient EPO screened by long-term passage of cell lines producing EPO transformed with an expression vector comprising a dhfr (dihydrofolate reductase) gene and an EPO gene at low methotrexate (MTX) concentrations. It relates to a cell line to be produced, a method for producing the same, and a method for producing a high titer of EPO using a cell line of the present invention.

Description

Cell line producing erythropoietin (EPO), its preparation method and method of producing EPO using the same

The present invention relates to a cell line producing erythropoietin (hereinafter abbreviated as 'EPO') and a method for producing EPO using the same.

More specifically, the present invention provides a method for efficiently culturing EPO selected by long-term passage of a cell line producing EPO transformed with an expression vector including a dhfr (dihydrofolate reductase) gene and an EPO gene at low methotrexate (MTX) concentration. It relates to a cell line to be produced, a method for producing the same, and a method for producing a high titer of EPO using a cell line of the present invention.

Erythropoietin (EPO) is a glycoprotein that differentiates erythrocytes produced in bone marrow into erythrocytes. It is an acidic glycoprotein hormone composed of 166 amino acids, also called erythropoietin, and combined with carbohydrates. In healthy people, EPO is usually present in the blood at 10-20 mU / ml, and in the event of a massive bleeding caused by an accident, the production of EPO is stimulated when the oxygen concentration in the human body is reduced, increasing its productivity by at least 1,000 times. do. In addition, abnormalities in the kidneys may lead to decreased production of EPO, leading to severe anemia (Jacobson et al., Nature, 179, 633, 1957).

Therefore, EPO was approved as a treatment for pernicious anemia for use in hemophilia, physiological anemia and chronic renal failure in 1989. In 1991, EPO was approved for treatment for pernicious anemia for patients with acquired immunodeficiency using azacytidine (AZT). . In addition, EPO has been used to treat anemia, replace blood transfusion before and after surgery in rheumatoid arthritis patients or cancer patients undergoing drug treatment, and the scope of application is increasing.

Until now, clinically used EPO was mainly extracted from sheep plasma (US Pat. No. 3,033,753), human urine (US Pat. No. 3,865,801), and the amount thereof was not widely used. Therefore, many attempts have been made to produce EPO by genetic engineering method, specifically cDNA of human EPO gene has been cloned (US Pat. No. 4,703,008), and this gene has been derived from dhfr mutant strain derived from mammalian cell line CHO cell line (ATCC CRL 9096). And the like have been attempted to produce pure EPO by transformation (Jacobs et al., Nature, 313, 806, 1985; Lin et al., Proc. Natl. Acad. Sci. USA, 82, 7580, 1985).

The transformed CHO cells can efficiently increase the production of EPO by amplifying the EPO gene by gradually adapting to the medium to which methotrexate (MTX) is added (Malik et al., DNA and Cell Biol., 11, 453, 1992). Specifically, Kirin-Amgen's patent (International Patent Publication No. 85-2610) produced EPO at a concentration of 21 μg / 10 ⁶ cells / 48 hours from a cell line adapted at a concentration of 1 μM MTX. However, in order to adapt the cells stepwise to the concentration of 1μM MTX, a long culture period of 6 months or more is required, side effects of deterioration of the cell growth, and expensive to mass-produce EPO.

Meanwhile, in order to express a large amount of foreign genes from the CHO cell line using a low MTX concentration, the present inventors removed 128-270 sites of the SV40 promoter, linked the dhfr gene, and transformed the cell line to foreign cells at 20mM MTX concentration. Cell lines capable of expressing genes in large quantities have been developed. Specifically, the expression vector pCMV-dhfr (KCTC 8671 P) is obtained by inserting the dhfr gene in the plasmid vector pRc / CMV, and the expression vector pEpoG-dhfr (KCTC 0181 P) or pEpoC-dhfr ( KCTC 0180 P) was prepared. These expression vectors were transformed into dhfr mutant CHO cell lines to develop g2 (KCTC 0183 P) and EC1 (KCTC 0182 P) cell lines capable of amplifying genes even at low MTX concentrations, respectively (Korean Patent Application No. 95-22441). . In addition, the productivity of each cell line g2-20 and results of selected cell lines EC1-20 confirming the EPO productivity derived from the cell line EC1 or g2 were each 23μg / 10 ⁶ cells / 24 hours with 45μg / 10 ⁶ cells / 24 hours .

