RU2776108C2 - Lyophilized composition based on hgf - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the field of medicine, namely hepatology, and is a stable lyophilized composition that contains: (1) hepatocyte growth factor; (2) trehalose and (3) one or more compounds selected from a group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in which the mass ratio of hepatocyte and trehalose growth factor is from 1:4 to 1:460, and the mass ratio of hepatocyte growth factor and each of these one or more compounds is from 1:1 to 1:50 in a lyophilized composition. Also, the group of inventions relates to a method for obtaining a lyophilized composition and to a method for stabilizing the growth factor of hepatocytes.

EFFECT: improved stability during storage of hepatocyte growth factor in comparison with traditional lyophilized compositions.

10 cl, 18 tbl, 7 dwg

Description

FIELD OF TECHNOLOGY

[0001]

The present invention relates to a lyophilized composition containing a hepatocyte growth factor.

BACKGROUND OF THE INVENTION

[0002]

Hepatocyte growth factor (HGF) is a biologically active peptide with proliferative activity against liver parenchyma cells and found in various animal species. In humans, human hepatocyte growth factor (hHGF) has been found in the blood plasma of patients with fulminant hepatitis (patent source 1), and in recent years, recombinant human hepatocyte growth factor (rhHGF) can be produced in large quantities through biotechnology (non-patent source 1 ). It is assumed that such hepatocyte growth factors can be used as therapeutic and prophylactic agents effective against hepatitis and cirrhosis by increasing the number of normal parenchymal liver cells, as well as due to their various biological functions, as drugs for renal failure and pathological changes. in the stomach, duodenum, skin, etc., arising from the side effects of anticancer drugs.

[0003]

The lyophilized composition containing hepatocyte growth factor, which was disclosed in Patent Literature 2, contains hepatocyte growth factor and a stabilizing agent such as glycine, alanine, sorbitol, mannitol or dextran sulfate.

Patent Literature 3 discloses a lyophilized formulation containing hepatocyte growth factor at a concentration of less than 5 mg/ml, which is suitable for medicines, this formulation contains a stabilizing agent such as arginine, lysine, histidine, glutamic acid, or aspartic acid.

Patent Literature 4 discloses a lyophilized formulation containing a hepatocyte growth factor and a stabilizing agent such as purified sucrose.

LIST OF CITATIONS

PATENT LITERATURE

[0004]

[Patent Source 1] Patent Publication JP-A-S63-22526

[Patent Source 2] Patent Publication JP-A-H9-25241

[Patent source 3] WO 2000/072873

[Patent source 4] WO 2008/102849

NON-PAtent LITERATURE

[0005]

[Non-patent source 1] Nature, vol. 342, p. 440-443, 1989

BRIEF DESCRIPTION OF THE INVENTION

PROBLEMS SOLVED BY THE PRESENT INVENTION

[0006]

However, lyophilized formulations containing hepatocyte growth factor, which have been disclosed in patent publications JP-A-H9-25241, WO 2000/072873 and WO 2008/102849, which are intended to increase the stability of hepatocyte growth factor, are still not able to provide sufficient stability when storage.

In addition, it is not known how to reduce the formation of impurities along with hepatocyte growth factor, or how to maintain the pre-storage ratio of isoforms in freeze-dried formulations containing hepatocyte growth factor.

[0007]

Therefore, it is an object of the present invention to provide a lyophilized formulation having improved storage stability of hepatocyte growth factor compared to conventional lyophilized formulations.

MEANS FOR SOLVING THE PROBLEM

[0008]

The inventors of the present invention have made a thorough study to achieve the above object, and, consequently, it has been found that it is possible to achieve the goal with respect to a lyophilized composition containing (1) hepatocyte growth factor, (2) trehalose, and (3) one or more compounds selected from the group , consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts. Thus, the inventors of the present invention have realized the present invention.

[0009]

The present invention provides for the following:

[1] a lyophilized formulation containing (1) hepatocyte growth factor, (2) trehalose, and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine , creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts;

[2] a method for obtaining a lyophilized composition, including the steps:

(I) before adding (1) hepatocyte growth factor pH adjusting aqueous solution containing at least (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, to a pH of 4.5-6.5, and obtaining an aqueous solution containing (1) hepatocyte growth factor, (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts; and

(II) lyophilizing the aqueous solution containing the hepatocyte growth factor obtained in step (I);

[3] a method for stabilizing hepatocyte growth factor by incorporating (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris (hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized composition containing (1) hepatocyte growth factor;

[4] a method for stabilizing hepatocyte growth factor by incorporating (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris (hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized composition containing (1) hepatocyte growth factor, to reduce the formation of hepatocyte growth factor aggregates, reduce the formation of impurities along with hepatocyte growth factor and / or maintain the isoform ratio established before storage;

