Protein-containing aqueous solution

Abstract

To provide aqueous solution containing protein as an active ingredient, the aqueous solution being stable when stored in solution state, and having reduced pain due to injection.SOLUTION: Provided is an aqueous solution containing a phosphate buffer at a concentration of 1 to 20 mM and, as an active ingredient, protein. For example, provided is an aqueous solution containing a phosphate buffer having a concentration of 1 to 20 mM as a buffer and human growth hormone as an active ingredient, and further containing poloxamer as a nonionic surfactant and phenol as tonicity agent.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an aqueous liquid preparation containing a protein as an active ingredient, which is storage stable in a solution state and reduces pain upon injection, and more specifically, to an aqueous liquid preparation in which the active ingredient is a protein and which contains a phosphate buffer at a concentration of 1 to 20 mM as a buffer. [Background technology]

[0002] Various preparations have been developed whose active ingredients are proteins. Many of these proteins are recombinant proteins produced using gene recombination technology. Protein preparations, which are high molecular weight substances, cannot be absorbed by oral administration, so they are administered exclusively by subcutaneous injection, intramuscular injection, or intravenous injection. Therefore, administration of these preparations is accompanied by pain due to the injection needle. The ingredients contained in the preparation affect the level of pain.

[0003] Human growth hormone (hGH) is a single-chain polypeptide hormone consisting of 191 amino acid residues in its natural form. hGH is used as a therapeutic agent for short stature due to growth hormone deficiency, short stature in Turner syndrome, adult growth hormone deficiency, etc. (Non-Patent Document 1).

[0004] Treatment with hGH usually takes place over a long period of time, measured in years, during which hGH must be injected intramuscularly 2 to 4 times a week, or subcutaneously 6 to 7 times a week. Therefore, in order to reduce the burden of hospital visits for patients, self-injection at home is generally used for hGH treatment. In addition, hGH was traditionally prepared as a freeze-dried preparation, but as freeze-dried preparations must be dissolved in a dissolving solution when used, aqueous liquid preparations in which hGH is dissolved in advance have been developed to improve patient convenience (Non-Patent Document 1).

[0005] hGH is administered to patients by intramuscular or subcutaneous injection. Many patients are children, and therefore it is highly desirable for hGH preparations to be less painful when injected. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Growject Subcutaneous Injection 6 mg / Growject Subcutaneous Injection 12 mg Package Insert (2017) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an aqueous liquid preparation containing a protein as an active ingredient, which is stable in storage in a solution state and reduces pain when injected, and in particular, an aqueous liquid preparation containing a growth hormone as an active ingredient. [Means for solving the problem]

[0008] In the course of research aimed at the above-mentioned object, the present inventors discovered that by using a phosphate buffer with a concentration of 1 to 20 mM as the buffer contained in the aqueous solution, it is possible to maintain the stability of the growth hormone in the aqueous solution while reducing the pain during injection, and thus completed the present invention. That is, the present invention includes the following. An aqueous liquid preparation containing a phosphate buffer at a concentration of 1.1 to 20 mM and a protein as an active ingredient. 2. The aqueous liquid preparation according to 1 above, wherein the concentration of the phosphate buffer is 5 to 16 mM. 3. The aqueous liquid preparation according to 2 above, wherein the concentration of the phosphate buffer is 8 to 12 mM. 4. Any of the aqueous liquid preparations according to 1 to 3 above, wherein the protein has a concentration of 1 to 50 mg / mL. 5. Any of the aqueous liquid preparations according to 1 to 4 above, further comprising a nonionic surfactant. 6. The aqueous liquid preparation according to 5 above, wherein the nonionic surfactant is polysorbate or poloxamer. 7. The aqueous liquid preparation according to 5 above, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188. 8. Any of the aqueous liquid preparations according to 5 to 7 above, wherein the concentration of the nonionic surfactant is 1 to 10 mg / mL. 9. Any of the aqueous liquid preparations according to 1 to 8 above, further containing a preservative. 10. The aqueous liquid preparation according to claim 9, wherein the preservative is benzyl alcohol or phenol. 11. The aqueous liquid preparation according to claim 9, wherein the preservative is benzyl alcohol and the concentration of the benzyl alcohol is 2 to 20 mg / mL. 12. The aqueous liquid preparation according to claim 9, wherein the preservative is phenol and the concentration of the phenol is 1 to 10 mg / mL. 13. Any of the aqueous liquid preparations according to 1 to 12 above, further comprising an isotonicity agent. 14. The aqueous liquid preparation according to claim 13, wherein the isotonicity agent is a neutral salt or a sugar alcohol. 15. The aqueous liquid preparation according to claim 13, wherein the isotonicity agent is sodium chloride or D-mannitol. 16. Any of the aqueous liquid preparations according to 1 to 15 above, having a pH of 5.5 to 7.2. 2. An aqueous liquid preparation according to claim 1, comprising 17.1-50 mg / mL protein, 5-16 mM phosphate buffer, 1.8-2.2 mg / mL poloxamer 188, 2.8-3.8 mg / mL phenol, and 35-45 mg / mL D-mannitol, and having a pH of 6.0-6.4. 18. The protein is growth hormone, somatomedin, insulin, glucagon, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin, darbepoietin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, DNasel, thyroid-stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor. 17. The aqueous liquid preparation according to any one of 1 to 17 above, wherein the agent is selected from the group consisting of fibroblast growth factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), an enzyme having an activity of degrading beta-amyloid, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and GLP-1 receptor agonist. 19. The aqueous liquid preparation according to any one of 1 to 17 above, wherein the protein is selected from the group consisting of a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, and a human antibody. 20. The aqueous liquid preparation according to 1 to 17 above, wherein the protein is selected from the group consisting of anti-IL-6 antibody, anti-beta amyloid antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-HER2 antibody, anti-PCSK9 antibody, and anti-TNF-α antibody. 21. The protein is a lysosomal enzyme, and the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, iduronate 2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine 1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. 18. The aqueous liquid preparation according to 1 to 17 above, which is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, and hyaluronidase-1. 22. The protein is a fusion protein of an antibody and another protein, and the other protein is a growth hormone, a lysosomal enzyme, a cytokine, a lymphokine, a blood coagulation factor, an antibody, a fusion protein of an antibody and another protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), erythropoietin, darbepoietin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone, DNasel, thyroid-stimulating hormone (TSH), nerve growth factor (NGF), ciliary neurotrophic factor (CN TF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon α, interferon β, interferon γ, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor α receptor (TNF-α receptor), and an enzyme having an activity of degrading beta-amyloid. 23. The protein is a fusion protein of an antibody and a lysosomal enzyme, and the lysosomal enzyme is α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activating protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, α-galactosidase 18. The aqueous liquid preparation according to 1 to 17 above, which is selected from the group consisting of A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, and hyaluronidase-1. 24. An aqueous liquid preparation according to any one of 1 to 23 above, which is administered by subcutaneous injection or intramuscular injection. Effect of the Invention

