JP2009249292A - Erythropoietin solution preparation using polyphosphoric acid as stabilizer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stable erythropoietin solution preparation, especially a stable feline erythropoietin solution preparation where conventionally no case of a formulated preparation is available. <P>SOLUTION: This erythropoietin solution preparation containing a polyphosphoric acid as a stabilizer is effective as the erythropoietin solution preparation since it holds the activity of the erythropoietin and is stable for a long period of time. Further it is preferable that the concentration of the polyphosphoric acid is 1 μM to 1 mM, the citric acid buffer solution of pH 6.0 to 7.0 is contained, sodium chloride as an isotonifier is contained, polysorbate 80 as an adsorption inhibitor is contained and maltose as the stabilizer is further contained. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a stable erythropoietin solution formulation.

  Erythropoietin (hereinafter sometimes referred to as “EPO” in the present specification) is a hormone that promotes erythropoiesis and is produced mainly in the kidney. Human EPO is composed of 192 amino acids, of which 27 residues from the N-terminal are signal peptides and 165 residues are the amino acid sequences of mature proteins. N-linked and O-linked glycans It is a modified glycoprotein with a molecular weight of about 34,000 Da. When tissues are subjected to hypoxic stress, EPO increases red blood cell production by promoting the conversion of primitive progenitor cells in the bone marrow to pro-erythroblasts. Other than humans, EPO cDNA cloning of mice, rats, dogs, cats, etc. has been performed (Non-patent Document 1).

  EPO is effective in improving anemia caused by chronic renal failure, chemotherapeutic agents or surgery, and human EPO is used as a pharmaceutical. It should be noted that dogs and cats, which are companion animals, also show anemia similar to that of the above-mentioned humans, which is the main cause of death and disability of these animals. It is used as a medicine (Non-patent Document 2). However, when human EPO is administered to dogs and cats, neutralizing antibodies are expressed due to differences in species, and the effectiveness of EPO administered by these antibodies is diminished or cross-reacts with dogs and cats original EPO. Then, there are problems such as worsening anemia (Non-Patent Document 1).

As a human EPO preparation, there is a solution preparation containing an amino acid instead of a protein as a stabilizer. It is stored at 10 ° C. for 2 years, at 25 ° C. for 6 months or more, and at 40 ° C. for 2 weeks or more. A result of 95% or more is shown (Patent Document 1). However, Patent Document 1 does not describe that polyphosphoric acid can be used as a stabilizer for an EPO solution preparation, and Patent Document 1 does not describe a cat EPO solution preparation.
Japanese Patent Laid-Open No. 10-182481 Ven et al: Blood, 82, 1507 (1993) Cokel et al .: Journal American Better Medical Association (J. Am. Vet. Med Assc), 15, 214 (1998)

  It is an object of the present invention to provide a stable erythropoietin solution preparation, particularly a stable cat erythropoietin solution preparation that has not been formulated so far.

  As a result of intensive studies, the present inventors have newly found that polyphosphoric acid is effective as a stabilizer in an erythropoietin solution preparation, and have completed the invention.

That is, this invention consists of the following structures.
(1) A stable erythropoietin solution formulation comprising polyphosphoric acid as a stabilizer.
(2) The stable erythropoietin solution preparation according to (1), wherein the concentration of polyphosphoric acid is 1 μM to 1 mM.
(3) The stable erythropoietin solution preparation according to (1) or (2), comprising a citrate buffer solution having a pH of 6.0 to 7.0.
(4) The stable erythropoietin solution preparation according to any one of (1) to (3), which contains sodium chloride as an isotonic agent.
(5) The stable erythropoietin solution preparation according to any one of (1) to (4), comprising polysorbate 80 as an adsorption inhibitor.
(6) The stable erythropoietin solution formulation according to any one of (1) to (5), further comprising maltose as a stabilizer.
(7) The stable erythropoietin solution preparation according to any one of (1) to (6), wherein erythropoietin is produced in a recombinant animal cell.
(8) The stable erythropoietin solution preparation according to any one of (1) to (7), wherein the erythropoietin is cat erythropoietin.

