JP6050289B2 - Selection method of cell lines expressing high levels of recombinant proteins - Google Patents

Description

  The present invention relates to a simple method for selecting a cell line that highly expresses a recombinant protein. Specifically, the recombinant protein contained in the culture supernatant of a recombinant protein-expressing cell line cultured in a serum-free medium is subjected to reverse phase chromatography. The present invention relates to a method for selecting a cell line that highly expresses a recombinant protein by analyzing by chromatography.

  A method for producing a recombinant protein using mammalian cells transformed with an expression vector incorporating a gene encoding a desired protein is a widely used technique in industrial fields such as pharmaceutical production, and α-galactosidase. A, lysosomal enzymes such as iduronic acid 2-sulfatase, glucocerebrosidase, galsulfase, α-L-iduronidase, acid α-glucosidase, tissue plasminogen activator (t-PA), blood coagulation factor VII, blood coagulation factor Blood coagulation factors such as factor VIII and blood coagulation factor IX, erythropoietin, interferon, thrombomodulin, follicle stimulating hormone, granulocyte colony stimulating factor (G-CSF), various antibody drugs, etc. are manufactured using this technology, It is commercially available.

  When producing recombinant proteins using mammalian cells, in order to increase the production efficiency of the recombinant protein, recombination is carried out from mammalian cells transformed with an expression vector incorporating a gene encoding the desired protein. It is necessary to select cells that highly express the protein. Such selection includes, as a selection marker, an enzyme that degrades drugs such as puromycin and neomycin (drug resistance marker) incorporated into an expression vector, and cells transformed with such an expression vector have a drug corresponding to the selection marker. It is generally performed by culturing in a medium containing a constant concentration. By culturing in the presence of a drug corresponding to the selectable marker, only cells that express the drug selectable marker at a high level proliferate and are selected. Cells expressing a drug selectable marker at a high level tend to express a gene encoding a desired protein simultaneously incorporated into an expression vector at a high level. As a result, a mammal expressing a desired protein at a high level Cells are obtained.

  The cells selected by the selectable marker tend to express the desired protein at a high level, but the expression level varies from individual cell to individual cell. Therefore, in order to further increase the production efficiency of the recombinant protein, it is necessary to further select cells that express the desired protein at a higher level from the cells selected by the selection marker. Such further selection of cells is generally carried out by culturing cells and quantifying recombinant proteins secreted into the medium for each cell line by ELISA (Patent Document 1). However, quantification by the ELISA method requires the preparation of an antibody against the recombinant protein.

  In addition, as a method for selecting cells that express a desired protein at a higher level, there is a method that utilizes the affinity of a recombinant protein with a receptor (Patent Document 2). However, a method using affinity with a receptor requires that a receptor protein is separately prepared using a gene recombination technique or the like.

  Japanese Patent No. 2657816   International Publication No. 2009/020142

  Under the above background, an object of the present invention is to provide a simple method for selecting a cell line that highly expresses a recombinant protein.

In the research aimed at the above purpose, the present inventors analyzed the recombinant protein contained in the culture supernatant of a recombinant protein-expressing cell line cultured in a serum-free medium by reverse-phase chromatography, thereby recombining the recombinant protein. The present inventors have found that a cell line that highly expresses a protein can be selected, thereby completing the present invention. That is, the present invention provides the following.
(1) A method for selecting a cell line that highly expresses a recombinant protein,
(A) transforming a cell with an expression vector incorporating a gene encoding a desired protein and a selectable marker to obtain a transformed cell;
(B) selecting a plurality of cell lines by selectively culturing the transformed cells in the presence of a drug corresponding to the selection marker;
(C) culturing each of the cell lines in a serum-free medium, and collecting the culture supernatant of the cell line after the culturing,
(D) Each of the collected culture supernatants of the cell line is subjected to reverse phase chromatography, and then the effluent from the reverse phase column is continuously guided to the flow path, and the effluent flowing in the flow path Continuously measuring the absorbance of
(E) identifying an absorbance peak corresponding to the recombinant protein by the measurement, and comparing the area of the peak obtained for each culture supernatant of the cell line;
(F) selecting a cell line showing the relatively large area from the cell lines.
(2) The method according to (1) above, wherein the cell is a mammalian cell.
(3) The method according to (2) above, wherein the mammalian cell is a CHO cell.
(4) 20-40% propanol containing 0.08-0.12% trifluoroacetic acid, the stationary phase of which is chemically bonded porous spherical silica gel whose surface is modified with octadecylsilyl groups. An aqueous solution is used as the first mobile phase, and an aqueous solution with the same concentration of trifluoroacetic acid as compared with the first mobile phase, but with an increased concentration of propanol is used as the second mobile phase. The method according to any one of (1) to (3) above.
(5) The recombinant protein is a lysosomal enzyme such as α-galactosidase A, iduronic acid 2-sulfatase, glucocerebrosidase, galsulfase, α-L-iduronidase, acid α-glucosidase, tissue plasminogen activator (t- PA), blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor such as blood coagulation factor IX, erythropoietin, darbepoetin, interferon, thrombomodulin, follicle stimulating hormone, granulocyte colony stimulating factor (G-CSF), antibody Or the method according to any one of (1) to (4) above, which is selected from the group consisting of these analogs.
(6) The method according to any one of (1) to (4) above, wherein the recombinant protein is selected from the group consisting of erythropoietin, darbepoetin, or analogs thereof.

