JP2013153770A - Method for producing recombinant human fsh - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for refining a recombinant human FSH (Follicle-Stimulating Hormone) in high yield and high purity from a culture fluid of recombinant human FSH-producing cells using only steps without use of any organic solvent.SOLUTION: A method for producing the recombinant human FSH includes: a step (a) of culturing recombinant human FSH-producing mammalian cells in a serum-free medium and secreting a recombinant human FSH in a culture fluid; a step (b) of preparing a culture supernatant; a step (c) of subjecting the culture supernatant to cation-exchange column chromatography to retrieve a human FSH-active fraction; a step (d) of subjecting the fraction to dye affinity column chromatography to retrieve a human FSH-active fraction; a step (e) of subjecting the fraction to hydrophobic column chromatography to retrieve a human FSH-active fraction; and then a step (f) of subjecting the fraction to gel filtration column chromatography to retrieve the final recombinant human FSH-active fraction.

Description

  The present invention relates to a method for producing recombinant human FSH, more specifically, a method for producing recombinant human FSH by culturing recombinant human FSH-producing mammalian cells using a serum-free medium, and The present invention relates to a production method for purifying the recombinant human FSH obtained in the culture supernatant to a high purity that can be used as a medicine as it is.

  Follicle-stimulating hormone (FSH) is a glycoprotein with a molecular weight of 34 kD composed of one α and one β subunit, and has an activity of promoting the production and secretion of estrogens in the ovary. A medicine mainly composed of human FSH is used as a therapeutic agent for infertility, and human FSH contained therein was originally purified from human urine. Recently, the sale of recombinant human FSH produced using genetic recombination technology has been recognized, including “regulated ovarian stimulation for multifollicular development” and “hypothalism associated with hypothalamic-pituitary dysfunction and It is used as a fertility treatment using “ovulation induction in rare ovulation” as its usage.

  Genes encoding the α subunit and β subunit of human FSH have been cloned. Among these, the α subunit is the same as the α subunit of human chorionic gonadotropin (hCG) (Patent Document 1 and non-patent document). Reference 1).

  A method for producing recombinant human FSH using mammalian cells (such as CHO cells) transformed with an expression vector incorporating a gene encoding two subunits of human FSH has been disclosed (Patent Document 2). See). Here, genes encoding two subunits of human FSH are incorporated into separate expression vectors and introduced into the same CHO cell. Then, the two subunits expressed in separate expression vectors constitute a heterodimer in the cell, and human FSH having activity is obtained. However, there is no specific description about the purification method of human FSH.

  As a method for purifying recombinant human FSH, blue pigment, hydrophobic, and reversed-phase column chromatography are used in this order from the culture supernatant of recombinant human FSH-producing cells cultured in serum-containing or serum-free medium. (See Patent Document 3). Here, a method for purifying recombinant human FSH by using anion exchange column chromatography following the above chromatography is also disclosed. In any of these methods, 2-propanol is used in the elution of FSH from reverse phase column chromatography, but an organic solvent such as 2-propanol may denature the protein. The use of such an organic solvent is not preferable from the viewpoint of the environment, and a facility for processing the waste liquid containing the organic solvent is required, so that it is not preferable from the economical viewpoint in industrial production.

Moreover, as a method for purifying recombinant human FSH, a method using anion exchange, immobilized metal ion adsorption, hydrophobicity, and reverse phase column chromatography in this order is disclosed (Patent Document 4). Also in this method, 2-propanol is used for elution of recombinant human FSH from reversed-phase column chromatography, and for the same reason as described above, a preferable purification method from the environmental and economic viewpoints. I can't say that. In addition, although it is human FSH derived from urine, a method using anti-human FSH antibody affinity and reverse phase column chromatography is disclosed as a method for purifying human FSH (Patent Document 5). Even in this method, 2-propanol is used in the elution of human FSH from reversed-phase column chromatography, which has the same problem as the above method.

  On the other hand, as a method for purifying recombinant human FSH without using an organic solvent, a method using dye, weak anion exchange, hydrophobicity, strong anion exchange, and hydrophobic column chromatography in this order is disclosed ( Patent Document 6). However, in this document, examples are disclosed only for mutant proteins constructed by inserting other amino acid sequence fragments into wild-type human FSH, instead of wild-type human FSH.

  In addition, a method for purifying human FSH from human urine has been conventionally known (Patent Document 7), but includes a step of using an organic solvent (ethanol), and has the same problems as described above.

