JP4454571B2 - Protein-free formulation - Google Patents

Description

The present invention relates to a granulocyte colony stimulating factor-containing preparation, and in particular, a granulocyte colony stimulating factor-containing preparation which is stabilized by preventing loss or inactivation of active ingredients due to adsorption or association onto a container wall, polymerization, oxidation, etc. About.

  Granulocyte colony-stimulating factor (hereinafter sometimes referred to as G-CSF) is a glycoprotein having a molecular weight of about 20,000 that acts on neutrophil progenitor cells and promotes their proliferation and differentiation and maturation.

  Since the applicant has purified high-purity human G-CSF by culturing cell lines collected from tumor cells of oral floor cancer patients, this has led to the successful cloning of the human G-CSF gene. At present, it has become possible to produce recombinant human G-CSF in large quantities in animal cells by genetic engineering methods. The applicant of the present application has succeeded in formulating the purified G-CSF, and supplies the product to the market as an infection-protecting agent (Japanese Patent No. 2165515).

  G-CSF is used in a very small amount, and usually a preparation containing 0.1 to 1000 μg (preferably 5 to 500 μg) of G-CSF is administered at a rate of 1 to 7 times / week per adult. However, this G-CSF exhibits adsorptivity to the walls of, for example, injection ampoules and syringes. G-CSF is unstable and susceptible to external factors, causing physical and chemical changes such as association, polymerization or oxidation due to temperature, humidity, oxygen, ultraviolet rays, etc. It causes a decrease in activity.

  Therefore, various prescription designs have been made in order to supply a stable G-CSF preparation to the market. For example, a preparation containing a buffer selected from acetic acid, lactic acid, citric acid, maleic acid, phosphoric acid and a salt thereof, or arginine and a salt thereof (Japanese Patent Publication No. 8-505610) has been proposed. Further, there is a G-CSF preparation containing 1 part by weight to 10,000 parts by weight of a surfactant with respect to 1 part by weight of G-CSF as a stabilizer (Japanese Patent Laid-Open No. 63-146826). This publication describes that in a G-CSF-containing solution formulation, the amount of surfactant added, particularly its lower limit, is critical to prevent loss of G-CSF and achieve stabilization. ing.

An object of the present invention is to provide a preparation of G-CSF that reduces the complexity of the production process and is more stable for long-term storage.

  As a result of earnest research to achieve the above object, the present inventors have found that even when a very small amount of a surfactant is added as a stabilizer, a stable G-CSF solution preparation can be obtained. Completed the invention.

  That is, the present invention comprises granulocyte colony stimulating factor and 0.0001 to 1 part by weight of at least one pharmaceutically acceptable surfactant with respect to 1 part by weight of granulocyte colony stimulating factor, at a pH of 7 or less. There is provided a stable granulocyte colony stimulating factor-containing preparation.

In this specification, the term “stabilized” means that the G-CSF-containing solution preparation has a residual ratio of G-CSF of 95% or more after 6 months storage at 25 ° C. or 2 weeks under storage at 40 ° C. It means that the G-CSF remaining rate after storage is maintained at 75% or more.

  The G-CSF used in the solution preparation of the present invention can be any human G-CSF purified to a high purity. Specifically, those having substantially the same biological activity as that of mammals, particularly human G-CSF, include those derived from nature and those obtained by gene recombination methods. G-CSF obtained by a genetic recombination method has the same amino acid sequence as that of natural G-CSF, or has one or more of the amino acid sequences deleted, substituted, or added, and exhibits the biological activity. Including what you have. The G-CSF in the present invention may be produced by any method, and a human tumor cell line is cultured, extracted from various methods and separated and purified, or E. coli, yeast, Those produced by Chinese hamster ovary (CHO) cells, C127 cells, etc., extracted by various methods, separated and purified are used. Most preferred are those produced by CHO cells using genetic recombination methods.