At this time, the EPO productivity of the EC1-20 cell line was confirmed to be higher than the productivity of the cell line adapted to the concentration of 1μM MTX developed by the conventional Kirin-Amgen.

In addition, the present inventors repeat the process of cell culture, cell subcloning, cell clone expansion using the EC1-20 cell line obtained above using a low MTX concentration cell medium in order to develop a cell line producing EPO more efficiently. The present invention was completed by selecting a cell line EC1-71-1C8 that efficiently produces EPO through long-term passage and mass production of high titer EPO using the cell line EC1-71-1C8.

An object of the present invention is to provide a cell line EC1-71-1C8 (Accession Number: KCTC 0296 BP) that efficiently produces erythropoietin.

In addition, the present invention provides a method for selecting a cell line that efficiently produces EPO through long-term passage that repeats the process of cell culture, cell subcloning, cell clone expansion using a cell medium of low MTX concentration. There is this.

At this time, the MTX concentration is preferably 20 nM, and passage culture is preferably 5 months or longer.

In addition, the present invention provides a method for producing a high titer of EPO in large quantities by culturing the cell line EC1-71-1C8.

1 is a comparison of the EPO productivity in the existing cell lines EC1, EC1-20 and EC1-71-1C8 cell line of the present invention,

Figure 2 shows the amount of EPO mRNA produced in the existing cell lines EC1, EC1-20 and cell lines EC1-71-1C8 of the present invention,

Figure 3a is labeled with the existing cell lines EC1, EC1-20 and cell lines EC1-71-1C8 of the present invention [S ³⁵ ] -radioactive methionine, respectively, secreted into the cell supernatant after 10, 20 and 60 minutes. EPO amount is shown by SDS-polyacrylamide gel electrophoresis,

Figure 3b is labeled with the existing cell lines EC1, EC1-20 and cell lines EC1-71-1C8 of the present invention [S ³⁵ ] -radioactive methionine, respectively, present in the cells after 0, 10, 20, 60 minutes The amount of EPO expressed in Western blot,

Figure 4 shows the result of Western blotting (B) by 15% SDS-polyacrylamide gel electrophoresis (A) of EPO separated and purified by the method of the present invention,

Figure 5 shows the biological activity of the purified EPO by the method of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a cell line for efficiently mass producing EPO. Specifically, the present invention provides a cell line EC1-71-1C8 (Accession Number: KCTC 0296 BP) that produces high titers of EPO.

EC1-20 cells (Korean Patent Application No. 95-22441, which describes the preparation method thereof) which have been previously reported are exchanged at regular intervals in a medium to which dialysis serum, low concentration G418 has been added After incubation for about 1 month and analyzed by FACScan using mouse anti-human EPO antibody, it was observed that various cells with different levels of EPO production were mixed. These results show that the cell lines containing the expression vector using gene amplification vary in the degree to which each cell adapts to the MTX concentration when cultured for a long period of time under a selection condition such as a medium containing MTX. . As a result, some cells produce more EPO by producing more genes, while others lose their amplified genes, which reduces their productivity.

Reference Example 1 Preparation of Expression Vectors pEpoG-dhfr and pEpoC-dhfr

The plasmid vector pCMV-dhfr was digested with restriction enzymes HindIII and ApaI and modified with klenow to obtain the expression vector pEpoG-dhfr by addition of the EPO genomic gene EpoG . In addition, the expression vector pEpoC-dhfr was obtained by linking EPO cDNA gene EpoC to the plasmid vector pCMV-dhfr.

