KR20050023875A - Composition for stabilized liquid formulation of human growth hormone - Google Patents

Abstract

PURPOSE: A composition for stabilized liquid formulation of human growth hormone is provided, which composition minimizes deamidation, polymer formation and oxidative dissociation of human growth hormone(hGH), so that the stability of the liquid formulation can be improved. CONSTITUTION: The composition for stabilized liquid formulation of human growth hormone comprises 1 to 10 mg/ml of human growth hormone(hGH), 5 to 100 mM of buffering agent, 0.001 to 20 mg/ml of polyethylene glycol(PEG) and 5 to 100 mg/ml of tonicity adjustment agent, wherein the human growth hormone is recombinant methionyl human growth hormone or recombinant natural human growth hormone; the buffering agent is sodium citrate; the tonicity adjustment agent comprises sodium chloride, mannitol and a mixture thereof; and the composition has pH 5.5 to 6.5.

Description

COMPOSITION FOR STABILIZED LIQUID FORMULATION OF HUMAN GROWTH HORMONE}

The present invention relates to a composition for stabilizing liquid formulations of human growth hormone containing human growth hormone, isotonic agents and polyethylene glycol as active ingredients in a buffer without the addition of preservatives.

Human growth hormone (hGH) is an unglycosylated peptide hormone secreted from the anterior pituitary gland, which binds to receptors in various tissues of the human body and indirectly promotes or directly acts on the secretion of other growth factors, thereby promoting growth in various parts of the human body. It is known to promote. After the growth hormone extracted from human pituitary gland was found to be effective in treating pituitary dysplasia, the demand for hGH exploded, but the amount of growth hormone extracted from human pituitary gland was very limited. A child who received hGH from the human pituitary gland died of the Creutsfelt-Jacob Disease, a fatal neurological disease caused by a virus contaminated during growth hormone extraction, and died in the US Food and Drug Administration (FDA) The use of hormones extracted and purified from the human pituitary gland was inhibited (Roger, L., Science 234: 22, 1986).

Recently, hGH biosynthesized in Escherichia coli by Genentech Co., Ltd. has been approved as a rare medicine by FDA, and then recombinant hGH by Eli Lilly, Nordisk, Denmark, etc. Is commercialized and sold.

Generally, a problem with liquid formulations of pharmaceutical proteins, including hGH, is instability during the storage period. Long-term preservation of aqueous solutions is known to result in progressive degradation products due to deamidation, polymer formation, oxidation of methionine groups, and cleavage of peptide bonds. Removal can reduce its rate of degradation (Baffi, RA, Dev. Biol. Stand. 74: 181, 1992; Hageman, MJ, Drug Dev. Ind. Phar . 14: 2047, 1988). Due to these problems, hGH, which is currently used in practice in the clinic, is generally in the form of a lyophilized preparation that is dissolved and used before injection.

When using a hGH lyophilized formulation, the formulation is dissolved in the accompanying solvent and then injected subcutaneously or intramuscularly. In general, lyophilized formulations maintain long-term stability, but there are problems in reconstituting them into dissolution aids and injections. It is required. As part of this research, Genentech developed a liquid hGH formulation containing hGH, mannitol, buffers and nonionic surfactants (USP 6,448,225), suggesting a citrate solution as the preferred buffer and maintaining a pH of 5-7. It is illustrated that it is preferable. In addition, WO93 / 19776 discloses stable hGH prepared by selecting citrate salt as a buffer and optionally using amino acids and / or mannitol or other carbohydrates and preservatives such as benzyl alcohol, such as glycine, alanine and histidine. A liquid composition is disclosed.

In particular, WO 94/03198 discloses stable liquid compositions containing hGH, buffers, nonionic surfactants and optionally neutral salts, mannitol or preservatives, the buffers being citrate, sodium chloride and The composition containing polysorbate 20 (Tween 20) is specified. However, nonionic surfactants and preservatives contained in the composition can stabilize the aggregation reaction of hGH, but result in a significant amount of deamidated hGH, lowering the structural stability of hGH.

Therefore, liquid hGH can be obtained by setting various conditions of buffer, storage temperature, and additives, which are important stabilization factors for controlling degradation product formation rate by deamidation, polymer formation and oxidation of hGH. Screening for formulation conditions is desired. Accordingly, the present inventors have completed the present invention by developing a composition for human growth hormone liquid formulations having improved stability and safety by setting conditions that are non-ionic surfactants and preservatives that can potentially be toxic to the human body.