  According to the present invention, a cell line that highly expresses a recombinant protein can be easily selected.

The chromatogram which shows the analysis result by the reverse phase chromatography of the culture supernatant of a cell strain (cell strain 1), and a control solution. The broken line shows the culture supernatant of the cell line, and the solid line shows the chromatogram of the control solution. The vertical axis represents absorbance, and the horizontal axis represents elapsed time (minutes) after completion of sample loading. (A) shows a peak derived from darbepoetin. The chromatogram which shows the analysis result by the reverse phase chromatography of the culture supernatant of the cell lines 1-3. (1) shows the chromatogram of the culture supernatant of cell line 1, (2) is the cell line 2, and (3) is the cell line 3. The vertical axis represents absorbance, and the horizontal axis represents elapsed time (minutes) after completion of sample loading.

  In the present invention, the term “cell” is not particularly limited as long as it is a cell capable of expressing a recombinant protein by transformation with an expression vector incorporating a gene encoding a desired recombinant protein. , Preferably yeast, insect cells, plant cells such as callus, and mammalian cells. When the cell is a mammalian cell, the type of the cell is not particularly limited, but is preferably a cell derived from human, mouse, or hamster, and CHO cell or COS cell can be preferably used.

  In the present invention, the term “cell line” is derived from a single cell obtained by transforming a cell with an expression vector incorporating a gene encoding a desired recombinant protein, followed by selective culture. A cell with uniform (or almost uniform) character and stability. In the present invention, “cell line that highly expresses a recombinant protein” refers to a cell line that has a relatively high expression level when the expression level of the recombinant protein is compared among a plurality of cell lines. For example, when the expression levels of 100 cell lines are compared, the cell lines are preferably those having the highest level of expression in the top 1 to 10 levels, more preferably the cell lines having the level of expression of the top 1 to 5 levels. More preferably, it is a cell line with the highest expression level.

  In the present invention, an expression vector used for transforming a cell can express a gene encoding a desired protein incorporated in the vector and a selectable marker in the cell into which the expression vector has been introduced. There is no particular limitation. A gene encoding a desired protein is placed under the control of a promoter (gene expression control site), which is a region on DNA that can control (regulate) the frequency of transcription of a gene present downstream thereof. The gene expression control site that can be used at this time is not particularly limited as long as the transcription of the gene incorporated downstream of the cell is a mammalian cell and can be strongly induced in the mammalian cell. However, a promoter derived from cytomegalovirus (CMV), a promoter derived from a virus such as the SV40 early promoter, and an elongation factor 1α (EF-1α) promoter are preferred.

  In the present invention, a gene that can be incorporated into an expression vector as a gene encoding a desired recombinant protein is particularly selected as long as a cell line that highly expresses the recombinant protein can be selected by the method of the present invention. Although not limited, α-galactosidase A, iduronic acid 2-sulfatase, glucocerebrosidase, galsulfase, α-L-iduronidase, lysosomal enzymes such as acid α-glucosidase, tissue plasminogen activator (t-PA), blood Coagulation factor VII, blood coagulation factor VIII, blood coagulation factor such as blood coagulation factor IX, erythropoietin, darbepoetin, interferon, thrombomodulin, follicle stimulating hormone, granulocyte colony stimulating factor (G-CSF), granulocyte monocyte colony Stimulation factor (GM-CSF , Macrophage colony stimulating factor (M-CSF), a gene such as that encoding antibodies, or those analogues. In particular, the method of the present invention can be suitably used to select cell lines that highly express erythropoietin or darbepoetin, or analogs thereof.