  Examples of the surfactant used to obtain the stable G-CSF-containing preparation of the present invention include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate; glycerin Glycerin fatty acid esters such as monocaprylate, glycerin monomylate and glycerin monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate , Polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, Polyoxyethylene sorbitan fatty acid esters such as oxyethylene sorbitan tristearate; Polyoxyethylene sorbite fatty acid esters such as polyoxyethylene sorbite tetrastearate and polyoxyethylene sorbit tetraoleate; Polyoxy such as polyoxyethylene glyceryl monostearate Ethylene glycol fatty acid ester; polyethylene glycol fatty acid ester such as polyethylene glycol distearate; polyoxyethylene alkyl ether such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxy Polyoxyethylene poly, such as ethylene polyoxypropylene cetyl ether Xoxypropylene alkyl ether; polyoxyethylene alkylphenyl ether such as polyoxyethylenelenyl phenyl ether; polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogen castor oil); Polyoxyethylene beeswax derivatives such as polyoxyethylene sorbit beeswax; Polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; Polyoxyethylene fatty acid amides such as polyoxyethylene stearamide; HLB 6-18 having an anion interface; An activator, for example, an alkyl sulfate having an alkyl group having 10 to 18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate; Polyoxyethylene alkyl ether sulfates having an average added mole number of ethylene oxide of 2 to 4 and an alkyl group of 10 to 18 carbon atoms such as sodium rilsulfate; carbon atoms of the alkyl group such as sodium lauryl sulfosuccinate Typical examples include alkylsulfosuccinic acid ester salts having 8 to 18 numbers; natural surfactants such as lecithin, glycerophospholipid; fingophospholipids such as sphingomyelin; and sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. Can be mentioned. One or more of these surfactants can be added to the solution preparation of the present invention in combination.

  Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, particularly preferred are polysorbates 20, 21, 40, 60, 65, 80, 81, 85, and most preferred are polysorbates 20 and 80.

  The amount of the surfactant added to the G-CSF-containing preparation of the present invention is generally 0.0001 to 1 part by weight with respect to 1 part by weight of G-CSF, preferably 0 with respect to 1 part by weight of G-CSF. 0.01 to 1 part by weight, more preferably 0.2 to 1 part by weight with respect to 1 part by weight of G-CSF, more preferably 0.2 to 0.8 part by weight with respect to 1 part by weight of G-CSF. And most preferred is 0.4 to 0.8 parts by weight per 1 part by weight of G-CSF. In particular, when the content of G-CSF per 1 mL of the preparation is 125 μg or 250 μg, the addition amount of the surfactant is preferably 100 μg. Therefore, the addition amount of the surfactant of 0.4 part by weight or 0.8 part by weight per 1 part by weight of G-CSF is particularly preferable. When a protein such as albumin is not added as a stabilizer, if the added amount of the surfactant exceeds 1 part by weight relative to 1 part by weight of G-CSF, the residual ratio of G-CSF after long-term storage tends to decrease. It was observed. Moreover, even if it is a case where the addition amount of surfactant is 1 weight part or less with respect to 1 weight part of G-CSF, adsorption | suction to the container of G-CSF can fully be suppressed.

  Preferred G-CSF containing formulations of the present invention are substantially free of protein as a stabilizer. Some products currently on the market are supplemented with proteins such as human serum albumin or purified gelatin as stabilizers in order to suppress chemical and physical changes in G-CSF. However, the addition of a protein as a stabilizer has a problem that a very complicated process is required to remove viral contamination.

  The G-CSF-containing preparation of the present invention has a pH of 7 or less, preferably a pH of 5 to 7, more preferably a pH of 6 to 6.8, and most preferably a pH of 6.2 to 6. 8. As will be described later, the residual rate of G-CSF after the accelerated test at 40 ° C. for 2 weeks is stable at a pH of 7 or less. From this viewpoint, the pH is preferably about 7.0 or less. Moreover, when the production | generation ratio of the glycolysis body of G-CSF was measured after 40 degreeC-2 weeks acceleration test, when pH became 4 or less, it was observed that the content of a sugar chain degradation body increases rapidly. Therefore, from this viewpoint, the pH is preferably about 5 or more. Furthermore, when administered as an injection, it is preferably near neutral with little irritation to the human body, and when these are combined, it is most preferable that the pH be 6.2 to 6.8.