It is an object of the present invention to provide a composition for the liquid preparation of human growth hormone which can ensure safety with high stability by minimizing degradation by deamidation, polymer formation and oxidation of human growth hormone.

In order to achieve the above object, the present invention provides a composition for the liquid preparation of human growth hormone containing human growth hormone, buffers, polyethylene glycol and isotonic agents as active ingredients.

The composition of the present invention does not contain a preservative, thereby improving safety and exhibiting further improved stability, including polyethylene glycol (PEG) instead of nonionic surfactants used as stabilizers in conventional human growth hormone liquid formulations.

More preferably, the composition of the present invention comprises 1 to 10 mg / ml human growth hormone, 5 to 100 mg / ml sodium chloride (NaCl), mannitol as isotonic agent in 5 to 100 mM sodium citrate buffer. And mixtures thereof and 0.001 to 20 mg / ml polyethylene glycol, wherein the pH of the solution is preferably weakly or slightly acidic, with a pH of 5.5 to 6.5 being preferred.

Although the composition of the present invention is used as a stabilizer in the conventional hGH liquid formulation, nontoxic and non-immunogenic PEG is selected as a stabilizer instead of polysorbate, which is a nonionic surfactant that may act as a potential risk to the human body, and benzyl alcohol. By not adding a preservative such as phenol, it is possible to secure stability and safety for 12 months or more under low temperature (2 to 8 ° C) storage conditions.

Human growth hormone useful in the present invention can also be obtained from prokaryotic or eukaryotic organisms transformed with DNA encoding human growth hormone using well-known genetic recombination techniques, for example human growth using Escherichia coli or yeast as a host. The method for producing a hormone may be prepared according to the method described in Korean Patent No. 25013 or Korean Patent No. 316347. The human growth hormone of the present invention thus prepared may be recombinant methionyl human growth hormone or recombinant natural human growth hormone.

The buffer used in the present invention is to adjust the pH of the liquid formulation, and uses an organic acid such as citric acid or acetic acid that does not adversely affect human growth hormone, and the pH of the solution is 5.0 to 7.0, preferably to obtain stability. pH 5.5-6.5, most preferably pH 6.0. The concentration of buffer is 5-100 mM, preferably 5-50 mM, most preferably 5-20 mM.

In the present invention, polysorbate, a nonionic surfactant used as a stabilizer in a conventional liquid formulation, is potentially toxic to humans, so PEG, which exhibits non-toxicity and non-immunogenicity, is used as a stabilizer to rule out such risks. .

PEG is a useful polymer that has the property of dissolving in various organic solvents as well as water solubility. It is non-toxic and can be quickly removed from the body, and thus can be useful for stable pharmaceutical compositions (WO01 / 26692). ).

 It should be aqueous so as not to precipitate in an aqueous environment, such as a physiological environment, and the molecular weight of PEG is not limited, but 200 to 10,000 Da is preferred, and 3,000 to 8,000 Da are particularly preferred. PEG may or may not be branched. The concentration of PEG in the composition of the present invention is preferably 0.001 to 20 mg / ml.

In addition, the isotonic agent used in the present invention is to bring the solution into the isotonic state, and sodium chloride, mannitol, sucrose, sudrose, dextrose, sorbitol, or a mixture thereof may be used, preferably sodium chloride, Mannitol, or a mixture thereof, wherein the mixture is preferably mixed with sodium chloride and mannitol in a ratio of 1: 1 to 1: 2. It is preferable that the concentration of the tonicity agent in the composition of the present invention is 5 to 100 mg / ml.

The composition of the present invention may further comprise amino acids such as glycine, histidine. The amino acid acts as a stabilizer to impart stability to heat and is preferably included at a concentration of 0.01 to 10 mg / ml.

The present invention preferably comprises human growth hormone at a concentration of 3 mg / ml in 10 mM sodium citrate buffer; Sodium chloride at a concentration of 40 mg / ml; And PEG at a concentration of 2 mg / ml, the composition for liquid formulation of stabilized human growth hormone having a simple composition with a minimum pH of 6.0.

The present invention also provides human growth hormone at a concentration of 3 mg / ml in 10 mM sodium citrate buffer; Mannitol at a concentration of 40 mg / ml; And a composition for liquid formulation of stabilized human growth hormone having a pH of 6.0, consisting of PEG at a concentration of 2 mg / ml.