[0009] According to the present invention, it is possible to reduce pain during injection of a preparation whose active ingredient is a protein. [Brief description of the drawings]

[0010] [Figure 1] The gauge used in Examples 1 and 2 to have subjects express pain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to an aqueous liquid preparation whose active ingredient is a protein. The type of protein as the active ingredient is not particularly limited, but examples thereof include growth hormone, somatomedin (including somatomedin A, B, and C), insulin, glucagon, lysosomal enzymes, cytokines, lymphokines, blood coagulation factors (including blood coagulation factors VII, VIII, and IX), antibodies, fusion proteins of antibodies and other proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin, darbepoetin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), gonadotropin, DNasel, thyrotropin, and thyrotropin. and neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), or an enzyme with beta-amyloid degrading activity, etanercept, pegvisomant, metreleptin, abatacept, asfotase, and glucagon-like peptide-1 receptor agonists.

[0012] When the active ingredient is an antibody, there is no particular limitation on the biological species of the antibody, for example, a mouse antibody, a humanized antibody, a human-mouse chimeric antibody, or a human antibody. In addition, there is no particular limitation on the antigen to which the antibody specifically binds, for example, an anti-IL-6 antibody, an anti-beta amyloid antibody, an anti-BACE antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-HER2 antibody, an anti-PCSK9 antibody, or an anti-TNF-α antibody. In addition, the antibody is not limited to those having a basic structure consisting of a total of four polypeptide chains, two immunoglobulin light chains and two immunoglobulin heavy chains, and may be a single chain antibody, Fab, F(ab'), F(ab') antibody, or any other antibody that retains specificity for a specific antigen. 2 , or an antigen-binding fragment.

[0013] When the active ingredient is a lysosomal enzyme, the type of lysosomal enzyme is not particularly limited, but examples thereof include α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, or hyaluronidase-1.

[0014] When the active ingredient is a fusion protein of an antibody and another protein, the type of the other protein is not particularly limited, but may be, for example, growth hormone, lysosomal enzyme, cytokine, lymphokine, blood coagulation factor, antibody, fusion protein of antibody and other protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), erythropoietin, darbepoietin, tissue plasminogen activator (t-PA), thrombomodulin, follicle-stimulating hormone, DNasel, thyroid-stimulating hormone (TSH), nerve growth factor stimulating factor (GF-CSF), erythropoietin, thyroid-stimulating hormone (TSH), thyroid-stimulating hormone (TH ... factor (NGF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF), neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, neuregulin 1, activin, basic fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), interferon alpha, interferon beta, interferon gamma, interleukin 6, PD-1, PD-1 ligand, tumor necrosis factor alpha receptor (TNF-alpha receptor), or an enzyme with beta-amyloid degrading activity.

[0015] When the other protein is a lysosomal enzyme, the type of the lysosomal enzyme is not particularly limited, and examples thereof include α-L-iduronidase, iduronate-2-sulfatase, glucocerebrosidase, β-galactosidase, GM2 activator protein, β-hexosaminidase A, β-hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase, β-mannosidase, galactosylceramidase, saposin C, arylsulfatase A, α-L-fucosidase, aspartylglucosaminidase, α-N-acetylgalactosaminidase, acid sphingomyelinase, and α-galactosidase. A, β-glucuronidase, heparan N-sulfatase, α-N-acetylglucosaminidase, acetyl-CoA α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, or hyaluronidase-1.

[0016] The biological species of the protein contained in the aqueous liquid formulation as an active ingredient is not particularly limited, but is preferably a human protein. The protein can be produced as a recombinant protein using gene recombination technology. The recombinant protein can be produced, for example, by incorporating a gene encoding the protein into an expression vector, using the vector to transform mammalian cells (e.g., CHO cells derived from Chinese hamster ovary), E. coli, etc., and culturing the transformed cells.

[0017] In the present invention, the term "growth hormone" refers particularly to human growth hormone, but is not limited to this and also includes growth hormones from mammals, including livestock such as cows and horses, and pets such as dogs and cats. "Growth hormone" also includes analogs of growth hormone in which one or more amino acid residues constituting the growth hormone have been substituted, deleted, or inserted.