Japanese Patent No. 2008444 Japanese Patent No. 2559196 WO2006 / 051070 WO2005 / 063811 Japanese Patent No. 2523843 WO2007 / 065918 JP 2001-323000 A Nature, 286: 684-687 (1980)

  Under the background described above, the present invention has a high purity in which recombinant human FSH can be used as a medicine as it is from the culture medium of recombinant human FSH-producing cells, using only the process that excludes the use of organic solvents. To provide a method for purification with high yield.

  The inventors of the present invention used recombinant human FSH contained in a culture supernatant of a recombinant human FSH-producing cell cultured in a serum-free medium, by cation exchange column chromatography, dye affinity column chromatography. It was found that recombinant human FSH with extremely high purity can be purified with extremely high efficiency by purification by a combination of hydrophobic column chromatography and gel filtration column chromatography. The present invention has been completed by further studies based on these findings.

That is, the present invention provides the following.
1. A method for producing recombinant human FSH comprising:
(A) culturing recombinant human FSH-producing mammalian cells in a serum-free medium to secrete recombinant human FSH into the culture medium;
(B) preparing a culture supernatant by removing the cells from the culture solution;
(C) subjecting the culture supernatant obtained in step (b) to cation exchange column chromatography to recover a recombinant human FSH active fraction;
(D) subjecting the fraction collected in step (c) to dye affinity column chromatography to recover a recombinant human FSH active fraction;
(E) subjecting the fraction collected in step (d) to hydrophobic column chromatography to recover a recombinant human FSH active fraction;
(F) subjecting the fraction collected in step (e) to gel filtration column chromatography to recover a recombinant human FSH active fraction;
A manufacturing method comprising in this order.
2. The above 1. in which the ion exchanger of the cation exchange column chromatography is a weak cation exchanger. Manufacturing method.
3. 3. The production method according to 2 above, wherein the weak cation exchanger has both selectivity based on hydrophobic interaction and hydrogen bond.
4). Item 4. The method according to Item 2 or 3, wherein the weak cation exchanger has a phenyl group, an amide bond, and a carboxyl group.
5. 5. The production method according to any one of 1 to 4 above, wherein the dye of the dye affinity column chromatography is a blue triazine dye.

  According to the present invention, since recombinant human FSH can be produced in cell culture without using serum, recombinant human FSH free from contamination by viruses and prions derived from the use of serum is produced. Can do. For this reason, the recombinant human FSH obtained by the present invention can be administered to the human body as a safe infertility therapeutic agent that completely eliminates the risk of infection by these contaminants. In addition, according to the present invention, since recombinant human FSH can be purified without using an organic solvent, the possibility of denaturation of recombinant human FSH due to contact with an organic solvent is eliminated. Since the organic solvent is not contained in the waste liquid generated in step (1), it is preferable in terms of the environment, and equipment for treating the organic solvent in the waste liquid is not required, which is preferable in terms of economy.

  In the present invention, “recombinant human FSH-producing mammalian cells” are artificially expressed so that genes encoding two subunits of human FSH (α subunit and β subunit) are expressed or strongly expressed. Mammalian cells that have undergone various manipulations. The gene to be strongly expressed at this time is generally introduced into the mammalian cell by transformation with an expression vector incorporating the gene, but is not limited thereto, and the endogenous gene is strongly expressed. It may be an artificially modified one. As a means of artificially modifying the endogenous gene so that it can be strongly expressed, a method can be used in which the promoter existing upstream of the endogenous gene is replaced with a promoter that induces strong gene expression. I can't. The mammalian cells are not particularly limited, but cells derived from humans, mice and hamsters are preferred, and CHO cells derived from Chinese hamster ovary cells are particularly preferred.

  In the present invention, “recombinant human FSH” refers to human FSH that is secreted into the medium when the above-described recombinant human FSH-producing mammalian cells are cultured.

In the present invention, the serum-free medium for culturing recombinant human FSH-producing mammalian cells includes, 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.

  As a serum-free medium, a DMEM / F12 medium (mixed medium of DMEM and F12) may be used as a basic medium, and each of these mediums 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 CDoptiCHO, CHO-S-SFM II or CD CHO (Invitrogen), IS CHO-V or IS CHO-V-GS (Irvine), EX-CELL302 or EX-CELL305 ( JRH) may be used as the basic medium.