[5] a method for stabilizing hepatocyte growth factor by incorporating (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris (hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized composition containing (1) hepatocyte growth factor, to reduce the formation of hepatocyte growth factor aggregates;

[6] a method for stabilizing hepatocyte growth factor by incorporating (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris (hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized composition containing (1) hepatocyte growth factor, in order to reduce the formation of impurities along with hepatocyte growth factor;

[7] a method for stabilizing hepatocyte growth factor by incorporating (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris (hydroxymethyl)methylamine and their pharmaceutically acceptable salts in a lyophilized composition containing (1) hepatocyte growth factor, in order to maintain the ratio of hepatocyte growth factor isoforms established before storage; and

[8] items [1]-[7] described above, wherein the hepatocyte growth factor comprises the amino acid sequence represented by SEQ ID NO: 1.

BENEFICIAL EFFECT

[0010]

With the present invention, it is possible to obtain a lyophilized formulation having improved storage stability of hepatocyte growth factor compared to conventional lyophilized formulations.

BRIEF DESCRIPTION OF GRAPHICS

[0011]

Fig. 1 is an exemplary chromatogram of impurities along with hepatocyte growth factor;

fig. 2 is an exemplary chromatogram of hepatocyte growth factor isoforms;

in fig. 3 shows the amount of soluble aggregates in the lyophilized formulations tested in Test Example 2, as is and after storage at 60° C. for 1 month;

in fig. 4 shows the total amount of impurities in the lyophilized formulations tested in Test Example 3, in the initial state and after storage at 60°C for 1 month;

in fig. 5 shows the ratio of peak B versus isoforms in the lyophilized formulations tested in Test Example 4, as is and after storage at 60° C. for 1 month;

in fig. 6 shows the correlation between trehalose content and peak B ratio with respect to isoforms in the lyophilized formulations tested in Test Example 4; and

in fig. 7 shows the correlation between arginine content and B peak ratio versus isoforms in the lyophilized formulations tested in Test Example 4.

DESCRIPTION OF EMBODIMENTS

[0012]

Embodiments of the present invention are specifically described later in this document. The present invention is not limited to the embodiments described below, and can be modified in various ways within the scope of the present invention.

[0013]

The lyophilized formulation of the present invention contains (1) hepatocyte growth factor, (2) trehalose, and (3) one or more compounds (hereinafter also referred to as "compound (3)") selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts.

The hepatocyte growth factor for use in the present invention is a protein having an activity of promoting the growth of liver parenchyma cells, and is not specifically limited to the degree of purification in which it can be used as a drug. For example, Patent Publication JP-A-H11-56382 can be cited as a reference.

[0014]

The hepatocyte growth factor may be any naturally occurring in mammalian organ tissues such as the liver, spleen and kidneys, or in fluid tissues such as plasma and serum, or modified with a variant amino acid sequence and/or variant carbohydrate chains. Examples of mammals include humans, rats, rabbits, cows, horses, sheep, and the like, among which humans are preferred. Exemplary human hepatocyte growth factor amino acid sequences include SEQ ID NO: 1.

Hepatocyte growth factor can be extracted and purified from mammalian organ tissues such as liver, spleen and kidneys and from liquid tissues such as blood plasma and serum, or isolated from hepatocyte growth factor producing cells.

The hepatocyte growth factor can be obtained by introducing into an appropriate host cell an appropriate vector into which a polynucleotide encoding a hepatocyte growth factor has been introduced using a genetic engineering technique. The vector is not specifically limited in that it provides for the expression of a polynucleotide encoding a hepatocyte growth factor. Examples of the vector include a vector containing a selectable marker that is an antibiotic resistance gene such as neomycin, a vector containing a gene encoding dihydrofolate reductase (DHFR) or glutamine synthetase (GS), which can be amplified in animal cells, and the like, among which pCI vectors (available from Promega) and pcDNA vectors (available from Invitrogen) are preferred. The host cell is not particularly limited in that it allows transformation with the vector and expression of the recombinant hepatocyte growth factor. Examples of the host cell include Escherichia coli , Bacillus subtilis , yeast, filamentous fungi, plant cells, insect cells, animal cells, and the like, among which animal cells are preferred, and Chinese hamster ovary (CHO) cells are particularly preferred. .

The hepatocyte growth factor obtained by the genetic engineering technique may be, for example, a recombinant hepatocyte growth factor without particular limitation. For example, Patent Publication JP-A-H3-285693 can be cited as a reference. The hepatocyte growth factor used can be, for example, recombinant human hepatocyte growth factor (rhHGF) derived from a host cell that is a CHO cell.