  According to the present invention, it is possible to prepare an EPO solution preparation that is stable and has few insoluble fine particles and can be prepared as an injection.

  The EPO used in the present invention is not particularly limited as long as it is derived from a mammal, but is preferably human, dog or cat EPO, more preferably cat EPO. And may have substantially the same biological activity as that of naturally occurring EPO obtained by genetic recombination or naturally derived EPO. EPO obtained by recombination may have the same amino acid sequence as natural EPO, or may have one or more of the amino acid sequences deleted, substituted, or added and having biological activity. . Further, the EPO used in the present invention may be a chemically modified EPO such as polyethylene glycol (PEG).

  The EPO used in the present invention may be produced by any method. For example, EPO may be produced by extraction / separation / purification from urine by various methods, recombinant animal cells obtained by genetic engineering techniques, Although it may be extracted / separated and purified by the method, it is preferably produced from recombinant animal cells, more preferably produced from recombinant CHO cells.

  A production method for producing EPO used in the present invention using recombinant animal cells is described below.

  Examples of the method for producing a recombinant animal cell include a method in which a plasmid incorporating an EPO-encoding DNA, that is, an expression vector, is prepared and transformed into an animal cell by a conventional method. The promoter incorporated into the expression vector may be any promoter that can finally obtain active EPO, such as SV40 early, late, and hCMV, by host animal cells. As the marker gene, neomycin (neo) gene, dihydrofolate reductase (dhfr) gene or the like is used. Examples of the additive substance for selection include G-418 when the marker gene is the neo gene, and methotrexate when the marker gene is the dhfr gene. For transformation into host cells, known methods such as calcium phosphate method and DEAE dextran method are used. A precipitation method such as a lipofectin method, a protoplast polyethylene method, an electroporation method and the like can be used.

  The method for purifying EPO produced from the recombinant animal cells can be purified by culturing animal cells as described above and carrying out several types of column chromatography on the EPO obtained in the culture supernatant. For example, it can be purified by a combination of a copper chelate carrier, an anion exchanger and a gel filtration carrier.

Polyphosphoric acid is suitable as the stabilizer used in the present invention. A preferred polyphosphoric acid is a linear condensed polyphosphoric acid obtained by dehydration condensation of orthophosphoric acid. A straight chain structure in which two or more PO ₄ tetrahedrons share an oxygen atom at the apex and are connected in a straight chain. Examples include chain condensed polyphosphoric acid. The polyphosphoric acid herein may be a polyphosphate having a molecular structure in which hydrogen of the hydroxyl group of polyphosphoric acid is substituted with a metal, and examples of the metal include sodium and potassium. In addition, n showing the polymerization number of phosphoric acid of polyphosphoric acid becomes like this. Preferably it is 15-2000.

  Although it does not specifically limit as content of polyphosphoric acid which is a stabilizer in a formulation, Preferably it is 1 micromol-1 mM. The solution preparation of the present invention is stable even if it contains substantially no protein or amino acid other than EPO, such as serum albumin or gelatin, and therefore preferably does not contain the protein or amino acid.

  In the present invention, it is also one of preferred embodiments that maltose is contained as a stabilizer in addition to the polyphosphoric acid. Maltose is also called maltose, and is a reducing disaccharide in which two α-glucose molecules are α1-4 glycosidic bonds. The maltose content is preferably 110 mg / mL to 440 mg / mL, more preferably 150 mg / mL to 400 mg / mL. When the solution formulation contained 1 μM to 1 mM polyphosphoric acid and 150 mg / mL to 400 mg / mL maltose as stabilizers, the highest residual EPO activity was shown in an accelerated test at 40 ° C. for one month. .

  The solution preparation of the present invention can be preferably prepared by dissolving in a buffer known in the field of solution preparations such as Tris buffer and citrate buffer, but citrate buffer is preferably used from the viewpoint of stability. Is done. The citrate buffer is preferably a sodium citrate buffer. The preferred pH of the buffer is 5.0 to 8.0, more preferably 6.0 to 7.0.