[0018] In the present invention, growth hormone can be produced using recombinant gene technology. A method for producing biologically active growth hormone by inserting a growth hormone gene into an expression vector, transforming mammalian cells (e.g., CHO cells derived from Chinese hamster ovary) or Escherichia coli with the vector, and culturing the transformed cells is well known to those skilled in the art (US2010 / 0227819, US4342832, etc.).

[0019] In the present invention, there is no particular limitation on the concentration of the protein contained in the aqueous liquid preparation. However, the concentration is preferably 1 to 50 mg / mL, more preferably 1 to 10 mg / mL, and even more preferably 2 to 8 mg / mL. A suitable protein concentration is, for example, 4 mg / mL or 8 mg / mL. When the protein is a growth hormone, these concentrations are particularly preferred.

[0020] The aqueous liquid preparation of the protein of the present invention contains a phosphate buffer as a buffer. The concentration of the phosphate buffer contained in the aqueous liquid preparation is preferably 1 to 20 mM, more preferably 5 to 16 mM, even more preferably 8 to 15 mM, and even more preferably 8 to 12 mM. For example, the final concentration in the aqueous liquid preparation is adjusted to 10 mM, 11 mM, or 12 mM. The pH of the aqueous liquid preparation containing the buffer is preferably 5.5 to 7.2, more preferably 6.0 to 6.4, even more preferably 6.0 to 6.3, and particularly preferably 6.2.

[0021] The aqueous liquid preparation of the protein of the present invention further contains a nonionic surfactant. The nonionic surfactant contained in the aqueous liquid preparation may be polysorbate, poloxamer, or the like, which may be used alone or in combination. As the polysorbate, polysorbate 20 and polysorbate 80 are particularly preferred, and as the poloxamer, poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is particularly preferred. The concentration of the nonionic surfactant contained in the aqueous liquid preparation is preferably 1 to 10 mg / mL, more preferably 1 to 5 mg / mL, even more preferably 1 to 3 mg / mL, even more preferably 1.5 to 2.5 mg / mL, even more preferably 1.8 to 2.2 mg / mL, and is adjusted to, for example, 2 mg / mL. When poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is used as the nonionic surfactant, its concentration in the aqueous solution is preferably 1 to 10 mg / mL, more preferably 1 to 5 mg / mL, even more preferably 1 to 3 mg / mL, even more preferably 1.5 to 2.5 mg / mL, and even more preferably 1.8 to 2.2 mg / mL, and is adjusted to, for example, 2 mg / mL.

[0022] It is also assumed that the aqueous solution of protein will not be disposable, but will be used repeatedly after opening rather than once. In such a case, it is desirable to be able to prevent deterioration of quality due to bacterial contamination during repeated use. Therefore, the aqueous solution of protein of the present invention may contain a preservative. There is no particular limitation on the preservative contained in the aqueous solution as long as it is pharma- ceutical acceptable, but benzyl alcohol, phenol, benzoic acid, or a mixture thereof can be preferably used.

[0023] When phenol is used as a preservative, its concentration in the aqueous solution is preferably 1 to 10 mg / mL, more preferably 2.5 to 4.0 mg / mL, even more preferably 2.8 to 3.8 mg / mL, for example, 3.3 mg / mL. When benzyl alcohol is used as a preservative, its concentration in the aqueous solution is preferably 2 to 20 mg / mL, more preferably 7 to 12 mg / mL, even more preferably 8 to 10 mg / mL, for example, 9 mg / mL. When benzoic acid is used as a preservative, its concentration in the aqueous solution is preferably 1 to 20 mg / mL.

[0024] The aqueous liquid preparation of the protein of the present invention may also contain an isotonicity agent. The isotonicity agent contained in the aqueous liquid preparation is not particularly limited as long as it is pharma- ceutical acceptable, but sugar alcohols, neutral salts, or mixtures thereof can be suitably used. D-mannitol is a suitable sugar alcohol, and sodium chloride is a suitable neutral salt. When D-mannitol is used as the isotonicity agent, its concentration in the aqueous liquid preparation is preferably 35 to 70 mg / mL, more preferably 35 to 45 mg / mL, for example 40 mg / mL. When sodium chloride is used as the isotonicity agent, its concentration in the aqueous liquid preparation is preferably 5.5 to 11.5 mg / mL, more preferably 5.5 to 7.5 mg / mL, for example 6.5 mg / mL. However, the concentration of the isotonicity agent is not limited to these, and should be appropriately adjusted in relation to the concentrations of other components in the aqueous liquid preparation. Usually, the concentration of the isotonicity agent is adjusted so that the osmotic pressure of the aqueous liquid preparation is 0.9 to 1.6, more preferably 0.9 to 1.1, when physiological saline is used as a control. In the present invention, the osmotic pressure ratio refers to the osmotic pressure ratio to physiological saline.

[0025] Preferred examples of the aqueous liquid formulation of the protein of the present invention are given below. (Preferred Example 1) An aqueous liquid preparation containing 1-50 mg / mL protein, 5-16 mM phosphate buffer, 1.8-2.2 mg / mL poloxamer 188, 2.8-3.8 mg / mL phenol, and 35-45 mg / mL D-mannitol, and having a pH of 6.0-6.4. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9-1.1. Also, 8-10 mg / mL benzyl alcohol may be added instead of phenol. (Preferred Example 2) An aqueous liquid preparation containing 1-10 mg / mL protein, 8-15 mM phosphate buffer, 1.8-2.2 mg / mL poloxamer 188, 2.8-3.8 mg / mL phenol, and 35-45 mg / mL D-mannitol, and having a pH of 6.0-6.4. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9-1.1. Also, 8-10 mg / mL benzyl alcohol may be added instead of phenol. (Preferred Example 3) An aqueous liquid preparation containing 2-8 mg / mL protein, 10 mM phosphate buffer, 2 mg / mL poloxamer 188, 3.3 mg / mL phenol, and 40 mg / mL D-mannitol, and having a pH of 6.2. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9-1.1. Also, 8-10 mg / mL benzyl alcohol may be added instead of phenol.