  The G-CSF-containing preparation of the present invention contains a diluent, a solubilizer, an isotonic agent, an excipient, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant and the like. May be. For example, as the isotonic agent, saccharides such as polyethylene glycol; dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, raffinose and the like can be used. Examples of sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and carbon. Examples thereof include those having a sulfhydryl group such as thioalkanoic acid having 1 to 7 atoms. Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbic acid stearate, sodium bisulfite And chelating agents such as sodium sulfite, triamyl gallate, propyl gallate or disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate. As excipients, amino acids such as glycine, cysteine, threonine, cystine, tryptophan, methionine, lysine, hydroxylysine, histidine, arginine may be added. Furthermore, it is usually added to solution preparations such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate; and organic salts such as sodium citrate, potassium citrate, sodium acetate. Ingredients may be included.

  The amount of G-CSF contained in the solution preparation of the present invention can be determined according to the type of disease to be treated, the severity of the disease, the age of the patient, etc., but is generally 1-1000 μg / mL, preferably 10 It is -800 microgram / mL, More preferably, it is 50-500 microgram / mL.

  The solution formulation of the present invention can be prepared by dissolving these components in an aqueous buffer known in the field of solution formulation such as phosphate and / or citrate buffer. The phosphate buffer is preferably sodium monohydrogen phosphate-sodium dihydrogen phosphate, and the citrate buffer is preferably a sodium citrate buffer.

  The stabilized G-CSF-containing preparation of the present invention is usually administered by a parenteral route, for example, injection (subcutaneous injection, intravenous injection or intramuscular injection), transdermal, transmucosal, nasal, transpulmonary, etc. However, oral administration is also possible.

  The G-CSF-containing preparation of the present invention is usually stored in a sealed or sterilized plastic or glass container. The container can be supplied in a prescribed dosage form such as an ampoule, vial or disposable syringe, or it can be provided in a large dosage form such as an injection bag or bottle. Preferably, it is supplied as a G-CSF-containing preparation filled in a vial, an ampule for syringe or a prefilled syringe.

  As shown in the examples below, the G-CSF-containing preparation of the present invention exhibits an extremely good G-CSF residual rate even after an accelerated test at 40 ° C. for 2 weeks or storage at 25 ° C. for 6 months. Show. G-CSF has 1 to 2 sialic acids at the end of the sugar chain, but may produce a degraded product in which sialic acid is decomposed during long-term storage. The G-CSF-containing preparation of the present invention was shown to keep the degradation product formation ratio low even after an accelerated test at 40 ° C. for 2 weeks. Further, the G-CSF-containing preparation of the present invention can sufficiently suppress the adsorption to the container, and after an accelerated test at 40 ° C. for 2 weeks regardless of the shape of the container such as a vial-filled preparation or a syringe-filled preparation. And a very good G-CSF residual rate after storage at 25 ° C. for 6 months.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Test method 250 mg of G-CSF, 0.1 g of polysorbate 20, 30 g of D-mannitol were weighed and adjusted with sodium phosphate buffer so as to have various pH values shown in Table 1 below. Was 1 L.

Each preparation solution was prepared aseptically, filtered, and aseptically filled in 1 mL vials and sealed to produce a G-CSF solution formulation.
The preparation containing G-CSF 250 μg / mL aseptically prepared as described above was left in a constant temperature bath at 40 ° C. for 2 weeks.