Reference Example 2 Preparation of ECl Cell Line

Dhfr mutant CHO cell line (ATCC CRL 9096) cells were transformed with the expression vector pEpoG-dhfr or pEpoC-dhfr to obtain transformant ECl having human EPO generating ability. These cells were cultured in α-MEM medium containing MTX.

Reference Example 3 Culture of dhfr Mutant CHO Cell Line

Culture medium-10μg / ml hypoxanthin, 10μg / ml thymidine, 50μg / ml glycine, glutamine in DMEM / F12 medium (Gibco Inc.) with 10% calf serum 587 μg / ml, glucose 4.5 mg / ml, penicillin-streptomycin 100 IU / ml (Sigma) were added-to dhfr mutations passaged at 37 ° C. to maintain 5% carbonic acid in CHO cells were harvested using a centrifuge. A dish of 6 cm diameter was inoculated to 5 × 10 ⁵ cells / ml, and washed twice with OPTI MEM I (manufactured by Gibco Co.) the next day.

Reference Example 4 Preparation of Transformed ECl Cell Line

5 μg of the plasmid was diluted in 1.5 ml of OPTI MEM I, 20 μg of lipofectin (Lipofectin, Gibco Co., Ltd.) diluted in the same amount was mixed and left for 10 minutes, and then evenly placed on the prepared CHO cells. The cells were incubated at 37 ° C. for 5 hours in a 5% carbon dioxide incubator, and 3 ml of DMEM / F12 medium containing 20% of calf serum was added, followed by further incubation for 24 hours. Cells obtained by treatment with 0.25% trypsin (Gibco Corporation) and centrifuged were aliquoted into 96-well plates to 2 × 10 ⁴ cells per well. Cultured by adding α-MEM medium (Gibco Corporation) containing 10% of dialyzed serum (26300-020 from Gibcosa), 550 μg / ml of G418, was incubated for 2 weeks while changing medium at 4 day intervals. Dozens of surviving clones were obtained, and a hybridoma culture of mouse anti-human EPO antibody (ATCC CRL8164) was isolated and purified in our laboratory to measure the EPO production capacity of each clone. The indirect Elisa method (Engvall Perlman, J. Immunol. 109, 129, 1972) was performed using a goat anti-mouse IgG antibody (manufactured by Sigma), resulting in a trait containing the expression vector pEpoG-dhfr or pEpoC-dhfr. Among the transformants, the cell line ECl producing the most EPO was selected.

Each of the selected cell lines ECl was taken 1 × 10 ⁶ , transferred to a 10 cm diameter dish, and incubated for 2 weeks with medium exchange at α-MEM medium containing 20 nM methotrexate (MTX) at 4 day intervals. The cells adapted to this concentration of MTX were named ECl-20, respectively. EPO production of this ECl-20 was measured and showed a specific productivity of 45 μg / 10 ⁶ cells / 24 hours.

Therefore, the present invention adapts the cells to low MTX concentrations by long-term passage of the EC1-20 cell line (KCTC 0182 BP), and then selects a cell line that efficiently mass-produces EPO by subcloning the cell clones. . The EC1-20 cell line is adapted for MTX concentrations by long-term passages for at least 5 months, specifically 2 weeks culture in cell containing 20 nM MTX-cell subcloning-cell clone expansion of 2 weeks or more 2 weeks culture-cell subcloning-2 weeks or more cell clone expansion-2 weeks culture-cell cloning is repeated EC1-71- Isolate 1C8 cell line. EC1-71-1C8 cell line of the present invention is shown to significantly improve the EPO mRNA level, EPO production, EPO secretion rate and the like.

The screening method of the present invention is different from simply subcloning a cell, and is a new method very different from a method of adapting a cell to MTX while increasing a known concentration of MTX.

In addition, the EPO produced from the EC1-71-1C8 cell line of the present invention was isolated and purified by gel filtration, etc., and the results were then subjected to Western blotting and SDS-polyacrylamide gel electrophoresis ( PAGE) and the like (see FIG. 4), and their biological activity is verified (see FIG. 5).