  In the present invention, the term “selection marker” refers to a gene incorporated in an expression vector or a protein encoded by the gene that functions to select cells transformed with the expression vector. The most common selection marker is an enzyme (drug resistance marker) that degrades drugs such as puromycin and neomycin. In addition, dihydrofolate reductase (DHFR) and glutamine synthetase (GS) can also be used as selection markers. When an enzyme (drug resistance marker) that degrades a drug such as puromycin or neomycin is used as a selection marker, the drug corresponding to the selection marker is a cytotoxic drug that is degraded by these enzymes. If the selectable marker is a puromycin resistance gene, it is puromycin. When the selection marker is dihydrofolate reductase (DHFR), methotrexate (MTX) is used, and when the selection marker is glutamine synthetase (GS), methionine sulphoximine (MSX) is used as a drug corresponding to the selection marker. Can do.

  In the present invention, the culture for selecting cells into which the selection marker has been introduced is referred to as “selective culture”, and the medium used for selecting the introduced cells is referred to as “selection medium”. In selective culture, in order to obtain a cell line derived from a single cell, the cells are diluted and seeded so that each well of a multi-well plate (such as a 96-well plate) contains one transformed cell. To incubate. In selective culture, multiple cell lines are selected. Although the number of cell lines to be selected is arbitrary, approximately 10 to 100 types of cell lines are selected.

Cells selected by selective culture are cultured in serum-free medium. There are no particular limitations on the serum-free medium that can be used at this time, for example:
Amino acids ... 3-700mg / L,
Vitamins ... 0.001-50mg / L,
Monosaccharides 0.3 to 10 g / L,
Inorganic salt: 0.1 to 10,000 mg / L,
Trace elements: 0.001 to 0.1 mg / L,
Nucleoside: 0.1 to 50 mg / L,
Fatty acid ... 0.001-10mg / L,
Biotin ... 0.01-1mg / L,
Hydrocortisone ... 0.1-20 μg / L,
Insulin ... 0.1-20mg / L,
Vitamin B ₁₂ ... 0.1-10mg / L,
Putrescine 0.01 ~ 1mg / L,
A medium containing sodium pyruvate... 10 to 500 mg / L and a water-soluble iron compound is preferably used. If desired, thymidine, hypoxanthine, conventional pH indicators and antibiotics may be added.

  Further, as a serum-free medium, DMEM / F12 medium (mixed medium of DMEM and F12) may be used as a basic medium, and each of these media is well known to those skilled in the art. Furthermore, the serum-free medium contains sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, iron (II) sulfate, asparagine, aspartic acid, serine and polyvinyl alcohol, DMEM ( HG) HAM modified (R5) media may be used. Furthermore, commercially available serum-free media such as CDoptioCHO, CHO-S-SFMII or CD CHO (Invitrogen), IS CHO-V or IS CHO-V-GS (Irvine), EX-CELL302 or EX-CELL305 (JRH) Etc.) may be used as the basic medium.

  The cell line is cultured in a serum-free medium under the same conditions including the cell density at the start of culture for all the cell lines, and preferably, all the cell lines are simultaneously cultured. The culture supernatant collected after completion of the culture is filtered through a filter and then subjected to analysis by reverse phase chromatography. The pore size of the filter used at this time is preferably 0.1 to 2.0 μm, for example, 0.45 μm. In addition, the culture supernatant can be frozen and stored for later analysis, rather than immediately subjected to analysis.

In the present invention, the stationary phase that can be used in reversed-phase chromatography for analyzing the culture supernatant is one that can be adsorbed with a recombinant protein when equilibrated with the following first mobile phase. As long as it can be used without particular limitation, it is preferably a chemically bonded porous spherical silica gel whose surface is modified with an Octa Decyl Silyl group (C ₁₈ H ₃₇ Si).

  In the reverse phase chromatography, both the solution A and the solution B, which are mixed or independently form a mobile phase, are aqueous solutions. The ratio of solution B can be increased continuously or intermittently, but is preferably increased continuously, and particularly preferably linearly (ie continuously at a constant rate). To) enhanced. In the present invention, the “first mobile phase” is an equilibration liquid for equilibrating a column before loading a sample. The “second mobile phase” refers to a mobile phase that is passed through the column in order to elute the recombinant protein from the column after passing the first mobile phase through the column. When simply referred to as “mobile phase”, the first mobile phase and the second mobile phase 2 are shown without distinction. In the present invention, a preferred example of the mobile phase is that the first mobile phase is 0.08 to 0.12% trifluoroacetic acid and a 20 to 40% propanol aqueous solution, and the second mobile phase is the first mobile phase. An aqueous solution with the same concentration of trifluoroacetic acid as compared to the phase but with an increased concentration of propanol. In particular, the second mobile phase is compared to the first mobile phase in reverse phase chromatography. Thus, the concentration of propanol is an aqueous solution that can be linearly increased between 20% and 80%. A further preferred example of the mobile phase is that the first mobile phase is 0.08 to 0.12% trifluoroacetic acid and 35% propanol aqueous solution, and the second mobile phase is the first phase in reverse phase chromatography. Compared with the mobile phase, the concentration of propanol is an aqueous solution that is linearly increased from 35% to 70%.