The G-CSF content in the vial was measured based on Method 1 below. Further, the content of G-CSF sugar chain degradation product in the vial was measured based on the following method 2.
Method 1
A C4 reverse phase column (4.6 mm × 250 mm, 300 Å) was used, and pure water, acetonitrile, and trifluoroacetic acid were used for the mobile phase. The G-CSF content was measured by reverse phase high performance liquid chromatography. An amount equivalent to 5 μg was injected as G-CSF, and G-CSF was eluted with a gradient of acetonitrile, and spectroscopically detected at a wavelength of 215 nm.

  Based on the G-CSF content measured by this method, the residual rate (%) after acceleration at 40 ° C. for 2 weeks was calculated based on the following formula.

Method 2
By anion exchange high performance liquid chromatography, a G-CSF sugar chain degradation product (one in which all sialic acid present at the sugar chain terminal was degraded) and G-CSF (unmodified product) were detected. That is, using an anion exchange column (TSKgel DEAE), using 20 mM Tris-HCl buffer (pH 7.4) as a mobile phase, eluting with a NaCl gradient (0-500 mM), spectroscopically detecting at a wavelength of 215 nm. .

  Using the measured values of the G-CSF glycan degradation product and the unchanged G-CSF product measured by this method, the ratio of G-CSF glycan degradation product (%) after acceleration at 40 ° C. for 2 weeks based on the following formula: ) Was calculated.

Example 1: Effect of various pHs on G-CSF remaining rate G-CSF remaining rate after performing an accelerated test at 40 ° C. for 2 weeks for solution preparations prepared at various pHs shown in Table 1 It was calculated by the formula described in. The obtained results are shown in FIG.

When the pH was 7 or less, the residual rate of G-CSF was 75% or more.

Example 2 Effect on G-CSF Glycodegradation Product Formation at Various pHs G-CSF Glycolate after Accelerated Test at 40 ° C. for 2 Weeks for Solution Formulations Prepared at Various pHs listed in Table 1 The decomposition product formation ratio was calculated by the formula described in Method 2. The obtained results are shown in FIG.

When the pH was in the range of about 5 to 7, the production ratio of G-CSF sugar chain degradation product was extremely low.

Example 3: Effect of G-CSF adsorption on container with surfactant concentration
After adding 250 mg of G-CSF and 5.844 g of sodium chloride, add each polysorbate 20 concentration shown in Table 2 below, adjust the pH to 6.5 with sodium phosphate buffer, and adjust the total amount. 1L.

  Aseptically prepared and filtered, each G-CSF preparation solution listed in Table 2 was filled 1 mL into a vial (untreated white glass vial (5 mL) manufactured by Murase Glass Co., Ltd.). And 24 hours after filling, the G-CSF content was measured by the reverse phase high performance liquid chromatography described in Method 1.

  Using the G-CSF content measured by this method, the adsorption inhibition rate (%) after 24 hours from the filling was calculated based on the following formula.

  The obtained results are shown in Table 3 and FIG.

The adsorption inhibition rate was sufficient even when the polysorbate concentration relative to 1 part by weight of G-CSF was 1 part by weight or less.

Example 4: Stability of a vial or syringe-filled preparation A total amount of 1 L prepared by weighing 250 mg of G-CSF, 0.1 g of polysorbate 20, 7 g of sodium chloride, and adjusting the pH to 6.5 with sodium phosphate buffer. After aseptically preparing and filtering, a vial (same as above) or syringe (Nihon Becton Dickinson Hipack SFC, 1 mL long) was filled and sealed in an aseptic manner. The indicated G-CSF solution formulation was prepared.

The preparation containing G-CSF 250 μg / mL aseptically prepared in this manner was left in a constant temperature bath at 40 ° C. for 2 weeks or in a constant temperature bath at 25 ° C. for 6 months.
G-CSF content in vial or syringe is measured based on method 1, and G-CSF residual rate after acceleration at 40 ° C for 2 weeks and G-CSF residual rate after storage at 25 ° C for 6 months It was calculated by the formula described in 1.

  The results obtained are shown in Table 5.