EC1-71-1C8 cell line of the present invention was deposited on January 18, 1997 to the Gene Bank of Korea Institute of Science and Technology Biotechnology Research Institute (Accession Number: KCTC 0296 BP).

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

However, the embodiments are only illustrative of the present invention and the present invention is not limited by the embodiments.

Example 1 Long-term passage of EC1-20 cells

10 ⁶ existing EC1-20 cell lines (KCTC 0182P) were added to 10% dialyzed calf serum (Gibcos, 26300-020), α-MEM medium with 20 nM MTX, 550 μg / ml G418 (Gibcos). The plate was transferred to a 10 cm diameter dish, which was incubated for 2 weeks while exchanging medium at 4 ° C. in a 37 ° C., 5% carbon dioxide incubator. These cells were plated in 96 well plates at 0.5-1 cells per well. After about three weeks, the amount of EPO in the cell clone culture grown was measured by ELISA (Enzyme Linked Immunosorbent Assay) method to select clone # 7 having the highest optical density (OD). Continue culturing these cells for at least 2 weeks to increase the number of cells, then subculture 10 ⁶ cells in the same medium for 2 weeks, then divide them into 96 well plates at 0.5-1 cells per well and grow up about 3 weeks later. Clonal culture was measured by ELISA to select clone # 71 with the highest OD value. The cells were repeated again and again to isolate the cell line EC1-71-1C8 with the highest EPO productivity.

In other words, two-week culture-cell subcloning- over two-week cell clone expansion- two-week culture-cell subcloning- over two-week cell clone expansion- two-week culture-cell cloning in a medium containing 20nM MTX The highly productive EC1-71-1C8 cell line was isolated. This method is different from simply subcloning cells and adapting the cells to MTX while increasing the concentration of MTX.

Example 2 Isolation of EC1-71-1C8 Cell Line

In order to measure EPO productivity, ⁵ × 10 ⁵ cells were dispensed into 6-well plates, medium was changed after 24 hours, and cell culture was taken again after 24 hours to measure the amount of secreted EPO.

At this time, in order to measure the EPO production of various cell lines, the ELISA method established in the laboratory was used. That is, EPO was bound by adding a cell culture solution diluted at various concentrations to an ELISA plate coated with 3 μg of purified anti-EPO mouse monoclonal antibody (which was isolated and purified from ATCC CRL8164 hybridoma culture solution). A rat monoclonal antibody (BF-11, obtained from Dr. Ryuzo Sasaki of Kyoto University, Japan) was added to human EPO biotinylated using Amersham Biotinylation Kit. Next, the peroxidase-bound straptavidin was added at a dilution of 1: 1000, followed by color development, and the absorbance was measured at 495 nm. In order to obtain a standard curve, purified human EPO (manufactured by Boehringer Mannheim, Germany) at various concentrations (0-500 U / ml) was used instead of the cell culture in the sandwich ELISA method, followed by ELISA. It was. As a result, a standard curve was obtained in a straight line between the concentrations of 100-400 U / ml. Within this range, the EC1-71-1C8 cell line of the present invention having the highest productivity of EPO was isolated by measuring the amount of EPO in the cell culture sample.

Example 3 Measurement of EPO Specific Productivity of Cell Lines

In order to accurately measure the EPO specific productivity of the existing EC1, EC1-20 cell lines and EC1-71-1C8 cell lines of the present invention, the EC1-71-1C8, EC1, EC1-20 cell lines were each 5x10 ⁵ cells. Six wells of the cell lines were dispensed, and after 24 hours, the medium was changed, and again after 24 hours, the cell culture was taken to quantify the EPO production. Boringer Mannheim's EPO-ELISA kit was used to quantify the amount of EPO. Cell cultures were added at several concentrations to the plate coated with antibodies to EPO, and EPO monoclonal antibodies bound to peroxidase were added. At this time, in order to quantify the amount of EPO present in the culture medium, the standard amount of purified EPO provided in the kit is quantified as in the culture medium samples, and a standard curve is obtained from them, and the corresponding EISA value of the sample EPO is obtained. The amount of EPO was determined. As a result EC1-20 cell line and confirmed that 37.1μg / 10 ⁶ cells / 24 hours, EC1-71-1C8 cell line of the invention is producing EPO to 129.9 μg / 10 ⁶ cells / 24 hours (see Fig. 1) .