  Depending on the situation, each chromatography for purification of recombinant human FSH can be performed in the presence of a nonionic surfactant to prevent nonspecific adsorption of proteins. The nonionic surfactant is not particularly limited, but a polysorbate surfactant is preferably used, and polysorbate 80 is more preferably used. The concentration of the nonionic surfactant is preferably 0.005% (w / v) to 0.015% (w / v), more preferably 0.01% (w / v).

  Human FSH purification can be performed in a room temperature or low temperature environment, but is preferably performed in a low temperature environment, particularly 1 to 10 ° C.

  In the first chromatography step of purification, recombinant human FSH is bound to a cation exchange column equilibrated with a phosphate buffer to which salts have been added. At this time, the pH of the phosphate buffer is preferably adjusted to 5 to 6.5, more preferably about 5.5 to 6.0. The salt added to the phosphate buffer at this time is not particularly limited, but is preferably sodium chloride, and its concentration is preferably 50 to 250 mM.

  After washing the cation exchange column to which the recombinant human FSH is bound, the recombinant human FSH is eluted with a phosphate buffer having an increased salt concentration. At this time, the pH of the phosphate buffer is preferably adjusted to 5.5 to 6.5, more preferably around 6.0. The salt added to the phosphate buffer at this time is not particularly limited, but is preferably sodium chloride, and its concentration is preferably 300 to 900 mM, more preferably 400 to 800 mM.

  The ion exchanger used for cation exchange column chromatography is not particularly limited, but is preferably a weak cation exchanger, and more preferably a weak cation exchanger having both hydrophobic interaction and selectivity based on hydrogen bonds. For example, CaptoMMC (GE Healthcare) or the like can be used as a weak cation exchanger having a phenyl group, an amide bond, and a carboxyl group, and having selectivity based on hydrophobic interaction and hydrogen bond.

Dye affinity chromatography in the second step of purification is for removing contaminants by utilizing the strong affinity of human FSH for a specific dye. Blue triazine dyes are preferably used, but other triazine dyes are also suitable. Particularly preferred is the blue Cibacron ^™ Blue F3GA, covalently immobilized on a Sepharose 6 Fast Flow matrix.
Sepharose 6 FF, Fast Flow (GE Healthcare).

  The dye affinity chromatography column is equilibrated to near neutral, preferably pH 7.8 to 8.2 with a buffer, and this is subjected to the elution fraction of the first step. At that time, the elution fraction in the first step is previously adjusted with a buffer solution to adjust the pH to near neutral, preferably pH 7.8 to 8.2. Elution can be performed by increasing the salt concentration. The salt used for elution is not particularly limited, but is preferably potassium chloride, and its concentration is preferably 1.8 to 2.2 mol / L, particularly preferably about 2 mol / L.

  Hydrophobic chromatography in the third step of purification is for removing contaminating proteins and the like. The hydrophobic chromatography exchange resin is not particularly limited, but Phenyl-Sepharose can be suitably used.

  It is necessary to adjust the salt concentration of the fraction containing human FSH to be subjected to hydrophobic chromatography. The salt used at this time is not particularly limited, but is preferably sodium chloride or potassium chloride. The concentration is preferably 2.5 to 3.5 mol / L, more preferably 2.8 to 3.2 mol / L in terms of sodium chloride ion concentration. The column is adjusted to near neutral, preferably pH 7.8 to 8.2, more preferably about 8 with a buffer solution to which salt is added. The salt added at this time is not particularly limited, but is preferably sodium chloride, and its concentration is preferably 2 to 3 mol / L, more preferably 2.3 to 2.7 mol / L, and particularly preferably about 2.5 mol. / L.

  Elution can be performed by reducing the salt concentration. The salt used for elution is not particularly limited, but is preferably sodium chloride, and the concentration is preferably 1.2 to 1.8 mol / L, more preferably about 1.4 to 1.6 mol / L.

  In the fourth step of purification, gel filtration column chromatography is for removing low-molecular impurities such as endotoxin, multimers of human FSH, degradation products, etc. Human FSH is obtained.

  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.

[Construction of human FSH expression vector]
The pEF / myc / nuc vector (Invitrogen) was digested with KpnI and NcoI, and the region containing the EF-1α promoter and its first intron was excised.
It was blunt-ended with DNA polymerase. pCI-neo (Invitrogen) was digested with BglII and EcoRI to excise the region containing the CMV enhancer / promoter and intron, and then blunt-ended with T4 DNA polymerase. A region containing the EF-1α promoter and its first intron was inserted into this to construct a pE-neo vector (FIGS. 1-1 and 1-2).