The hepatocyte growth factor may be a modified hepatocyte growth factor that is modified by substitution, deletion, addition or insertion, or a combination thereof, using one or more, preferably 1-10 amino acid residues in the amino acid sequence of a naturally occurring hepatocyte growth factor in such a range, so that the modified amino acid sequence retains the activity of the hepatocyte growth factor. The modified hepatocyte growth factor may be one having, for example, 80%, preferably 85%, more preferably 90%, and most preferably 95% homology to the amino acid sequence of the naturally occurring hepatocyte growth factor. For example, patent publication WO 90/010651, patent publication JP-A-H4-030000 and patent publication JP-A-H5-111383 may be cited as reference.

[0015]

The concentration of hepatocyte growth factor before lyophilization is not particularly limited and may be, for example, 5 mg/ml or less, preferably 3 mg/ml or less, and more preferably 1 mg/ml or less.

[0016]

The lyophilized formulation of the present invention is administered parenterally to patients.

When the lyophilized formulation is used for inhalation administration, the lyophilized formulation of the present invention is dissolved prior to use and administered to a patient via a nebulizer.

When the lyophilized formulation is used for injection, the lyophilized formulation of the present invention is dissolved prior to use and administered intravenously, subcutaneously or intramuscularly to a patient.

The concentration of hepatocyte growth factor in the aqueous solution obtained by dissolving the lyophilized composition is not particularly limited, and may be, for example, 5 mg/ml or less. The water for dissolving the lyophilized composition of the present invention is not particularly limited as long as it is water used in medical practice, and examples of which include water for injection, sterilized purified water, saline, and the like.

[0017]

The trehalose for use in the present invention is not specifically limited to the degree of purification in which it can be pharmaceutically acceptable, and examples thereof include trehalose anhydride, trehalose hydroxide, and the like. Examples of trehalose hydroxide include, without particular limitation, trehalose hydroxide (trehalose dihydrate) listed in Japanese Pharmacopoeia 16th edition, and the like.

The amount of trehalose added to the lyophilized formulation is such that the weight ratio of hepatocyte growth factor to trehalose (hereinafter, in this section in terms of an equivalent amount of trehalose anhydride) is, for example, from 1:4 to 1:460, preferably 1:4 to 1:370 and more preferably 1:8 to 1:280. Alternatively, the total weight of the lyophilized composition on solids is 100.0 parts by weight, trehalose is, for example, 20.0-95.0 parts by weight and preferably 30.0-94.0 parts by weight. Also, in an aqueous solution containing 1.0 mg/ml of hepatocyte growth factor obtained by dissolving the lyophilized formulation, the concentration of trehalose is, for example, from 4.0 mg/ml to 460.0 mg/ml, and preferably from 8.0 mg /ml to 280.0 mg/ml.

[0018]

The compound (3) for use in the present invention is selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine, and pharmaceutically acceptable salts thereof.

Compound (3) is not specifically limited to the degree of purification in which it can be pharmaceutically acceptable. One or two or more types of compounds may be used (3).

Compound (3) preferably, in view of reducing the formation of aggregates and impurities, is one or more selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts.

Compound (3) includes arginine, histidine, lysine and meglumine as the main substance, and one or more of arginine, histidine and lysine is preferred. The amount of compound (3) that is added to the lyophilized composition may be in such a concentration range that will allow the hepatocyte growth factor to be dissolved without aggregation in an aqueous solution containing a hepatocyte growth factor, which is used to obtain a lyophilized composition, or in an aqueous solution containing a growth factor hepatocytes obtained by dissolving the lyophilized composition.

The amount of compound (3) added to the lyophilized formulation is such that the weight ratio of hepatocyte growth factor and compound (3) (hereinafter, in this section in terms of the equivalent amount of the free form) is, for example, from 1:1 up to 1:50, preferably from 1:1 to 1:40 and more preferably from 1:2 to 1:35. Alternatively, the total weight of the lyophilized composition in terms of solid particles is 100.0 parts by mass. The amount of compound (3) is, for example, 1.5-60.0 parts by mass, and preferably 3.0-59.0 parts by mass . Also, in an aqueous solution containing 1.0 mg/ml of hepatocyte growth factor obtained by dissolving the lyophilized composition, the amount of compound (3) is, for example, 1.0 mg/ml to 50.0 mg/ml, preferably 1 0 mg/ml to 40.0 mg/ml and more preferably 2.0 mg/ml to 35.0 mg/ml.

[0019]

Compound (3) of the present invention may be a pharmaceutically acceptable salt thereof. Examples of the pharmaceutically acceptable salt include hydrochloride, acetate salt, sodium salt, potassium salt, magnesium salt, and the like.

[0020]

In the present invention, in a lyophilized composition containing (1) hepatocyte growth factor, additionally containing (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, hepatocyte growth factor storage stability can be improved compared to conventional lyophilized formulations. Storage stability as used herein refers to a reduction in the formation of hepatocyte growth factor aggregates, a reduction in the formation of contaminants along with hepatocyte growth factor, and/or maintaining a pre-storage isoform ratio.