Example 4 Quantitative Analysis of EPO mRNA

In order to confirm whether the EC1-71-1C8 cell line of the present invention increases the level of mRNA by amplifying more genes compared with the existing EC1-20 cell line, the EC1-71-1C8 cell line of the present invention and the control group EC1-71 Isolate total RNA from 20 and EC1 cell lines, deploy each of these RNAs to 10 μg 1% agarose gel, then dry at 68 ° C. for 30 minutes and allow hybridization solution [5X Denhardt's solution, 5X SSPE, 0.1% SDS, 50% formamide, 100 μg / ml salmon sperm DNA] was added to a plastic bag and reacted at 42 ° C. for 2 hours. The radiolabeled EPO cDNA was added again, and further reacted for 24 hours, followed by washing for 30 minutes at 68 ° C. with 0.1 × SSC and 0.1% SDS solution. The gel thus washed was subjected to X-ray film to compare the concentration of EPO mRNA. As a result, it can be seen that the EPO mRNA level produced in the EC1-71-1C8 cell line of the present invention is more than twice higher than that of the conventional EC1-20 cell line (see FIG. 2).

Example 5 Comparison of EPO Secretion

Since the EC1-71-1C8 cell line of the present invention has a higher amount of EPO mRNA than the existing EC1-20 cell line, the EC1-71-1C8 cell line performs more translation of EPO than the EC1-20 cell line. EPO secretion kinetics of each cell was measured to demonstrate that EPO is also expected to secrete more. That is, the EC1, EC1-20, EC1-71-1C8 cell lines were inoculated in 6 well plates 6 by 2 X 10 ⁵ number, respectively, and then incubated for 30 minutes in medium without methionine when actively growing. Thereafter, the cells were replaced with a medium containing 100 μCi of [S ³⁵ ] -radioactive methionine and incubated for 3 hours to induce labeling of all proteins with radioisotopes. After replacement with non-radioactive medium, the cell supernatant was recovered after 10, 20, and 60 minutes, and the cells were also recovered and disrupted solution (0.1 M Tris-Cl, pH 8.0, 0.1 M NaCl, 0.1 M EDTA). , 2% triton X-100, 0.1% SDS) and centrifuged to recover the cell extracts. The antibody to EPO was added to the cell supernatant and the cell extracts, and then reacted at 4 ° C. overnight. Then, Protein A-Sepharose was added thereto, followed by further reaction at 4 ° C. for 1 hour. The reaction solution was centrifuged and the precipitate was separated into tris-triton solution (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 2% Triton X-100, 0.1 mM EDTA), and 10 mM Tris-Cl (pH 8.0). After washing well with, the precipitate was suspended in 40 μl of SDS-PAGE sample solution, boiled and 12.5% SDS-PAGE was performed. The gel was dried well to be exposed to an X-ray film and the results are shown in Figs. 3a and 3b.

3A shows EPO secreted into cell supernatants 10, 20 and 60 minutes after labeling EC1-20 and EC1-71-1C8 cells with [S ³⁵ ] -radioactive methionine, respectively. Herein, lane 1 shows a sample of EC1 cells, lane 2 for EC1-20 cells, and lane 3 for EC1-71-1C8 cells. As shown in this figure, the EPO secretion of EC1-71-1C8 cells is higher than that of EC1 cells. 3B shows the EPO amounts of cell extracts at 0, 10, 20 and 60 minutes after labeling each EC1, EC1-20 and EC1-71-1C8 cells with [S ³⁵ ] -radioactive methionine. As shown in this figure, the amount of EPO in EC1-71-1C8 cells is higher than that of EC1 cells.