  The pE-neo vector was digested with SfiI and BstXI, and an approximately 1 kbp region containing the neomycin resistance gene was excised (FIG. 2-1). Using pcDNA3.1 / Hygro (+) (Invitrogen) as a template, primer Hyg-Sfi (5'-GAGGCCGCCTCGGCCTCTGA-3 '; SEQ ID NO: 1) and primer Hyg-BstX (5'-AACCATCGTGATGGGTGCTATTCCTTTGC-3'; SEQ ID NO: 2 ) Was used to amplify the hygromycin gene by PCR reaction (FIG. 2-2). The amplified hygromycin gene was digested with SfiI and BstXI and inserted into the above pE-neo vector to construct a pE-hygr vector (FIG. 2-3).

Using human placenta cDNA library (Takara Bio) as a template, primer HCG-AF (5'-ATCCTGCAAAAAGCCCAGAG-3 '; SEQ ID NO: 3) and primer HCG-AR
A primary PCR reaction was performed using (5′-CTTGAAGCGTGTCAAAGTGG-3 ′; SEQ ID NO: 4). Furthermore, using the obtained PCR product as a template, a primer HCGA-ORF-F (5'-GCGAATTCGCCACCATGGATTACTACAGAA-3 '; SEQ ID NO: 5) having a sequence slightly downstream from the primary reaction primer HCG-AF, and primary Using a primer HCGA-ORF-R (5'-GCGAATTCTTAAGATTTGTGATAAT-3 'SEQ ID NO: 6) having a sequence slightly upstream from the reaction plumer HCG-AR, a secondary PCR reaction was carried out to obtain the cDNA of the human FSH α chain. Amplified. Similarly, the primary plumer FSH-F (5'-GACCACAGGTGAGTCTTGGC-3 '; SEQ ID NO: 7) and FSH-R (5'-TGGTCCTTCAGGACAAGGGT-) using human pituitary cDNA library (Takara Bio) as a template. PCR reaction using 3 ′; SEQ ID NO: 8) and secondary primer FSH-F2 (5′-GCGAATTCGCCACCATGAAGACACTCCAGT-3 ′; SEQ ID NO: 9) and FSH-R2 (5′-TAAGAATGCGGCCGCCCACTGATCTTTATT-3 ′; SEQ ID NO: 10) Was used to amplify cDNA of human FSH β chain.

  The primary PCR reaction of the human FSHα chain was carried out for 40 cycles, using 100 ng of human placenta cDNA library as a template and (95 ° C./10 seconds, 55 ° C./10 seconds, 72 ° C./10 seconds) as one cycle. In the secondary PCR reaction, 1 μL of the reaction solution of the primary PCR reaction was used as a template, and (95 ° C./10 seconds, 60 ° C./10, 72 ° C./10 seconds) was used as a cycle for 30 cycles. The primary PCR reaction of human FSH β chain was carried out for 40 cycles with 10 ng of human pituitary cDNA library as template and (98 ° C / 2 seconds, 60 ° C / 10, 72 ° C / 10 seconds) as one cycle. It was. In the secondary PCR reaction, 1 μL of the reaction solution of the primary PCR reaction was used as a template, and (98 ° C./2 seconds, 60 ° C./10, 72 ° C./10 seconds) was used as a cycle for 30 cycles.

  The amplified human FSH α chain cDNA was cleaved with EcoRI and then inserted into the EcoRI site of pBluescriptIISK (−) (pBS: Stratagene) digested with EcoRI. The obtained plasmid DNA was digested with XbaI and EcoRV, and the human FSHα chain cDNA was excised and inserted into the pE-neo vector digested with XbaI and Smal to obtain a human FSHα chain expression vector pE-neo (hCGα). (Figure 3).

Amplified human FSH β chain cDNA was digested with EcoRI and NotI after digestion with EcoRI and NotI.
It was inserted into pBluescriptIISK (-). The resulting plasmid DNA was digested with EcoRI and T4
After blunting treatment with DNA polymerase, digestion with NotI cut out human FSH β chain cDNA. The pE-hygr vector was digested with XbaI, blunt-ended with T4 DNA polymerase, and then digested with NotI. Human FSHβ chain cDNA was inserted into this pE-hygr vector to obtain a human FSHβ chain expression vector pE-hygr (FSHβ) (FIG. 4).