In addition, the present invention provides human growth hormone at a concentration of 3 mg / ml in 10 mM sodium citrate buffer; Mannitol at a concentration of 40 mg / ml and sodium chloride at a concentration of 26 mg / ml; PEG at a concentration of 2 mg / ml; And glycine at a concentration of 6 mg / ml, and a liquid formulation for stabilized human growth hormone having a pH of 6.0.

The stability of the hGH composition according to the present invention can be confirmed through a high performance liquid chromatography analysis method. Changes in physical properties of hGH are known to form dimers, polymers and deamides, the former two being identified by size exclusion HPLC (SE-HPLC) and the latter by reversed phase HPLC (RP-HPLC). In addition, since the relationship between the peak area of the hGH monomer obtained by size exclusion HPLC and the biological activity has been observed, the physicochemical measurement of the hGH content using the known biological activity as a standard is regarded as an alternative measuring method suitable for hGH biological analysis. (Yuki et al., Iyakuhin Kenkyu , 25: 383, 1994). Therefore, hGH evaluation using these two HPLCs provides for the assessment of the monomeric and deamide bodies as well as the biological activity of hGH. As a result, in the composition for hGH liquid preparation according to the present invention, a deamidated body is formed within 11% for 12 months under storage conditions of 4 ° C and pH 5.5 to 6.5, and the monomer content can be maintained at 99% or more. It turns out that. This result is that the composition for hGH liquid preparation according to the present invention can be safely supplied as a new form of hGH liquid formulation without the need for reconstitution of a conventional lyophilized formulation into a dissolving agent because its activity is kept stable for a long time under conditions of low temperature storage. It is present.

As described above, the composition for liquid formulation of hGH according to the present invention greatly improves stability and safety by using PEG instead of polysorbate, which is a nonionic surfactant, without adding a preservative used in a conventional liquid formulation. It can be usefully used, and in fact, it has been confirmed that they have a stability and safety of 12 months or more at a low temperature (2 to 8 ℃) storage conditions.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Reference Example 1 Analysis Method

Size-exclusion HPLC and reverse-phase HPLC were performed according to the European Pharmacopoeia EP method ( Europian Pharmacopia ).

Size exclusion HPLC was performed using a TSK gel G3000 SWXL (7.8 mm x 30 cm) column (Tosho, Japan) at room temperature with an eluate of 63 mM phosphate buffer (3-97, pH 7.0) containing 2-propanol at a flow rate of 0.6 ml. / Min. The eluate obtained therefrom was measured for absorbance at 214 nm to detect the formation of dimers and polymers due to changes in physical properties of hGH.

Reversed phase HPLC was performed using a Vydac 214TP54 (4.6 mm × 25 cm) column (Vydac, USA) with an eluent of 50 mM Tris HCl buffer (29: 71, pH 7.5) containing n-propanol at a flow rate of 0.5 ml / min. It was carried out by. At this time, the column temperature was 45 ℃, the absorbance of the eluate was measured at a wavelength of 220 nm to detect the formation of the deamide body due to the change in physical properties of hGH.

Preparation Example 1 Preparation of Composition for hGH Liquid Formulation

In order to improve the stability of hGH and the resulting degradation by the kind and concentration of additives such as buffers, stabilizers, isotonic agents, minimal simple combinations thereof, liquid compositions for various formulations as shown in Table 1 were prepared. . The hGH needed to prepare the solution formulation of the present invention was prepared according to the method disclosed in Korean Patent No. 316347. The hGH prepared as described above was a recombinant E. coli-derived expression product, and it was confirmed to have a purity of 99% or more in reversed phase chromatography (RP-HPLC) through various purification processes.

Liquid formulations of various compositions according to the present invention are dispensed with a solution of hGH in 10 mM sodium citrate buffer in a sterile bench to each sample glass vial, followed by addition of stabilizer, surfactant or PEG, amino acid, preservative at 4 ° C. By mixing them.

First, the compositions for each of the 20 liquid formulations shown in Table 1 were tested in two buffers, that is, compositions dissolved in 10 mM sodium citrate buffer (pH 6.0) and sodium acetate buffer (pH 6.0). At this time, PEG was 3,350 molecular weight and hGH was added at a concentration of 3 mg / ml.

As a result of an accelerated experiment of storing the above-prepared compositions for various times in an oven set at a temperature of 40 ° C. and checking the degree of proteolytic degradation products, a large amount of desorbed from the sodium acetate buffer composition than the sodium citrate buffer composition was observed. It was confirmed that proteolytic products such as amide bodies and dimers were produced, and the subsequent experiments were based on the composition of sodium citrate buffer.