[0026] The aqueous liquid preparation of the protein of the present invention is one in which the protein does not deteriorate even when stored for a long period of time at 2 to 8° C. Here, deterioration of the protein refers to a state in which the quantitative value of the protein is 98% or less, for example 95% or less, compared to immediately after the aqueous liquid preparation is filled into a vial or syringe as a formulation. In addition, the protein is also said to have deteriorated when the activity inherent to the protein is 98% or less, for example 95% or less, compared to immediately after the aqueous liquid preparation is filled into a vial or syringe as a formulation.

[0027] The period during which the protein can be stored without degradation is preferably at least 18 months, more preferably 24 months, even more preferably 30 months, and even more preferably 36 months.

[0028] The aqueous solution of the protein of the present invention is administered to patients by intramuscular injection, subcutaneous injection, etc. If the injection is painful, the patient's compliance with medication may decrease, resulting in insufficient therapeutic effect. The aqueous solution of the present invention reduces the pain felt by the patient during injection, thereby increasing the patient's compliance with medication and preventing the patient from avoiding medication.

[0029] The pain felt during an injection is subjective, but the visual analog scale (VAS) is commonly used as a method for objectively evaluating it. The VAS method generally involves the following steps: (1) Present the subject with a 100 mm line, with the left end representing no pain and the right end representing the worst pain imaginable, (2) indicate the pain the subject feels on the line, and (3) convert the pain indicated by the subject into a numerical value and tabulate the results.

[0030] Another method for objectively assessing pain is the Numeric Rating Scale (NRS). The Numeric Rating Scale is a method that roughly includes the following steps: (1) the subject is presented with a line divided into 11 levels ranging from no pain to the worst pain, (2) the subject expresses the pain he or she is feeling by using the numbers shown on the line, and (3) the numbers are tallied.

[0031] Another method for objectively assessing pain is the Faces Pain Scale. While VAS and NRS involve subjects expressing the pain they feel by using the numbers shown on a gauge, the Faces Pain Scale involves subjects expressing the pain they feel by using human facial expressions corresponding to the pain shown on the gauge.

[0032] Methods for objectively assessing pain may include, but are not limited to, an appropriate combination or modification of VAS, NRS, and face scale.

[0033] The aqueous liquid preparation of the present invention is one in which, when the pain felt by a subject upon subcutaneous injection is evaluated using the above-mentioned method for objectively evaluating pain, the pain felt is equal to or less than that felt upon subcutaneous injection of saline, for example.

[0034] The case where the protein contained in the aqueous liquid preparation is growth hormone will be described in detail below.

[0035] In the present invention, there is no particular limitation on the concentration of growth hormone contained in the aqueous liquid preparation. However, the concentration is preferably 1 to 12 mg / mL, more preferably 2 to 8 mg / mL. A suitable concentration of growth hormone is, for example, 4 mg / mL or 8 mg / mL.

[0036] The aqueous liquid preparation of growth hormone of the present invention contains a phosphate buffer as a buffer. The concentration of the phosphate buffer contained in the aqueous liquid preparation is preferably 1 to 20 mM, more preferably 5 to 16 mM, even more preferably 8 to 15 mM, and even more preferably 8 to 12 mM. For example, the final concentration in the aqueous liquid preparation is adjusted to 10 mM, 11 mM, or 12 mM. The pH of the aqueous liquid preparation containing the buffer is preferably 5.5 to 7.2, more preferably 6.0 to 6.4, even more preferably 6.0 to 6.3, even more preferably 6.0 to 6.2, and particularly 6.2.

[0037] The aqueous liquid preparation of growth hormone of the present invention further contains a nonionic surfactant. The nonionic surfactant contained in the aqueous liquid preparation may be polysorbate, poloxamer, or the like, which may be used alone or in combination. As the polysorbate, polysorbate 20 and polysorbate 80 are particularly suitable, and as the poloxamer, poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is particularly suitable. The concentration of the nonionic surfactant contained in the aqueous liquid preparation is preferably 1 to 10 mg / mL, more preferably 1 to 5 mg / mL, even more preferably 1 to 3 mg / mL, even more preferably 1.5 to 2.5 mg / mL, and even more preferably 1.8 to 2.2 mg / mL, and is adjusted to, for example, 2 mg / mL. When poloxamer 188 (polyoxyethylene (160) polyoxypropylene (30) glycol) is used as the nonionic surfactant, its concentration in the aqueous solution is preferably 1 to 5 mg / mL, more preferably 1 to 3 mg / mL, even more preferably 1.5 to 2.5 mg / mL, and even more preferably 1.8 to 2.2 mg / mL, and is adjusted to, for example, 2 mg / mL.

[0038] It is also assumed that the aqueous solution of growth hormone will not be disposable, but will be used repeatedly after opening rather than once. In such a case, it is desirable to be able to prevent deterioration of quality due to bacterial contamination during repeated use. Therefore, the aqueous solution of growth hormone of the present invention may contain a preservative. There is no particular limitation on the preservative contained in the aqueous solution as long as it is pharma- ceutical acceptable, but benzyl alcohol, phenol, benzoic acid, or a mixture thereof can be preferably used.