[Preparation of recombinant cells for human FSH expression]
The expression vectors pE-neo (hCGα) and pE-hygr (FSHβ) were transfected into CHO cells (CHO-K1: purchased from American Type Culture Collection) using Lipofectamine 2000 (lnvitrogen) by the following method. . That is, CHO-K1 cells were seeded in a 3.5 cm culture dish so that the cell density was close to confluence on the day before transfection, and cultured overnight at 37 ° C. in a 5% carbon dioxide aeration. The next day, after washing twice with PBS (−), the serum was replaced with 1 mL of D-MEM / F12 medium (Invitrogen) without serum. 200 μL of Lipofectamine 2000 solution diluted 20-fold in Opti-MEM I medium (Invitrogen) and plasmid DNA solution (pE-neo (hCGα) 13.2 μg / mL / pE-hygr (FSHβ) 6.6 μg / mL) Added and transfected at 37 ° C. for 5 hours.

  After transfection, the medium was replaced with a 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 culture medium was replaced with D-MEM / F12 / 5% FCS medium containing 0.6 mg / ml G418 and 0.4 mg / ml hygromycin B, 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 no more than one cell per well was seeded by limiting dilution, and cultured for about 10 days to form a single colony. The culture supernatant in the hole where a single colony was formed was collected, the FSH expression level was examined by ELISA, and a high expression strain of human FSH was selected.

In order to acclimate the selected cell line to serum-free floating cells, L-glutamine 4 mM, hypoxanthine 10 mg / L, thymidine 4 mg / L, G418 120 mg / L, hygromycin B 80 mg / L Subculture was performed until the cell growth was stabilized in the serum-free medium EX-CELL302 medium (JRH). Furthermore, the medium is switched to IS CHO-V-GS medium, and the culture is continued until the cell growth rate is stabilized. This is suspended in IS CHO-V-GS medium supplemented with 10% DMSO and liquid nitrogen is added. The seed cells were stored.

[Culture of recombinant cells for human FSH expression]
The seed cells were thawed, diluted to a concentration of 2 × 10 ⁵ cells / mL, and first cultured in IS CHO-V-GS medium for 3 days. Next, IS CHO-V-GS medium, CDoptiCHO (CD) supplemented with 8 mM L-glutamine, 10 mg / L hypoxanthine, 4 mg / L thymidine, 0.12 mg / mL G418 and 80 mg / L hygromycin B medium (Invitrogen) 1: 4 days by diluting 1 of the cell concentration in the mixed medium in a ratio to 2 × 10 ⁵ cells / mL, and diluting the cell concentration to 2 × 10 ⁵ cells / mL in yet CD medium The cells were statically cultured for 4 days at 37 ° C. in the presence of 5% CO ₂ and expanded.

The number of cells was counted, diluted with CD medium so that the cell concentration was 5 × 10 ⁵ cells / mL, and 5 L of the cell solution was transferred to an incubator and cultured with shaking for 3 days. Further, 20 L of fresh CD medium was added to the incubator and cultured with shaking for 3 days. The culture conditions at this time were: stirring speed 60 rpm, pH 7.2, dissolved oxygen 70%, temperature 37 ° C., top aeration (air) 500 mL / min, submerged aeration (C 0 ₂ ) 50 mL / min, submerged aeration (0 ₂ ) 50 mL / min.

Next, the cell culture medium of the above incubator is diluted with a CD medium not containing G418 and hygromycin B so as to be 2 × 10 ⁵ cells / mL, and 250 L of the cell culture medium is put into a 250 L incubator. The cells were transferred and cultured with shaking for 7 days. The culture conditions at this time were: stirring speed 120 rpm, pH 7.2, dissolved oxygen 70%, temperature 37 ° C., top aeration (air) 10 L / min, submerged aeration (air) 250 mL / min, submerged aeration (C 0 ₂ ) 2500 mL / min, liquid aeration (0 ₂ ) was 2500 mL / min. During the culture, sampling was performed every day, and the number of cells, survival rate, glucose concentration, lactate concentration, and expression level of human FSH were measured.

The cell culture was repeated three times (lot numbers 1 to 3). In either case, the viable cell density reached approximately 1 × 10 ⁷ cells / mL or more over the 6th to 7th days of culture, indicating that high-density cell culture was achieved (FIG. 5). When the concentration of human FSH secreted from the cells into the medium was measured by ELISA, the concentration increased with a slight time lag in the cell growth curve. L was reached (FIG. 6).