Liquid formulations Isotonic agent (40 mg / ml) Stabilizer (2 mg / ml) Amino acid (6 mg / ml) Preservative (3 mg / ml) One NaCl Polysorbate 20 2 PEG 3 Polysorbate 20 Benzyl alcohol 4 Mannitol Glycine 5 Polysorbate 20 6 Polysorbate 20 Glycine 7 Polysorbate 20 Histidine 8 PEG Glycine 9 Polysorbate 20 Glycine Benzyl alcohol 10 PEG Glycine Benzyl alcohol 11 Sucrose Polysorbate 20 Benzyl alcohol 12 Dextrose Polysorbate 20 Benzyl alcohol 13 Mannitol / NaCl (40/26 mg / ml) Glycine 14 Polysorbate 20 15 Polysorbate 20 Glycine 16 Polysorbate 20 Histidine Benzyl alcohol 17 Polysorbate 20 Glycine Benzyl alcohol 18 PEG Glycine 19 PEG Glycine Benzyl alcohol 20 Sorbitol Polysorbate 20 Glycine Benzyl alcohol

Example 1 Stabilizing Effect of Liquid hGH Formulations According to Stabilizers

In this embodiment, in order to investigate whether the stabilizer contained in the hGH liquid formulation can suppress the change in the physical properties of hGH present in the liquid phase, the liquid formulations 1 to 3 of Table 1 are mixed with 10 mM sodium citrate buffer pH 6.0 One liquid formulation was used to compare the effect of PEG with polysorbate 20, a nonionic surfactant used as a stabilizer.

The formation of the deamide and dimer decomposition products of hGH formed over time while storing the liquid formulations 1 to 3 at 40 ° C. was analyzed by high performance liquid chromatography according to Reference Example 1, and the liquid formulations 1 to 3 were analyzed. Table 2 shows the results of analyzing the stability at 40 ° C. storage by high-performance liquid chromatography, and the production rate constants of the proteolytic products calculated by the above-mentioned chromatography are shown in Table 4 , respectively. At this time, the production rate constant k of the proteolytic product was calculated by taking the commercial log from 100% minus the generation rate of the variants and calculating these values from the slope of the graph plotted against the retention time.

Liquid formulation 40 ℃ preservation Monomer Content (%) (SE-HPLC) Deamide Content (%) (RP-HPLC) At startup 3 days 7 days 28 days At startup 3 days 7 days 28 days One 99.8 99.4 99.2 96.6 6.1 8.5 13.3 33.5 2 99.8 99.6 99.7 99.1 4.5 5.1 9.6 30.8 3 99.8 98.9 97.7 93.9 5.5 8.1 13.4 30.7

Liquid formulation 40 ℃ storage per week Dimer creation rate constant in days ^-1 × 10 ^-4 Rate constant for deamidation formation in days ^-1 × 10 ^-3 One 3.74 4.95 2 0.62 3.40 3 13 5.42

As confirmed in Tables 2 and 3 , the formulation conditions under which the PEG was selected as the stabilizer under the same buffer and isotonic conditions and without the preservative (liquid formulation 2) using polysorbate 20 as the stabilizer at 40 ° C. ( The hGH stabilization effect was superior to that of the liquid formulation 1) or the preparation conditions further comprising benzyl alcohol as a preservative (liquid formulation 3).

Example 2 Stabilizing Effect of hGH Liquid Formulations According to Isotonic Agents

In this example, the hGH stabilization effect was compared according to the types of isotonic agents, such as mannitol or sodium chloride, which can be selected as isotonic agents in the liquid formulation containing hGH. The effect was also confirmed. Liquid formulations 8, 10, 18 and 19 of Table 1 were mixed in 10 mM sodium citrate buffer (pH 6.0) to prepare liquid formulations of various compositions, followed by deamidation of hGH formed over time, stored at 40 ° C. The sieve and dimer decomposition products were analyzed by high performance liquid chromatography in accordance with Reference Example 1, and the results are shown in Tables 4 and 5 .