  The analysis result of the culture supernatant of each cell line obtained as a chart by reverse phase chromatography is compared with the chart of serum-free medium analyzed as a control. When the chart of the cell line expressing the recombinant protein is compared with the chart of the serum-free medium, the peak that appears only in the chart of the cell line is identified as the peak of the recombinant protein. The area of this peak is compared for each cell line, and a cell having a relatively large peak area is selected as a high expression cell line. The selected high-expression cell line is further analyzed for the properties of the recombinant protein expressed as desired, and the optimal one is used as a cell for actual production of the recombinant protein.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, it is not intended that the present invention be limited to the examples.

[Preparation of darbepoetin expression vector]
As the expression vector, the pE-neo vector disclosed in the previous document (Japanese Patent Publication No. 2010-511378) incorporating a neomycin resistance gene as a selection marker was used. Darbepoetin (Nesp) is a mutant erythropoietin in which five mutations of Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr are introduced into 165 amino acid residues constituting the peptide chain of natural erythropoietin. Table Hei 08-506023).

  Human erythropoietin cDNA was obtained by PCR using a set of primers EPO-5 ′ (SEQ ID NO: 1) and primer EPO-3 ′ (SEQ ID NO: 2) and a human fetal liver-derived cDNA library (Clontech) as a template. Amplified with Next, mutations of Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr were introduced into the amplified human erythropoietin cDNA by site direct mutagenesis to obtain darbepoetin cDNA. The darbepoetin cDNA was digested with EcoRl and Xbal and inserted between EcoRl and Xbal of the pE-neo vector to obtain a darbepoetin expression vector (pE-neo (DAR)).

[Creation of a cell line expressing darbepoetin]
A darbepoetin expression vector (pE-neo (DAR)) was transfected into CHO cells (CHO-K1: purchased from American Type Culture Collection) using Lipofectamine 2000 (Invitrogen). After transfection, the cells were replaced with D-MEM / F12 (D-MEM / F12 / 5% FCS) medium containing 5% FCS and cultured at 37 ° C. for 2 days under aeration of 5% carbon dioxide gas. Thereafter, the medium was replaced with a D-MEM / F12 / 5% FCS medium containing 0.6 mg / mL G418, and selective culture was performed at 37 ° C. under aeration of 5% carbon dioxide. Cells that proliferated in the selective medium were passaged several times to obtain recombinant cells.

  Next, the recombinant cells were seeded in a 96-well plate under the condition that 1 or less cells per seed were seeded by limiting dilution, and cultured in a selective medium for about 10 days to form a single colony. . Here, three types of cells obtained from a single colony were selected and used as cell lines (cell lines 1 to 3).

[Preparation of culture supernatant]
Commercially available serum-free medium supplemented with 4 mM L-glutamine, 10 mg / L hypoxanthine, 4 mg / L thymidine and 120 mg / L G418 so that each cell line has a cell concentration of 2 × 10 ⁵ cells / mL The sample was diluted with EX-CELL302 medium (JRH), and statically cultured in the presence of 5% CO ₂ at 37 ° C. for 4 days. After culture, the culture supernatant was collected and filtered through a membrane filter having a pore size of 0.45 μm. At this time, only a serum-free medium to which no cells were added was allowed to stand under the same conditions, and this was cultured and filtered through a membrane filter having a hole diameter of 0.45 μm as a control solution.

[Analysis of culture supernatant by reversed-phase column chromatography]
(1) Apparatus Shimadzu HPLC system LC-20A (Shimadzu Corporation) is a reverse phase column, Aeros ^™ widepore XB-C18 (filler: non-porous silica gel coated with porous silica gel, pore size: 300 mm, Column size: 4.6 mm diameter × 250 mm length, Phenomenex) was set, and this column was placed in a 25 ° C. thermostat (column thermostat). In addition, an absorptiometer was installed downstream of the column so that the absorbance (measurement wavelength 215 nm) of the effluent from the column could be continuously measured.