Example 6 Separation and Purification of EPO

The transformed EC1-71-1C8 cell line obtained in Example 1 was incubated for 2 days in α-MEM medium containing 10% of calf serum dialyzed with dialysis, and replaced with serum-free medium, SFMII (Gibco Corporation) medium for 5 days. Incubated. The precipitate obtained by adding the same amount of saturated ammonium sulfate solution to the culture was dissolved in 10 mM Tris-HCl buffer (pH 8.6) and dialyzed in the same buffer for two days. EPO was first isolated using a buffer added to 0.25M salt by salt gradient in FPLC using a mono-Q column (from Pharmacia). In order to further purify the sample of the first separated EPO, it was injected again into the Sephacryl-200 column and washed with a washing solution (0.5 mM salt, 1 mM EDTA, 0.2 mM PMSF in 50 mM Tris buffer solution, pH 7.5) and gel filtration. (Gel filtration) was performed to purify the EPO. At this time, the degree of purification of EPO was confirmed by 15% SDS-PAGE (A) and Western blotting (B) (see FIG. 4).

In FIG. 4A, lane M is a reference for protein molecular weight, and lanes 1, 2, and 3 are amounts corresponding to 5, 3, and 1 μg of the final purified EPO, respectively. Figure 4b is Western blotting using a mouse anti-human EPO antibody (ATCC CRL8164 hybridoma culture was isolated and purified in our laboratory) and goat anti-mouse IgG antibody (Sigma) combined with peroxidase will be.

Example 7 Biological Activity Verification

MTT method (Tasuda et al., Acta Haematol. JPN, 53, 1045) using EP-FDC-P2 cells (Dr. Ryuzo Sasaki, Professor of Food Science and Technology, Kyoto University, Japan) as a cell line that responds and proliferates sensitively to EPO. , 1990) investigated the ex vivo cellular activity of EPO. The cells were suspended at 4 × 10 4 cells / well in 96 well plates and EPO standards (Behringer Mannheim) and EPO in the present invention were added to each well at various concentrations as in FIG. 5. After incubation for 20 hours at 37 ℃, CO ₂ incubator, MTT solution (5mg / ml) was added to each well of 0.01ml and incubated again for 4 hours. The supernatant was removed by centrifuging the plate at 1500 rpm for 5 minutes, and 0.1 ml of isopropanol solution dissolved in 0.04 N hydrochloric acid was added. After 20 minutes, the absorbance was measured at 540 nm by adding 3% SDS solution again. As a result, it was confirmed that the EPO obtained through the present invention had almost the same biological activity as the EPO standard (see FIG. 5).

EC1-71-1C8 cell line of the invention, the existing EC1-20 cells as compared to also produce EPO with 37.1μg / 10 concentration of ^{106 cells} / 24 hours in the EPO 129.9μg / 10 concentration of ^{106 cells} / 24 hours It can be produced and is very efficient for producing high potency EPO. Specifically, the amount of mRNA was more than two times higher than the existing EC1-20 cell line, the production of EPO was more than three times higher, and the secretion rate of EPO was also improved.

In addition, the cell line of the present invention selects cells by long-term passage that repeats cell culture, cell subcloning, and cell clone expansion by using a medium of low MTX concentration so as to mass-produce EPO even at low MTX concentration. Simple and low cost

Claims (5)

Cell line EC1-71-1C8 producing erythropoietin (EPO) (Accession No. KCTC 0296BP) A method for selecting a cell line of claim 1 through long-term passages in which cell culture, subcloning, and cell clone expansion are repeated using a low MTX concentration cell medium. The method of claim 2, wherein the MTX concentration is 20 nM. The method according to claim 2, characterized in that the subculture is carried out for 5 months or more. A method for mass production of EPO by culturing the cell line EC1-71-1C8 of claim 1