The G-CSF formulation of the present invention has a residual rate of 75% or more after acceleration at 40 ° C. for 2 weeks and a residual rate of 95% after storage at 25 ° C. for 6 months in both the vial-filled formulation and the syringe-filled formulation. % Or more, indicating excellent stability.

  The G-SCF-containing preparation of the present invention contains a very small amount of a surfactant of 1 part by weight or less with respect to 1 part by weight of G-CSF, so that the temperature, humidity and oxygen of G-CSF present in a trace amount in the preparation are included. In addition, it is possible to effectively solve problems relating to loss of active ingredients, decrease in activity, and the like that occur as a result of association based on external factors such as ultraviolet rays, polymerization, or oxidation or adsorption to the container wall. Accordingly, the present invention provides a solution formulation that reduces the complexity and cost of the production process and is stable for long-term storage.

FIG. 1 is a graph showing the relationship between pH and G-CSF residual rate after an accelerated test at 40 ° C. for 2 weeks. FIG. 2 is a graph showing the relationship between pH and G-CSF sugar chain degradation product formation rate after an accelerated test at 40 ° C. for 2 weeks. FIG. 3 is a graph showing the relationship between the weight part of polysorbate 20 and the adsorption inhibition rate with respect to 1 part by weight of G-CSF after 24 hours have elapsed after filling.

Claims (14)

A granulocyte colony-stimulating factor having a sugar chain and at least one pharmaceutically acceptable nonionic surfactant in an amount of 1 part by weight or less per 1 part by weight of the granulocyte colony-stimulating factor, and having a pH of 5 to 7. A stable granulocyte colony-stimulating factor-containing preparation. The granulocyte colony stimulating factor-containing preparation according to claim 1, comprising a surfactant in an amount within the range of 0.2 to 1 part by weight with respect to 1 part by weight of the granulocyte colony stimulating factor. The granulocyte colony stimulating factor-containing preparation according to claim 2, comprising a surfactant in an amount in the range of 0.2 to 0.8 parts by weight with respect to 1 part by weight of the granulocyte colony stimulating factor. The granulocyte colony stimulating factor-containing preparation according to claim 2, comprising a surfactant in an amount within the range of 0.4 to 0.8 parts by weight with respect to 1 part by weight of the granulocyte colony stimulating factor. The granulocyte colony stimulating factor-containing preparation according to claim 2, comprising 0.4 or 0.8 parts by weight of a surfactant per 1 part by weight of the granulocyte colony stimulating factor. The granulocyte colony-stimulating factor-containing preparation according to claim 1, which does not contain other proteins as a stabilizer. Nonionic surfactant is sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether At least one selected from the group consisting of polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene beeswax derivative, polyoxyethylene lanolin derivative, polyoxyethylene fatty acid amide The granulocyte colony-stimulating factor-containing preparation according to claim 1, which is a seed. The granulocyte colony-stimulating factor-containing preparation according to claim 7, wherein the polyoxyethylene sorbitan fatty acid ester is polysorbate 20 or polysorbate 80. 2. The granulocyte colony stimulating factor-containing preparation according to claim 1, which has a pH of 6 to 6.8. 2. The granulocyte colony stimulating factor-containing preparation according to claim 1, which has a pH of 6.2 to 6.8. 2. The granulocyte colony stimulating factor-containing preparation according to claim 1, which is filled in a vial, an ampule for a syringe, or a prefilled syringe. 2. The granulocyte colony-stimulating factor-containing preparation according to claim 1, wherein the residual rate of G-CSF after long-term storage is high and the production of sugar chain degradation products is small. G-CSF residual ratio after storage of G-CSF containing solution preparation at 25 ° C. for 6 months is 95% or more, or G-CSF residual ratio after storage at 40 ° C. for 2 weeks. The granulocyte colony stimulating factor-containing preparation according to claim 12, which is maintained at 75% or more. The granulocyte colony stimulating factor-containing preparation according to claim 1, wherein the granulocyte colony stimulating factor having a sugar chain is produced by CHO cells using a gene recombination method.

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