  In the solution preparation of the present invention, an isotonic agent is preferably used, and among them, a salt is preferably used. Specific examples of the salt used as the tonicity agent include sodium chloride or potassium chloride, but a preferred salt in the solution preparation of the present invention is sodium chloride. The preferable content of the tonicity agent is 5 mg to 10 g, more preferably 8 mg to 20 mg.

  Furthermore, an adsorption inhibitor is preferably used in the solution preparation of the present invention. As the adsorption inhibitor, a polyoxyethylene sorbitan alkyl ester surfactant is preferable, and specific examples include polysorbate 20, 21, 40, 60, 65, 80, or 85. Among them, polysorbate 80 is more preferable. A preferable addition amount of the adsorption inhibitor is 0.01 to 1 mg / mL, more preferably 0.02 to 0.1 mg / mL.

  The amount of cat EPO contained in the solution preparation of the present invention can be set according to the type, severity, age, etc. of the disease to be treated, but is generally 100 to 5000 U / mL, preferably 200 to 2000 U. / ML. The solution preparation of the present invention is administered orally, by injection (subcutaneous or intravenous injection), transdermally, transmucosally, nasally, and preferably administered by injection.

  In addition to EPO and polyphosphoric acid, the solution preparation of the present invention includes saccharides such as polyethylene glycol, dextran, D-mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, sucrose, sodium chloride, potassium chloride, chloride Ingredients usually added to solution preparations such as inorganic salts such as calcium, sodium phosphate, potassium phosphate and sodium hydrogen carbonate, organic salts such as sodium citrate, potassium citrate and sodium acetate can also be included. .

  The solution formulation of the present invention is usually contained in a sealed and sterilized plastic or glass container. In the case of an injection, the container is supplied in the form of an ampoule, a vial, a disposable syringe or the like.

  Whether or not the solution preparation of the present invention is a stable solution preparation can be confirmed by measuring the residual activity of EPO during or after the accelerated test at 40 ° C. for 2 months. The residual activity of EPO can be confirmed by measuring according to the method attached to human EPO ELISA kit (manufactured by R & D system) and examining the effect of addition.

  Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of these examples.

Reference Example 1 Preparation of recombinant CHO cells expressing feline EPO and production of feline EPO Inserted feline EPO gene (SEQ ID NO: 1) or mouse dhfr gene (SEQ ID NO: 2) into a commercially available pcDNA3.1 vector (manufactured by Invitrogen) 100 μg and 10 μg of the vector were prepared and mixed with CHO / dhfr − cells 1 × 10 ⁷ cells / 750 μL DMEM / F12 medium prepared. The mixture was allowed to stand at room temperature for 30 minutes, placed in an electroporation apparatus (manufactured by Bio-Rad), and energized at 330 μF and 400V. After standing for 5 minutes, the mixture was further left on ice for 10 minutes, suspended in 10 cc of IMDM medium (manufactured by Invitrogen) containing 10% FBS, hypoxanthine and thymidine, and spread on a 10 cm petri dish. The cells were cultured at 37 ° C. under 5% CO _{2 for} 2 days, and the cells were collected by centrifugation, suspended in 10 cc of IMDM medium (Invitrogen) containing 10% FBS and 0.3 mg / mL G-418, and 100 μL / It processed to the 96-well plate in the ratio of the well. Every 3 days, the medium was replaced with IMDM medium containing 10% FBS and 0.3 mg / mL G-418, and cultured for 15 days. After 15 days, microscopic observation was performed, surviving cells were selected, and colonies were isolated.

The obtained clone was grown in IMDM medium (manufactured by Invitrogen) containing 10% FBS and 0.3 mg / mL G-418 to prepare a 2 × 10 ⁵ cells / 10 cc / 10 cm petri dish, and a final concentration of 5 nM methotrexate (MTX ) Was added. Replace with the above medium containing 5 nM MTX every 3 days and continue culturing for 10-15 days. For cells that are alive and forming colonies, cells are recovered and 2 × 10 ⁵ cells / 10 cc / A 10 cm petri dish was prepared and a final concentration of 50 nM MTX was added. In the same manner, treatment was performed up to 500 nM MTX, and 2 clones that could confirm cell survival were selected.