[0039] When phenol is used as a preservative, its concentration in the aqueous solution is preferably 1 to 10 mg / mL, more preferably 2.5 to 4.0 mg / mL, more preferably 2.8 to 3.8 mg / mL, for example 3.3 mg / mL. When benzyl alcohol is used as a preservative, its concentration in the aqueous solution is preferably 2 to 20 mg / mL, more preferably 7 to 12 mg / mL, more preferably 8 to 10 mg / mL, for example 9 mg / mL. When benzoic acid is used as a preservative, its concentration in the aqueous solution is preferably 1 to 20 mg / mL.

[0040] The aqueous solution of growth hormone of the present invention may contain an isotonicity agent. The isotonicity agent contained in the aqueous solution is not particularly limited as long as it is pharma- ceutical acceptable, but sugar alcohols, neutral salts, or mixtures thereof can be suitably used. A suitable sugar alcohol is D-mannitol, and a suitable neutral salt is sodium chloride. When D-mannitol is used as the isotonicity agent, its concentration in the aqueous solution is preferably 35-70 mg / mL, more preferably 35-45 mg / mL, for example 40 mg / mL. When sodium chloride is used as the isotonicity agent, its concentration in the aqueous solution is preferably 25-35 mg / mL, more preferably 5.5-7.5 mg / mL, for example 6.5 mg / mL. However, the concentration of the isotonicity agent is not limited to these, and should be appropriately adjusted in relation to the concentrations of other components in the aqueous solution. Usually, the concentration of the isotonic agent is adjusted so that the osmotic pressure ratio of the aqueous liquid preparation to physiological saline is 0.9 to 1.6, more preferably 0.9 to 1.1.

[0041] Preferred examples of the aqueous liquid formulation of growth hormone of the present invention are given below. (Preferred Example 1') An aqueous liquid preparation containing 1-12 mg / mL growth hormone, 5-16 mM phosphate buffer, 1.8-2.2 mg / mL poloxamer 188, 2.8-3.8 mg / mL phenol, and 35-45 mg / mL D-mannitol, and having a pH of 6.0-6.3. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9-1.1. Also, 8-10 mg / mL benzyl alcohol may be added instead of phenol. (Preferred Example 2') An aqueous liquid preparation containing 4-8 mg / mL growth hormone, 5-16 mM phosphate buffer, 1.8-2.2 mg / mL poloxamer 188, 2.8-3.8 mg / mL phenol, and 35-45 mg / mL D-mannitol, and having a pH of 6.0-6.3. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9-1.1. Also, 8-10 mg / mL benzyl alcohol may be added instead of phenol. (Preferred Example 3') An aqueous liquid preparation containing 4-8 mg / mL growth hormone, 8-12 mM phosphate buffer, 2 mg / mL poloxamer 188, 3.3 mg / mL phenol, and 40 mg / mL D-mannitol, and having a pH of 6.2. However, phosphoric acid and / or sodium hydroxide may be further added to adjust the pH, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9-1.1. Also, 9 mg / mL benzyl alcohol may be added instead of phenol. (Preferred Example 4') An aqueous liquid preparation containing 4 mg / mL or 8 mg / mL growth hormone, 10 mM phosphate buffer, 2 mg / mL poloxamer 188, 3.3 mg / mL phenol, and 40 mg / mL D-mannitol, and having a pH of 6.2. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. Also, 9 mg / mL benzyl alcohol may be added instead of phenol. (Preferred Example 5') An aqueous liquid preparation containing 4 mg / mL or 8 mg / mL growth hormone, 10 mM phosphate buffer, 2 mg / mL poloxamer 188, 3.3 mg / mL phenol, and 40 mg / mL D-mannitol, and having a pH of 6.2. However, in order to adjust the pH, phosphoric acid and / or sodium hydroxide may be further added, and the concentration of D-mannitol may be increased or decreased so that the osmotic pressure ratio is 0.9 to 1.1. Also, 9 mg / mL benzyl alcohol may be added instead of phenol.

[0042] The aqueous liquid formulation of growth hormone of the present invention may be a formulation filled in a vial, or may be a prefilled syringe or cartridge-type formulation that is a syringe that is prefilled. When a prefilled syringe or cartridge-type formulation is used, the amount of liquid filled in one syringe is usually adjusted to 1 to 2 mL, and for example, one syringe is filled so that the indicated amount is 1.5 mL.

[0043] Immediately after preparation of growth hormone as an aqueous liquid, most of the growth hormone exists as a monomer in the aqueous solution. However, when stored as an aqueous liquid, dimers form over time, and the ratio of growth hormone existing as a monomer decreases. The quantitative value of growth hormone also decreases over time.

[0044] The aqueous liquid preparation of growth hormone of the present invention is one in which the growth hormone does not deteriorate even when stored for a long period of time at 2 to 8°C. Here, the deterioration of growth hormone refers to the fact that the ratio of growth hormone present as a monomer is 98% or less, for example 95% or less, compared to immediately after the aqueous liquid preparation is filled into a vial or syringe as a formulation. In addition, the growth hormone is also said to have deteriorated when the quantitative value of growth hormone is 98% or less, for example 95% or less, compared to immediately after the aqueous liquid preparation is filled into a vial or syringe as a formulation.

[0045] The period during which the growth hormone can be stored without degradation is preferably at least 18 months, more preferably 24 months, even more preferably 30 months, and even more preferably 36 months.