The cell culture solution was collected, the cells were removed using a Zeta Plus ^™ filter cartridge 90MZ03M (3M), and further filtered through a 0.22 μm filter to obtain a culture supernatant.

[Method for purifying human FSH]
6N HCl was added to the collected culture supernatant to adjust the pH of the culture supernatant to 5.5, and the precipitate was removed by filtration. This culture supernatant is equilibrated with a solution of 50 mM sodium phosphate / 100 mM NaCl (pH 5.5) 4 times the volume of the column, and is a cation exchange column that combines hydrophobic interaction and selectivity based on hydrogen bonding. A CaptoMMC column (column volume; about 10 L, bed height: about 20 cm) was loaded and adsorbed at a linear flow rate of 150 cm / hour. Subsequently, the column was washed with a 50 mM sodium phosphate / 100 mM NaCl (pH 5.5) solution of 4 times the column volume at the same flow rate, and then 5 mM 50 mM sodium phosphate / 400 mM NaCl (pH 6.0). ) The adsorbed protein was eluted with the solution.

  Next, as a virus inactivation step, tri-n-butyl phosphate (TNBP) and the above-mentioned captoMMC column elution fraction were added to the final concentrations of 0.3% (v / v) and 1% (w / v), respectively. Tween 80 was added and allowed to stand at room temperature for 6 hours.

About 40% by volume of 250 mM Tris-HCl (pH 8.5) was added to the fraction eluted from the above-mentioned virus-inactivated CaptoMMC column to adjust the conductivity to about 2.6 S / m. This solution was added to 4 column volumes of 50 mM Tris-HCl / 300 mM.
Blue, a dye column equilibrated with NaCl (pH 8.0) solution
A sepharose 6FF column (column volume: about 6 L, bed height: about 20 cm) was loaded and adsorbed at a linear flow rate of 60 cm / hour. Subsequently, at the same flow rate, the column volume is 4 times 50 mM Tris-HCl / 500 mM.
After washing the column with KCl (pH 8.0) solution, 50 mM of 5 column volumes
The adsorbed protein was eluted with a Tris-HCl / 2M KCl / 0.01% (w / v) Tween 80 (pH 8.0) solution.

Next, an equal amount of 4M NaCl solution was added to the blue sepharose 6FF column elution fraction to prepare a salt concentration. This solution was added to a column volume of 4 times 50 mM Tris-HCl / 2M.
A phenyl sepharose HP column (column volume: about 6 L, bed height: about 20 cm) equilibrated with a NaCl (pH 8.0) solution was loaded at a linear flow rate of 60 cm / hour for adsorption. Subsequently, the column was washed with 50 mM Tris-HCl, 2.5 M NaCl (pH 8.0) solution in a column volume of 4 times at the same flow rate, and then 5 column volume of 50 mM Tris-HCl, 1.6 M NaCl (pH 8.0). ) The adsorbed protein was eluted with the solution.

  Subsequently, the fraction eluted from the Phenyl sepharose HP column was sequentially concentrated with a Biomax 5 membrane (5 kDa cutoff, Millipore). This concentrated solution was added to 75 pg of Superdex peptide (column volume: about 4.8 L, bed height: about 60 cm) equilibrated with 20 mM sodium phosphate / 133 mM NaCl / 0.01% (w / v) Tween 80 (pH 7.4) solution. The sample was loaded at a linear flow rate of 14.9 cm / hour, and the absorbance peak at 280 nm (FIG. 7) was separated and collected as purified human FSH.

  The recovery rate of human FSH in each purification step is shown in Table 1. Here, the process FSH recovery rate refers to the ratio of the recovered human FSH amount to the loaded human FSH amount in each step, and the total FSH recovery rate is the human at the start of the purification process. The ratio of the amount of human FSH recovered in each process to the amount of FSH.

The amount of human FSH subjected to the cation column chromatography as the first purification step was 2256.1 mg, but 1043.5 mg of human FSH was finally recovered, and the recovery rate reached 43.9%. From this, it was found that a large amount of recombinant human FSH can be purified with a high yield by the above purification method.

[Analysis of purified human FSH]
The purified human FSH was subjected to SDS-PAGE electrophoresis under non-reducing and non-heating conditions. As a result of Coomassie staining of the gel, only a single band was observed at a molecular weight of about 45 kDa (FIG. 8). This band was stained by Western blotting using anti-human FSH antibody and was of human FSH (data not shown).