(2) Preparation of solution for mobile phase A solution obtained by adding pure water to 1 mL of trifluoroacetic acid was used as Solution A (0.1% TFA aqueous solution). A solution made up to 1 L by adding pure water to 1 mL of trifluoroacetic acid and 700 mL of 1-propanol was designated as Solution B (0.1% TFA, 70% propanol aqueous solution).

(3) Operational procedure Reverse phase column equilibrated with first mobile phase in which 100 μL each of culture supernatant and control solution of above-filtered cell line was mixed in equal volume of solution A and solution B Loaded. After loading, the volume ratio of solution B is increased linearly to 100% over 30 minutes at a flow rate of 0.2 mL / min, and solution B is allowed to flow at the same flow rate for 10 minutes, and then the first mobile phase is allowed to flow for 35 minutes. Flowed at the same flow rate. During this time, the absorbance (measurement wavelength 215 nm) of the effluent from the column was continuously measured to obtain a chart.

[Evaluation of analysis results]
The charts obtained by analyzing the culture supernatant of the cell line (cell line 1) and the control solution by reverse phase chromatography were compared (FIG. 1). As a result, in the culture supernatant of the cell line, a peak derived from darbepoetin (Peak A), which does not appear in the control solution, was detected, and when the cell line was cultured using a serum-free medium, It was found that cell lines expressing darbepoetin can be distinguished by analyzing the supernatant by reversed-phase column chromatography.

  Next, when the charts obtained by analyzing by reverse phase chromatography were compared for the three types of cell lines (cell lines 1 to 3) obtained by the selective culture, peak areas derived from darbepoetin for each cell line were compared. Differed (FIG. 2). Since the peak area is proportional to the expression level of darbepoetin, it was found that cell line 1, which has the largest peak area among the three cell lines, has the highest expression of darbepoetin. Thus, according to this method, by selecting a cell line that gave an analysis result having a large peak area, a high expression line of darbepoetin can be selected.

  According to the present invention, it is possible to select a cell line that highly expresses a recombinant protein that is used to produce a recombinant protein by culturing in a serum-free medium.

Sequence number 1: Primer EPO-5 ', synthetic sequence
Sequence number 2: Primer EPO-3 ', synthetic sequence
[Sequence Listing]
1189P

Claims (5)

A method for selecting a cell line that highly expresses a recombinant protein, comprising:
(A) transforming a cell with an expression vector incorporating a gene encoding a desired protein and a selectable marker to obtain a transformed cell;
(B) selecting a plurality of cell lines by selectively culturing the transformed cells in the presence of a drug corresponding to the selection marker;
(C) culturing each of the cell lines in a serum-free medium, and collecting the culture supernatant of the cell line after the culturing,
(D) Each of the collected culture supernatants of the cell line is subjected to reverse phase chromatography, and then the effluent from the reverse phase column is continuously guided to the flow path, and the effluent flowing in the flow path In this step , the reverse-phase chromatography is carried out using 0.08 to 0.12% tricyclic as a stationary phase with chemically bonded porous spherical silica gel whose surface is modified with octadecylsilyl groups. A 20 to 40% propanol aqueous solution containing fluoroacetic acid is used as the first mobile phase, and an aqueous solution in which the concentration of propanol is increased while the concentration of trifluoroacetic acid is the same as that of the first mobile phase. Performing as a second mobile phase ;
(E) identifying an absorbance peak corresponding to the recombinant protein by the measurement, and comparing the area of the peak obtained for each culture supernatant of the cell line;
(F) selecting a cell line showing the relatively large area from the cell lines;
Including methods.   The method of claim 1, wherein the cell is a mammalian cell.   The method of claim 2, wherein the mammalian cell is a CHO cell. The recombinant protein is lysosomal enzyme such as α-galactosidase A, iduronic acid 2-sulfatase, glucocerebrosidase, galsulfase, α-L-iduronidase, acid α-glucosidase, tissue plasminogen activator (t-PA), Blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor such as blood coagulation factor IX, erythropoietin, darbepoetin, interferon, thrombomodulin, follicle stimulating hormone, granulocyte colony stimulating factor (G-CSF), antibody, or these The method according to any one of claims 1 to 3 , wherein the method is selected from the group consisting of: The method according to any one of claims 1 to 3 , wherein the recombinant protein is selected from the group consisting of erythropoietin or darbepoetin, or an analog thereof.

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