Liquid formulation 40 ℃ preservation Monomer Content (%) (SE-HPLC) Deamide Content (%) (RP-HPLC) At startup 3 days 7 days 28 days At startup 3 days 7 days 28 days 8 99.8 99.7 99.7 99.2 4.5 7.1 15.9 48.8 10 99.8 99.6 99.4 98.2 4.3 7.0 16.0 48.7 18 99.8 99.7 99.6 99.1 4.2 5.1 8.2 33.2 19 99.8 99.6 99.4 97.9 3.7 5.0 8.2 33.1

Liquid formulation 40 ℃ storage per week Dimer creation rate constant in days ^-1 × 10 ^-4 Rate constant for deamidation formation in days ^-1 × 10 ^-3 8 0.62 7.89 10 2.49 8.09 18 1.24 2.65 19 2.49 2.97

As a result, as shown in Tables 4 and 5 , the stabilizing effect of hGH according to the type of isotonic agents such as mannitol or sodium chloride was 40/26 (w / w). It can be seen that the hGH is more stable when mixed in the ratio.

In addition, when the liquid formulations 8 and 10 and 18 and 19 were compared, hGH was more stable under the condition of no preservatives, and the accelerated experiments of the liquid formulations 15 and 16 showed that the stability was lowered compared to glycine when histidine was selected instead of glycine. It was.

Example 3 Stabilizing Effect of hGH Liquid Formulations According to Preservatives

In this example, the stabilizing effect of hGH according to the addition of preservatives in the liquid formulation containing hGH was compared. The liquid formulations 17 to 19 of Table 1 were mixed in 10 mM sodium citrate buffer (pH 6.0) to prepare liquid formulations of various compositions, and then stored at 40 ° C. The degradation product was analyzed by high performance liquid chromatography according to Reference Example 1, and the results are shown in Tables 6 and 7 .

Liquid formulation 40 ℃ preservation Monomer Content (%) (SE-HPLC) Deamide Content (%) (RP-HPLC) At startup 3 days 7 days 28 days At startup 3 days 7 days 28 days 17 99.8 99.2 98.4 93.8 5.2 7.6 11.0 32.9 18 99.8 99.7 99.6 99.1 4.2 5.1 8.2 33.2 19 99.8 99.6 99.4 97.9 3.7 5.0 8.2 33.1

Liquid formulation 40 ℃ storage per week Dimer creation rate constant in days ^-1 × 10 ^-4 Rate constant for deamidation formation in days ^-1 × 10 ^-3 17 8.76 3.90 18 1.24 2.65 19 2.49 2.97

As shown in Tables 6 and 7 , liquid formulations 18 and 19 with PEG as the stabilizer under the same buffer and isotonic conditions, in particular liquid formulation 18 without the addition of the preservative benzyl alcohol, exhibited the highest stabilizing effect of hGH at 40 ° C. I confirmed it.

<Example 4> Stabilization effect of hGH according to the molecular weight and concentration of PEG

In this example, the stabilizing effect of hGH in the liquid formulations was investigated according to the molecular weight and concentration of PEG selected as a stabilizer. PEG having a molecular weight of 3,350 and 8,000 Da was selected among the commonly used PEGs having a molecular weight of 200 to 10,000 Da for the composition of Liquid Formulation 2 of Table 1 dissolved in 10 mM sodium citrate buffer (pH 6.0). The formation of deamidated and dimer decomposed products of hGH over time, stored at 40 ° C. in a 20 mg / ml concentration range, was analyzed by high performance liquid chromatography in accordance with Reference Example 1.

Dimer Content (%) Deamide Content (%) PEG 3,350 (mg / ml) At startup 3rd day 7th day Day 14 At startup 3rd day 7th day Day 14 0.001 0.20 0.31 0.39 0.50 3.70 6.81 11.31 23.57 0.01 0.21 0.33 0.42 0.59 3.70 6.75 11.19 23.58 0.1 0.20 0.32 0.39 0.51 3.69 6.87 11.22 23.59 10 0.20 0.33 0.40 0.56 3.69 6.80 11.07 23.47 20 0.20 0.34 0.40 0.53 3.70 6.84 11.22 23.58 Dimer Content (%) Deamide Content (%) PEG 8,000 (mg / ml) At startup 3rd day 7th day Day 14 At startup 3rd day 7th day Day 14 0.001 0.20 0.31 0.36 0.47 3.73 7.10 11.72 24.72 0.01 0.20 0.33 0.40 0.53 3.74 6.94 11.43 24.28 0.1 0.20 0.33 0.38 0.52 3.69 6.67 11.11 23.20 10 0.23 0.34 0.42 0.56 3.70 6.67 10.95 23.22 20 0.25 0.38 0.46 0.63 3.77 6.52 10.84 22.87

As shown in Table 8 , the deamide and dimer analysis of hGH showed similar stability in the molecular weight of 3,350 and 8,000 Da in the concentration range of 0.001 to 20 mg / ㎖ of PEG.