  Moreover, when the above purified human FSH was analyzed by RP-HPLC, it was purified to a high purity although a peak 2 derived from the α chain and a peak 2 derived from the β chain showed a peak 2 with an area ratio of about 1%. It was found to be human FSH (FIG. 9). Peak 2 was found to be a peptide chain derived from the α chain from N-terminal amino acid sequence analysis and amino acid composition analysis.

The purified human FSH was analyzed by isoelectric focusing.
Multiple bands were observed between 3.5 and 5.5 (FIG. 10). This was similar to the previously reported isoelectric focusing pattern of recombinant FSH (Loumaye E., Human Reprod. Update 4: 862-881, 1998).

  When the specific activity of the purified human FSH was measured by the rat ovary weight increase method, it was 14,000 to 18,000 IU / mg. This value was equal to or higher than the specific activity of recombinant human FSH preparations already sold as pharmaceuticals in Japan (de Leeuw R., Mol. Hum. Reprod. 2: 361-369, 1996). .

[Quantification of human FSH by ELISA]
100 μL of anti-human FSHα subunit antibody (Leinco Technologies) diluted to 1 μg / mL with 0.05 M NaHCO ₃ solution was added to each well of a 96-well microtiter plate for EIA (NUNK) and left at room temperature for 2 hours or longer. And adsorbed. Next, after each well was washed three times with PBS containing 0.05% Tween 20 (PBS-T), each well was appropriately diluted with 0.5% BSA and 0.05% Tween 20 in PBS (PBS-BT) or 100 μL of human FSH standard (Sigma) was added and allowed to stand at room temperature for 2 hours or more. Next, after washing each well 3 times with PBS-T, 100 μL of PO-labeled anti-human FSH β chain antibody (Leinco Technologies) diluted 1000-fold with PBS-BT was added to each well, and at room temperature for 2 hours or more Left to stand. Next, each well was washed three times with PBS-T, and then 100 μL of the substrate solution was added to each well, and an enzyme reaction was performed at room temperature. As a substrate solution, 0.025 M citric acid and 0.05 M Na ₂ HPO ₄ were mixed to adjust the pH to 5.0, and 0.4 mg / mL orthophenylenediamine (OPD) and 0.008% H ₂ O ₂ were contained. What was added was used. Next, 100 μL of 2N H ₂ SO ₄ was added to each well to stop the reaction, and the absorbance at 492 nm of each well was measured with a 96-well plate reader.

[Analysis of human FSH by RP-HPLC method]
High performance liquid chromatography was performed using LC-20A System, SPD-20AV UV / VIS Detector (Shimadzu Corporation). TSKgel Octadecyl-4PW (4.6 mm diameter x 150 mm length, Tosoh Corporation) was equilibrated with a 4% acetonitrile solution containing 0.1% trifluoroacetic acid. This was loaded with a sample and eluted with a linear increase in acetonitrile concentration from 4% to 18%. For detection, absorbance at 214 nm was measured.

[Measurement of human FSH activity by rat ovary weight increase method]
Human FSH activity was measured by the rat ovary weight increase method (Steelman S., Endocrinology 53 (6): 604-616, 1953). Female SD rats (Nippon Charles River) weighing about 45-65 g were bred and acclimatized for 3 days after arrival. Rats received test sample or human FSH standard diluted to 0.2 mL with sample diluent, subcutaneously 6 times in the afternoon of 1st day, 2nd morning, noon, afternoon, 3rd morning and afternoon Administered. On the fifth day, the rat ovaries were removed and weighed. Human FSH standards include “First international standard for Follicule Stimulating Hormone, (FSH)
Recombinant, Human for Bioassay; NIBSC code 92/642; WHO ". The sample diluent was dissolved in 2 amps of the attached dissolution solution per ampoule (5000 IU / mL) in 3 ampules of hCG preparation (10000 IU, Japanese Pharmacopoeia), and 4.0 mL of the resulting solution and 3.0 g of bovine serum albumin Was dissolved in an appropriate amount of phosphate buffer (pH 7.1), adjusted to a total volume of 250 mL with the above buffer, and then filtered through a 0.22 μm membrane filter. Four test samples with different dilution ratios were administered to 5 rats per group and the titer was estimated, and then this test was performed. In this study, the ovarian mass estimated to be 95 to 105 mg is the high concentration group, the low concentration group is 1.5 to 2 times the dilution concentration of the high concentration group, 10 mice per group, 2 The activity of human FSH was measured by a -2 dose assay.