The obtained animal cell transformant 2 clones (rFeEPO / CHO1, rFeEPO / CHO2) were used to acclimate to a serum-free medium. As a serum-free medium, 20 μg / mL insulin and DMEM / F12 (manufactured by Invitrogen) supplemented with penicillin (100 U / mL) and streptomycin (100 μg / mL) as antibiotics were prepared. The cells subcultured in the IMDM medium (serum medium) containing 10% FBS in (2) above were conditioned by the following method. Serum-free medium: serum medium = 0: 100 (medium A), 50:50 (medium B), and 100: 0 (medium C) mixed solutions were prepared, respectively, and a cell density of 5 was obtained at the time of cell inoculation using a 250 mL Erlenmeyer flask. × was prepared so as to ¹⁰ 5 cells / mL, first 37 ° C. in a medium a, was carried out in the presence of 5% CO ₂ cell culture. Subculture was performed 4-6 times, and then cell culture was carried out on medium B in the presence of 37 ° C. and 5% CO ₂ . Subculture was performed 4-6 times, followed by cell culture in medium C at 37 ° C. in the presence of 5% CO ₂ and subculture was performed 4-6 times. As a result, the obtained cells were designated as rFeEPO / CHO1 ′ or rFeEPO / CHO2 ′, respectively. These cells were insulin-requiring cells that could grow in the presence of 20 μg / mL insulin, and were suspension cells.

Each of the obtained cells was prepared in a 250 mL Erlenmeyer flask so as to have an initial cell density of 5 × 10 ⁵ cells / mL, and cultured in the serum-free medium for 5 days under the conditions of 5% CO ₂ , 37 ° C. and 100 rpm. The productivity of the replacement cat EPO was about 10 mg / L.

Reference Example 2 Detection of Cat EPO Cat EPO in the culture supernatant obtained in Reference Example 1 was detected by Western blotting. The culture supernatant was subjected to SDS-PAGE in an acrylamide gel “Pagel” manufactured by Atoh Co., Ltd. Then, after blotting according to a conventional method on “Clear Blot Membrane” manufactured by Ato Co., Ltd., the membrane was added to a “Block Ace” (Dainippon Pharmaceutical Co., Ltd.) solution containing rabbit serum containing anti-human EPO polyclonal antibody. After reacting for 3 hours, washed with PBS containing 0.02% Tween 20 three times, and further reacted with Block Ace solution containing peroxidase-labeled goat anti-rabbit IgG (Biorat Co., Ltd.) for 6 hours and washed in the same manner Color was developed with Konica Immunostain HRP1000 manufactured by Konica. As a result, a band of about 30 to 35 kDa was detected from the cell culture supernatants of rFeEPO / CHO1 ′ and rFeEPO / CHO2 ′.

Using a human EPO ELISA kit (manufactured by R & D system), quantification of feline EPO in the culture broth was performed. As a result, the production rate of cat EPO was 800 to 1200 U / 10 ⁶ cells / 48 h. The estimated molecular weight of feline EPO is approximately 30 to 35 kDa, and as a result of measurement by human EPO ELISA kit, EPO could be quantified. Therefore, it was determined that feline EPO was expressed in the culture supernatant of Reference Example 1. did.

Reference Example 3 Purification of CHO cell producing cat EPO Concentration / buffer exchange by ultrafiltration method The cell culture supernatant containing feline EPO obtained in Reference Example 1 was removed by ultrafiltration device (Millipore) and a substance having a molecular weight of 10,000 or less and 10 mM Tris containing HCO60. The buffer solution (pH 7) was replaced with a buffer and concentrated 30 times. The membrane used for the ultrafiltration was YM10 (Millipore, molecular weight 10,000 cut).