Example 5 Stabilization Effect of hGH Liquid Formulations Under Various Temperature Conditions

Examples 1 to 4 show that the minimal, simple and human-safe composition for hGH liquid formulations is liquid formulation 2 consisting of hGH, NaCl and PEG in sodium citrate buffer and liquid formulation 18 consisting of hGH, mannitol / NaCl, PEG and glycine. I could confirm it. The liquid formulations 2 and 18 were stored at 40 ° C., 25 ° C., and 15 ° C. for 6 months, and analyzed for the degree of degradation products. To this end, the stability of hGH over time at each temperature condition was performed by chromatographic analysis according to Reference Example 1 to measure the production rate of the variants. Variant generation rate constant k was obtained from the slope of the graph plotted against retention time after taking commercial logarithms from 100% minus the generation rate of variants. Variant generation rate constants at 5 ° C. were extrapolated to the Arrhenius plot graph. At this time, as a comparative example, the currently marketed hGH liquid formulation Neutropin-Aquarus (Nutropin Aquous, Genentech) was analyzed and compared.

As a result, the degree of formation of hGH deamide in the compositions of Liquid Formulations 2 and 18 was 30-35%, and the degree of formation of dimers and polymers was 30-50% compared to the control at 40 ° C, 25 ° C and 15 ° C temperature conditions. Excellent stability was shown, and this result is also predictable from a plot of isomer formation rate constants, such as deamides, with temperature in FIGS. 1 and 2 . In addition, even when stored for 12 months under refrigerated conditions, it was confirmed that the amount of isomers in the composition of the liquid formulations 2 and 18 of the present invention did not exceed 11% and the amount of dimers and polymers did not exceed 1%.

As discussed above, the liquid formulation of hGH according to the present invention has to be reconstituted with a dissolution aid upon injection, as in the case of conventional lyophilized formulations, with a very stable composition that provides minimal additive conditions that potentially eliminate harmful factors. It exhibits better shelf life than conventional hGH liquid formulations by minimizing the degree of partial loss of activity due to inconvenience and regeneration of dimers.

1 shows an Arrhenius Plot of the rate of polymer production of human growth hormone,

2 shows an Arrhenius plot of the isomer production rate of human growth hormone.

Claims (15)

A composition for liquid preparation of human growth hormone, comprising human growth hormone (hGH), a buffer, polyethylene glycol (PEG), and an isotonic agent as an active ingredient. The method of claim 1, A composition comprising polyethylene glycol at a concentration of 0.001 to 20 mg / ml. The method of claim 1, The molecular weight of polyethylene glycol is 200 to 10,000 Da, characterized in that the composition. The method of claim 3, wherein The composition of the polyethylene glycol is characterized in that the molecular weight of 3,000 to 8,000 Da. The method of claim 1, A composition comprising human growth hormone at a concentration of 1 to 10 mg / ml. The method of claim 1, A composition characterized in that the human growth hormone is recombinant methionyl human growth hormone or recombinant natural human growth hormone. The method of claim 1, The buffer is a composition, characterized in that sodium citrate at a concentration of 5 to 100 mM. The method of claim 1, Isotonic agent is selected from the group consisting of sodium chloride, mannitol and mixtures thereof at a concentration of 5 to 100 mg / ml. The method of claim 8, Sodium chloride and mannitol are mixed in a ratio of 1: 1 to 1: 2. The method of claim 1, Composition is characterized in that the pH of the composition is 5.5 to 6.5. The method of claim 1, 3 mg / ml human growth hormone, 40 mg / ml sodium chloride and 2 mg / ml polyethylene glycol in 10 mM sodium citrate buffer. The method of claim 1, 3 mg / ml human growth hormone, 40 mg / ml mannitol and 2 mg / ml polyethylene glycol in 10 mM sodium citrate buffer. The method of claim 1, A composition comprising a amino acid at a concentration of 0.01 to 10 mg / ml as a stabilizer. The method of claim 13, A composition wherein the amino acid is glycine or histidine. The method of claim 13, 3 mg / ml human growth hormone, 26 mg / ml sodium chloride, 40 mg / ml mannitol, 2 mg / ml polyethylene glycol and 6 mg / ml glycine in 10 mM sodium citrate buffer Composition.