The method for producing recombinant human FSH of the present invention can prevent contamination of viruses by culturing recombinant human FSH-producing mammalian cells using a serum-free medium. Since the recombinant human FSH can be purified in high yield to a high purity that can be used as a medicine as it is, it has high utility as a production method of a recombinant human FSH.

The flow chart of the construction method of pE-neo vector is shown. The flow chart of the construction method of pE-neo vector is shown. The flow chart of the construction method of pE-hygr vector is shown. The flow chart of the construction method of pE-hygr vector is shown. The flow chart of the construction method of pE-hygr vector is shown. The structural diagram of human FSHα chain expression vector pE-neo (hCGα) is shown. The structural diagram of the human FSHβ chain expression vector pE-hygr (FSHβ) is shown. The living cell density of the recombinant cell for human FSH expression at the time of serum-free culture is shown. The concentration of human FSH in the medium during serum-free culture is shown. The pattern of absorbance at 280 nm in gel filtration columnography is shown. The pattern of the SDS-PAGE gel electrophoresis of the purified human FSH under non-reducing and non-heating conditions is shown. About 5 μg of human FSH was applied to each lane. In Lanes 1-3, the final purified products of lot numbers 1 to 3 were applied. An RP-HPLC chart of purified human FSH (lot number 1) is shown. 2 shows the isoelectric focusing pattern of purified human FSH. Approximately 10 μg of human FSH was applied to each lane. In Lanes 1-3, the final purified products of lot numbers 1 to 3 were applied.

Claims (7)

A method for producing recombinant human FSH that can be used as a medicine, comprising:
(A) an expression vector in which human FSHα chain cDNA is incorporated downstream of the EF-1α promoter and its first intron, and an expression vector in which human FSHβ chain cDNA is incorporated downstream of the EF-1α promoter and its first intron. Each building step,
(B) transfecting the expression vectors into mammalian cells to produce recombinant human FSH-producing mammalian cells;
(C) culturing the cells in a serum-free medium to secrete recombinant human FSH into the culture medium;
(D) preparing a culture supernatant by removing the cells from the culture solution;
(E) The culture supernatant obtained in the above step (d) is subjected to cation exchange column chromatography equilibrated with a buffer adjusted to pH 5 to 6.5 containing 50 to 250 mM salt, and then recombinant. Binding human FSH to the column and then recovering the recombinant human FSH active fraction with a buffer containing 300-900 mM salt and adjusted to pH 5-6.5;
(F) The fraction collected in the above step (e) is subjected to dye affinity column chromatography equilibrated with a buffer solution at pH 7.8 to 8.2 to adsorb recombinant human FSH to the column. And then recovering the recombinant human FSH active fraction with a buffer of pH 7.8-8.2 containing 1.8-2.2 M salt;
(G) The fraction collected in the above step (f) was subjected to phenyl sepharose column chromatography equilibrated with a buffer adjusted to pH 7.8 to 8.2 to give recombinant human FSH to the column. Recovering the recombinant human FSH active fraction with a buffer adjusted to pH 7.8-8.2 containing 1.2-1.8 M salt, and
(H) subjecting the fraction collected in step (g) to gel filtration column chromatography to recover a recombinant human FSH active fraction;
Ri name include in this order,
However I production method der in which organic solvent is not used in step (c) ~ (h),
The recombinant human FSH obtained by the production method has a purity that can be used as a medicine as it is, and has a specific activity of 14,000 to 18000 IU / mg.   The process according to claim 1, wherein the ion exchanger of the cation exchange column chromatography is a weak cation exchanger.   The process according to claim 2, wherein the weak cation exchanger has both hydrophobic interaction and selectivity based on hydrogen bonds.   The process according to claim 2 or 3, wherein the weak cation exchanger has a phenyl group, an amide bond and a carboxyl group.   The method according to any one of claims 1 to 4, wherein the dye of the dye affinity column chromatography is a blue triazine dye.   The purity which can be used as a medicament as it is is such that when the recombinant human FSH is analyzed by RP-HPLC, peaks other than the α chain peak and the β chain peak remain at about 1% by area ratio. Or the manufacturing method of any one of 5. The production method according to any one of claims 1 to 6, wherein the specific activity is measured by a rat ovary weight increase method.