[0046] The aqueous solution of growth hormone of the present invention is administered to patients by intramuscular or subcutaneous injection. It is assumed that administration to patients will be performed by doctors, as well as by the patients themselves or their guardians. Such types of drugs require the patients themselves or their guardians to comply with the administration schedule, and if the injection is painful, the patient's compliance with the medication may decrease, resulting in insufficient therapeutic effects. The aqueous solution of growth hormone of the present invention reduces the pain felt by patients during injection, so that patients' compliance with the medication will not decrease and all patients will be able to enjoy the intended therapeutic effects.

[0047] The pain felt during an injection is subjective, but the visual analog scale (VAS) is commonly used as a method for objectively evaluating it. The VAS method generally involves the following steps: (1) The subject is presented with a horizontal line, with the left end indicating no pain and the right end indicating the worst pain imaginable, (2) the subject indicates the pain he or she is feeling on the line, and (3) the pain indicated by the subject is converted into a numerical value and tabulated. The length of the line is generally 100 mm.

[0048] Another method for objectively assessing pain is the Numeric Rating Scale (NRS). The Numeric Rating Scale is a method that roughly includes the following steps: (1) the subject is presented with a line divided into 11 levels ranging from no pain to the worst pain, (2) the subject expresses the pain he or she is feeling by using the numbers shown on the line, and (3) the numbers are tallied.

[0049] Another method for objectively assessing pain is the Faces Pain Scale. While VAS and NRS involve subjects expressing the pain they feel by using the numbers shown on a gauge, the Faces Pain Scale involves subjects expressing the pain they feel by using human facial expressions corresponding to the pain shown on the gauge.

[0050] Methods for objectively assessing pain may include, but are not limited to, an appropriate combination or modification of VAS, NRS, and face scale.

[0051] The aqueous solution of growth hormone of the present invention is one in which the pain felt by a subject when it is injected subcutaneously is equivalent to or less than that felt when saline is injected subcutaneously, when evaluated using the above-mentioned method for objectively evaluating pain. EXAMPLES

[0052] The present invention will be described in more detail below with reference to examples, but it is not intended that the present invention be limited to the examples.

[0053] [Example 1] Evaluation of pain caused by aqueous liquid preparation (1) Aqueous liquid preparations of Formulation 1 and Formulation 2 containing the phosphate buffer and D-mannitol shown in Table 1 were prepared (Table 1). The phosphate buffer concentrations of Formulation 1 and Formulation 2 were 20 mM and 50 mM, respectively. The osmotic pressure ratios of both formulations were 1.0 to 1.1.

[0054] [Table 1]

[0055] The pain was evaluated using a gauge shown in Figure 1, on which schematic facial expressions expressing pain were displayed. Using this gauge, pain was quantified on a 10-point scale, with 1 representing "no pain" and 10 representing "the worst pain imaginable." The test was conducted using three subjects. The subjects (men in their 20s, 40s, and 50s) were administered Formulation 1, which had a phosphate buffer concentration of 20 mM, Formulation 2, which had a phosphate buffer concentration of 50 mM, and saline, once each, avoiding the same administration site. The administration was performed so that the subjects could not distinguish which solution was administered. The dosage for each was 125 μL, and the solution was administered subcutaneously to the thigh or upper arm of the subject using a syringe needle (BD micro-fine plus 31Gx5mm, Becton Dickinson). The gauge shown in Figure 1 was presented to the subjects immediately after administration, and they were asked to select the most appropriate facial expression that expressed the pain during injection from the schematic facial expressions displayed on the gauge. The pain scores were calculated by averaging the selected pain values. The pain scores were 3.0 for formula 1, 3.7 for formula 2, and 2.0 for saline. These results indicate that the pain experienced during injection of an aqueous solution can be reduced by reducing the concentration of phosphate buffer. In consideration of the safety of the subjects, the test was performed using an aqueous solution that did not contain protein components.

[0056] [Example 2] Evaluation of pain caused by aqueous liquid preparation (2) Aqueous liquid preparations of formulations 3 to 5 containing phosphate buffer, D-mannitol, poloxamer 188, and phenol shown in Table 2 were prepared (Table 2). However, only in formulation 5, an appropriate amount of sodium hydroxide was added to adjust the pH. The phosphate buffer concentrations in formulations 3, 4, and 5 were 10 mM, 20 mM, and 50 mM, respectively. The osmotic pressure ratios of all formulations were 1.0 to 1.1.

[0057] [Table 2]

[0058] The test was conducted using three subjects. The subjects (men in their 20s, 40s, and 50s) were administered once each of the following solutions, avoiding the same administration site: Formulation 3 with a phosphate buffer concentration of 10 mM, Formulation 4 with a phosphate buffer concentration of 15 mM, Formulation 5 with a phosphate buffer concentration of 50 mM, and physiological saline. The administration was conducted so that the subjects could not distinguish the administered solution. The dose of each solution was 125 μL, and it was administered subcutaneously to the thigh or upper arm of the subject using an injection needle (BD micro-fine plus 31Gx5mm, Becton Dickinson). Next, the pain score of each formulation was calculated using the same method as in Example 1. As a result, the pain score was 2.0 for Formulation 3, 2.0 for Formulation 4, 4.3 for Formulation 5, and 2.7 for physiological saline. These results indicate that the pain during injection of an aqueous liquid can be dramatically reduced by setting the concentration of the phosphate buffer to 15 mM or less. In particular, it is surprising that the pain during injection can be reduced by setting the concentration of the phosphate buffer to 15 mM or less, even compared to normal saline. In addition, in consideration of the safety of the subjects, the test was conducted using an aqueous liquid formulation that does not contain protein components.