2. Anion exchange column chromatography Next, DEAE Sepharose (manufactured by Amersham), which is an anion (anion) exchange carrier equilibrated with 10 mM Tris buffer (pH 7) containing HCO60, is prepared, and a solution containing cat EPO concentrated. Was introduced. After thoroughly washing the column, 10 mM Tris-AcOH buffer solution (pH 6) containing HCO60 and 10 mM Tris-AcOH (pH 5) containing HCO60 were passed through to lower the pH in the column to remove impurities. . Next, when pH 5 was reached, 50 mM NaCl / 10 mM Tris-AcOH (pH 5) /0.01% HCO 60, 100 mM NaCl / 10 mM Tris-AcOH (pH 5) /0.01% HCO 60, 200 mM NaCl / 10 mM Tris-AcOH (PH5) /0.01% HCO60, 500 mM NaCl / 10 mM Tris-AcOH (pH5) /0.01% HCO60 was eluted stepwise, and 200 mM NaCl / 10 mM Tris-AcOH (pH5) /0.01% HCO60 The fraction was subjected to the following copper chelate column chromatography.

  The activity recovery rate in this operation was measured by a human EPO ELISA kit and found to be about 50-70%.

3. Copper Chelate Column Chromatography Next, the above-mentioned purified fraction (2) was equilibrated with a 10 mM Tris buffer (pH 5) containing HCO60 in advance to a copper chelating Sepharose Fast Flow (Amersham) to which copper ions were chelated. Thereafter, the liquid was passed. When the column was passed through a column with 10 mM Tris-AcOH buffer (pH 5) containing HCO60, high molecular weight impurities other than cat EPO were removed, and these flow-throughs were collected.

  The activity recovery rate in this operation was measured by human EPO ELISA kit and found to be 70-80%.

4). Gel filtration Finally, the purified fraction (3) was introduced into a Sephacryl S-200 (Amersham) column for desalting. The column was developed with 20 mM sodium citrate / 150 mM sodium chloride / 0.01% HCO 60 at pH 6.8-7.0.

5. Purity, purification yield and specific activity of the obtained cat EPO The cat EPO sample obtained as a result of the above operations 1 to 4 was subjected to SDS-PAGE, and gel staining by silver staining as shown in lane 5 of FIG. The unification of cat EPO was confirmed. Further, as a result of a purity test using a gel stained with an image analyzer (manufactured by Bio-Rad), a purity of 90% could be confirmed. Further, the obtained cat EPO sample was applied to a C4 resin column (manufactured by VYDAC). The measurement conditions were a flow rate of 1 mL / min, gradient elution of 0-80% ethanol / 10 mM Tris-HCl (pH 7), and 230 nm UV absorbance measurement. As a result, elution was observed in a fraction having a retention time of 50-60 min and about 66-80% ethanol, and the purity of the obtained cat EPO was about 90%.

  Furthermore, the final purification yield of the feline EPO obtained as a result of the above operations 1 to 4 was 35-50% as a result of measurement by human EPO ELISA kit. Moreover, as a result of quantifying protein quantification of the gel filtration purification solution using BCA protein assay (manufactured by PIERCE), the specific activity was about 100,000 U / mg. The endotoxin content was 0.9 ng / mg protein.

Reference Example 4 Examination of pH stability using purified cat EPO Cat EPO500U, polysorbate 80 0.05 mg, and sodium chloride 8.5 mg were prepared in 1 mL with 10 mM Tris buffer or 10 mM citrate buffer. The pH of each buffer was adjusted to pH 4, 5, 6, 7, 8, and 9. Each prepared solution was filled in a 2 mL glass vial and left in a constant temperature bath at 40 ° C. for 1 month. The residual activity of feline EPO in the preparation was evaluated using a human EPO ELISA kit. The obtained results are shown in Table 1.

  From this result, it was found that the residual activity of cat EPO was highest at pH 6-7.

Example 1 Effect of Polyphosphate as Stabilizer Cat EPO 500U, polysorbate 80 0.05 mg, and sodium chloride 8.5 mg were prepared in 1 mL with 10 mM citrate buffer (pH 7.0). Furthermore, polyphosphoric acid (manufactured by SIGMA, P8510, polymerization number n = 12) was added so as to have a final concentration of 1 mM, 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM. Each prepared solution was filled in a 2 mL glass vial and left in a 40 ° C. constant temperature bath for 3 months. The residual activity of feline EPO in the preparation was evaluated using a human EPO ELISA kit. The obtained results are shown in Table 2.

  From this result, it was found that the addition of polyphosphoric acid improves the stability.