[0059] [Example 3] Evaluation of pain caused by aqueous liquid preparation (3) Aqueous liquid preparations of Formulations 6 to 7 were prepared (Table 3), containing phosphate buffer, D-mannitol, poloxamer 188, and phenol as shown in Table 3. However, only Formulation 6 contained an appropriate amount of sodium hydroxide to adjust the pH. The phosphate buffer concentrations of Formulations 6 and 7 were 10 mM and 50 mM, respectively. The osmotic pressure ratios of all formulations were 1.0 to 1.1.

[0060] [Table 3]

[0061] A comparison of pain between formulation 6, which has a phosphate buffer concentration of 10 mM, formulation 7, which has a phosphate buffer concentration of 50 mM, and saline was conducted using 31 subjects. Saline was administered first to the subjects (including men and women in their 20s to 70s), avoiding the same administration site, followed by formulations 6 and 7. The administration was conducted so that the subjects could not distinguish which solution was administered. The dosage for each was 125 μL, and the solution was administered subcutaneously to the thigh or upper arm of the subject using an injection needle (BD micro-fine plus 31Gx5mm, Becton Dickinson). Next, the pain score for each formulation was calculated using the same method as in Example 1. As a result, the pain score was 2.6 for formulation 6, 4.9 for formulation 7, and 2.7 for saline. It was shown that the pain during injection can be reduced by lowering the concentration of the phosphate buffer, and that the pain during injection can be reduced compared to saline in the case of 10 mM. In consideration of the safety of the subjects, the test was conducted using an aqueous liquid formulation that did not contain protein components.

[0062] [Example 4] Evaluation of the stability of aqueous liquid preparations of human growth hormone (1) Four types of aqueous solutions of growth hormone, GH Formulation 1 to GH Formulation 4, shown in Table 4, were prepared. The phosphate buffer concentrations of GH Formulation 1 and GH Formulation 2 were 50 mM and 16 mM, respectively. GH Formulation 3 and GH Formulation 4 differ only in the concentration of growth hormone, and the phosphate buffer concentration is 10 mM for both.

[0063] [Table 4]

[0064] 1.5 mL of each of the aqueous solutions of GH formulations 1 to 4 was filled into glass cartridges and stored in a dark place at 2 to 8 °C (long-term storage test) or at 25 °C (accelerated test). The pH, monomer (%), and quantitative (%) of the solution were determined over time during the storage period. The monomer (%) and quantitative (%) were determined by the method described in Example 6.

[0065] Table 5 shows the results of the stability evaluation of GH formulation 1 with a phosphate buffer concentration of 50 mM. In the long-term storage test, pH, monomer (%), and quantitative (%) were measured at the start of storage and after 3, 6, 9, 12, 18, and 24 months of storage. During the long-term storage test, pH and quantitative (%) hardly changed, and the monomer (%) was maintained at 99% or more. In the accelerated storage test, pH, monomer (%), and quantitative (%) were measured after 1, 2, and 3 months of storage. During the accelerated storage test, pH and quantitative (%) hardly changed, and the monomer (%) was maintained at 98% or more. These results indicate that GH formulation 1 is stable for at least 24 months at 2 to 8°C in a dark place. In addition, when the specification values ​​were set at 98% or more for the elemental amount (%) and quantitative amount (%), respectively, and 100% or more, it was predicted from the values ​​obtained in the long-term storage test that GH formulation 1 would meet the specification values ​​when stored in a dark place at a temperature of 2 to 8°C, even after 36 months of storage.

[0066] [Table 5]

[0067] Table 6 shows the results of the stability evaluation of GH formulation 2, which has a phosphate buffer concentration of 16 mM. In the long-term storage test, pH, monomer (%), and quantification (%) were measured at the start of storage, one month after storage, and three months after storage. The pH, monomer (%), and quantification (%) hardly changed during the storage period of the long-term storage test, and showed values ​​equivalent to those of GH formulation 1. In the accelerated test, pH, monomer (%), and quantification (%) were measured one month, two months, and three months after storage. The pH, monomer (%), and quantification (%) hardly changed during the storage period of the accelerated test, and showed values ​​equivalent to those of GH formulation 1. These results indicate that GH formulation 2 has the same stability as GH formulation 1, and that GH formulation 2 is predicted to be stable for at least 24 months at temperatures between 2 and 8°C in a dark place. In addition, if the standard values ​​are set at 98% or more for the elemental amount (%) and 100% or more for the quantitative amount (%), respectively, it is predicted that GH formulation 2 will meet the standard values ​​even after 36 months when stored in a dark place at a temperature of 2 to 8°C.

[0068] [Table 6]

[0069] Tables 7 and 8 show the results of the stability evaluation of GH formulation 3 and GH formulation 4, each with a phosphate buffer concentration of 10 mM. In the long-term storage test, pH, monomer (%), and quantitative (%) were measured at the start of storage and about one month (4 weeks) after the start of storage. In both formulations, pH, monomer (%), and quantitative (%) hardly changed during the storage period of the long-term storage test, and showed similar values ​​to GH formulation 1. In the accelerated test, pH, monomer (%), and quantitative (%) were measured about one month (4 weeks) after the start of storage. In both formulations, pH, monomer (%), and quantitative (%) hardly changed during the storage period of the accelerated test, and showed similar values ​​to GH formulation 1. These results indicate that GH formulations 3 and 4 have the same stability as GH formulation 1, and that GH formulations 3 and 4 are expected to be stable for at least 24 months at temperatures between 2 and 8°C in a dark place. In addition, if the standard values ​​are set at 98% or more for the elemental amount (%) and 100% or more for the quantitative amount (%), respectively, it is predicted that GH formulations 3 and 4 will meet the standard values ​​even after 36 months when stored at a temperature of 2 to 8°C in a dark place.