Example 2 Examination of Storage Stability of Cat EPO Solution Formulation Containing Stabilizers Polyphosphate and Maltose Cat EPO500U, Polysorbate 80 0.05 mg, Sodium Chloride 8.5 mg in 1 mL of 10 mM citrate buffer (pH 7.0) Prepared. Furthermore, maltose was added to a final concentration of 440 mg / mL, and 2 mL of a solution prepared such that polyphosphoric acid (manufactured by SIGMA, P8510, polymerization number n = 12) had a final concentration of 1 mM, 100 μM, and 10 μM was added. The glass vial was filled and left in a constant temperature bath at 40 ° C. for 4 months. The residual activity of feline EPO in the monthly preparation was evaluated using a human EPO ELISA kit. The obtained results are shown in FIG. The activity was maintained at about 100% over 4 months of storage.

Example 3 Acute toxicity test using mice Cat EPO solution preparation prepared in Example 2 (cat EPO 500U, polysorbate 80 0.05 mg, sodium chloride 8.5 mg, maltose 440 mg, 10 mM citrate buffer containing 100 μM polyphosphate) Acute toxicity test was performed using the solution (prepared to 1 mL with pH 7.0). Crj: CD-1 (ICR) mice were administered subcutaneously to 1 group of 5 mice (female). The dose was set at 5 doses of 1 μg / head, 10 μg / head, 100 μg / head, 1 mg / head, 10 mg / head and administered. After the administration, the general condition was observed and the body weight was measured once a week for 14 days, and an autopsy was conducted after 14 days. No abnormal symptoms were observed.

Example 4 Cat EPO Formulation Administration Test to Anemia Model Cat Cat EPO solution formulation prepared in Example 2 (cat EPO 500U, polysorbate 80 0.05 mg, sodium chloride 8.5 mg, maltose 440 mg, 10 mM containing 100 μM polyphosphate) Using citrate buffer (pH 7.0, adjusted to 1 mL), an administration test to an anemia model cat was performed. Anemia model cats were created by subcutaneously administering phenylhydrazine (PHZ) 7 mg / kg twice a week for 1 week before the administration of cat EPO. As a control, a group in which only PHZ was administered and a group in which PHZ and a human EPO preparation (500 U / kg) were administered was set, and a dosage group was set as a cat EPO preparation 100 U / kg administration group and a 500 U / kg administration group, respectively, and subcutaneously and intravenously. (Table 3).

In Control 1, no increase in the number of red blood cells was observed during the dosing period, but an increase in the number of red blood cells could be confirmed in all the treatment groups (including Control 2). In the third week after the start of the administration, the red blood cell count reached 1000 × 10 ⁴ μL, and it recovered to the same level as that of a healthy cat. In addition, the general state observation from the administration to the end and the body weight measurement were performed once a week and after 5 weeks, and no autopsy was observed. Was confirmed.

  According to the invention of the present application, it becomes possible to provide a stable EPO solution preparation, particularly a feline EPO solution preparation for the treatment of cat anemia, and a medicine such as an injection can be provided.

The transition of the cat EPO residual activity in the preservation | save 4 months in presence of polyphosphoric acid is shown.

Claims (8)

  A stable erythropoietin solution formulation comprising polyphosphoric acid as a stabilizer.   2. The stable erythropoietin solution preparation according to claim 1, wherein the concentration of polyphosphoric acid is 1 μM to 1 mM.   The stable erythropoietin solution preparation according to claim 1 or 2, comprising a citrate buffer having a pH of 6.0 to 7.0.   The stable erythropoietin solution formulation according to any one of claims 1 to 3, wherein sodium chloride is contained as an isotonic agent.   The stable erythropoietin solution formulation according to any one of claims 1 to 4, comprising polysorbate 80 as an adsorption inhibitor.   The stable erythropoietin solution preparation according to any one of claims 1 to 5, further comprising maltose as a stabilizer.   The stable erythropoietin solution preparation according to any one of claims 1 to 6, wherein erythropoietin is produced in a recombinant animal cell.   The stable erythropoietin solution preparation according to any one of claims 1 to 7, wherein the erythropoietin is cat erythropoietin.