[0070] [Table 7]

[0071] [Table 8]

[0072] [Example 5] Evaluation of the stability of aqueous liquid formulations of human growth hormone (2) For GH formulation 3 and GH formulation 4, which have a phosphate buffer concentration of 10 mM, a long-term storage test was conducted for 9 months from the start of storage, and an accelerated test was conducted for up to 3 months. Tables 9 and 10 show the results of the stability evaluation of GH formulation 3 and GH formulation 4, respectively. In the long-term storage test, pH, monomer (%), and quantitative (%) were measured at the start of storage, and 1 month, 2 months, 3 months, 6 months, and 9 months after the start of storage. In both formulations, pH, monomer (%), and quantitative (%) hardly changed during the storage period of the long-term storage test, and showed values ​​similar to those of GH formulation 1. In the accelerated test, pH, monomer (%), and quantitative (%) were measured 1 month, 2 months, and 3 months after the start of storage. In both formulations, pH, monomer (%), and quantitative (%) hardly changed during the storage period of the accelerated test, and showed values ​​similar to those of GH formulation 1. These results indicate that GH Formulations 3 and 4 have the same stability as GH Formulation 1, and that GH Formulations 3 and 4 are predicted to be stable for at least 24 months at 2-8°C in a dark place. In addition, if the specification values ​​are 98% or more for the unit amount (%) and 100% or more for the quantity (%), respectively, GH Formulations 3 and 4 are predicted to meet the specification values ​​even after 36 months when stored at 2-8°C in a dark place.

[0073] [Table 9]

[0074] [Table 10]

[0075] [Example 6] Measurement of growth hormone monomer (%) and quantification of growth hormone The monomer content (%) was measured by analyzing the samples using size-exclusion HPLC (SE-HPLC). A high-performance liquid chromatography system LC-20A (including a system controller CBM-20A, an online degassing unit DGU-20A5R, a liquid delivery unit LC-20AB, an autosampler SIL-20AC, a column oven CTO-20AC, and an ultraviolet-visible detector SPD-20AV or SPD-20A, Shimadzu Corporation) was equipped with an exclusion limit molecular weight of 5 × 10 5 A TSKgel G3000SWXL (inner diameter 7.8 mm × 30 cm, Tosoh Corporation), packed with hydrophilic silica gel for liquid chromatography with a particle size of 5 μm, was set. The column was equilibrated with the mobile phase (15.6 g of sodium dihydrogen phosphate dihydrate, 35.8 g of disodium hydrogen phosphate dodecahydrate, and 11.7 g of sodium chloride were dissolved in water to make 1000 mL, and then filtered through a membrane filter with a pore size of 0.22 μm). A sample diluted with pure water to a growth hormone concentration of 1 to 2 mg / mL was applied to the column, and the elution profile was created by monitoring the absorbance at 215 nm. The flow rate was 0.5 mL / min. The monomer percentage was calculated from the area of ​​the peak corresponding to the monomer (monomer peak area) and the area of ​​the peak corresponding to the dimer (dimer peak area) on the elution profile using the formula: monomer percentage (%) = monomer peak area / (monomer peak area + dimer peak area) × 100 (%).

[0076] In addition, growth hormone of known concentration was analyzed by SE-HPLC to prepare a calibration curve, and growth hormone present as a monomer in the sample was quantified by interpolating the monomer area onto this. The theoretical value of growth hormone contained in the solution at the start of storage, obtained from the calibration curve, was taken as 100%, and the quantitative value of each sample was calculated as the quantitative value (%). [Industrial Applicability]

[0077] According to the present invention, it is possible to provide an aqueous liquid preparation containing a protein as an active ingredient, which is stable in solution during storage and causes less pain when injected.

Claims

1. An aqueous liquid preparation comprising human growth hormone at a concentration of 1 to 50 mg / mL, a phosphate buffer at a concentration of 5 to 16 mM, a nonionic surfactant at a concentration of 1 to 10 mg / mL, a sugar alcohol, and a phenol, the nonionic surfactant being selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188, the osmotic pressure ratio being 0.9 to 1.1, and the preparation being free of sodium chloride.

2. 2. The aqueous liquid preparation according to claim 1, wherein the concentration of the phenol is 1 to 10 mg / mL.

3. 3. The aqueous liquid preparation according to claim 1, wherein the sugar alcohol is D-mannitol.

4. 4. The aqueous liquid preparation according to claim 1, having a pH of 5.5 to 7.

2.

5. An aqueous liquid preparation comprising human growth hormone at a concentration of 1 to 50 mg / mL, a phosphate buffer at a concentration of 5 to 16 mM, poloxamer 188 at a concentration of 1.8 to 2.2 mg / mL, phenol at a concentration of 2.8 to 3.8 mg / mL, and D-mannitol at a concentration of 35 to 45 mg / mL, having a pH of 6.0 to 6.4, an osmotic pressure ratio of 0.9 to 1.1, and not containing sodium chloride.

6. An aqueous liquid preparation comprising human growth hormone at a concentration of 4-8 mg / mL, a phosphate buffer at a concentration of 8-12 mM, poloxamer 188 at a concentration of 2 mg / mL, phenol at a concentration of 3.3 mg / mL, and D-mannitol at a concentration of 40 mg / mL, having a pH of 6.2, an osmotic pressure ratio of 0.9-1.1, and not containing sodium chloride.

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