The present invention provides a method for stabilizing hepatocyte growth factor by including (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized formulation containing (1) hepatocyte growth factor.

Namely, the present invention provides a method for stabilizing hepatocyte growth factor by including (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized formulation containing (1) hepatocyte growth factor, to reduce the formation of hepatocyte growth factor aggregates.

The present invention also provides a method for stabilizing hepatocyte growth factor by including (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine , tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in order to reduce the formation of impurities in the lyophilized composition containing (1) hepatocyte growth factor together with hepatocyte growth factor.

The present invention also provides a method for stabilizing hepatocyte growth factor by including (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine , tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in a lyophilized composition containing (1) hepatocyte growth factor, in order to maintain the ratio of hepatocyte growth factor isoforms established before storage.

[0021]

The term "aggregates" as used herein refers to hepatocyte growth factor aggregates, and examples thereof include soluble aggregates such as dimers and trimers, and insoluble aggregates formed through interactions.

In the present invention, the number of aggregates can be estimated, for example, by measuring the amount of soluble aggregates using size exclusion chromatography.

In the present invention, the amount of soluble aggregates, when, for example, the amount of aggregates is estimated for a lyophilized formulation after storage at 60°C for 1 month by size exclusion chromatography, is, for example, less than 1.4%, and preferably 1.0% or less . Alternatively, the number of aggregates measured for the lyophilized formulation after storage at 60°C for 1 month by size exclusion chromatography, for example, is a multiple of 4.6 or less, preferably a multiple of 4 or less, more preferably a multiple of 3.5 or less and even more preferably a multiple of 3 or less of the number of aggregates prior to storage.

The amount of insoluble aggregates can be assessed for large insoluble aggregates with a size of 1 µm or more using the light absorption method of an insoluble substance described in the Japanese Pharmacopoeia 16th edition or using a microflow imaging method, and for insoluble aggregates with a size of 1 µm or less by the light scattering method or the ultracentrifugation method.

[0022]

The term "impurity" or "impurities" as used herein refers to one or more substances corresponding to one or more peaks that are observed in reverse phase chromatography, with the exception of the hepatocyte growth factor peak.

The amount of impurities is estimated using reverse phase chromatography. For example, for the freeze-dried formulation used in the examples herein, two peaks (hereinafter referred to as "ImpA" and "ImpB", respectively) were observed at a retention time of approximately 10 minutes and other impurities other than ImpA and ImpB (hereinafter referred to as herein referred to as "other imp"), with a retention time of 6 to 7 minutes, as shown in FIG. one.

In the present invention, the amount of impurities, for example, when the amount of impurities is evaluated for the lyophilized composition after storage at 60°C for 1 month by reverse phase chromatography, is, for example, 1.7% or less, preferably 1.5% or less , more preferably 1.3% or less, and even more preferably 1.0% or less. Alternatively, the amount of impurities measured for the lyophilized formulation after storage at 60° C. for 1 month by reverse phase chromatography is a multiple, such as less than 9, more preferably 7 or less, more preferably 5 or less, and more preferably 3 or less to the amount of impurities prior to storage.

[0023]

The term "isoform" as used herein refers to different charge variants of hepatocyte growth factor and a number of proteins having different charge states.

Isoform ratios can be estimated, for example, using ion exchange chromatography. For example, for the lyophilized formulations used in the examples herein, two peaks (hereinafter referred to as "peak A" and "peak B", respectively) were observed with a retention time of 35 to 45 minutes as shown in FIG. 2. The term "isoform ratio" as used herein refers to the ratio of each isoform to the total number of isoforms.

[0024]

The biological activity of the hepatocyte growth factor in the present invention can be assessed according to a cellular assay that evaluates the proliferation of mink lung epithelial cells (Mv.1.Lu cells) or 4BMr5 cells in the presence of transformed growth factor β-1 (TGFβ-1) (see 258, pp. 1-11, 2001, etc.) and can be expressed as 50% effective concentration (EC50). The change in biological activity of a lyophilized formulation during storage can be expressed as a relative titer (%) obtained by dividing the EC50 before storage by the EC50 after storage.

[0025]

In order to maintain a constant pH of an aqueous solution containing a hepatocyte growth factor used to obtain a lyophilized composition, or an aqueous solution containing a hepatocyte growth factor obtained by dissolving a lyophilized composition, the lyophilized composition may contain a buffer agent.

The buffer agent for use in the present invention is not particularly limited insofar as it is pharmaceutically acceptable, and examples thereof include phosphoric acid, sodium dihydrogen phosphate, sodium dihydrogen phosphate monohydrate, citric acid hydrate, sodium citrate hydrate, disodium citrate, acetic acid, glacial acetic acid, hydrate sodium acetate, sodium carbonate hydrate, sodium hydrogen carbonate, glycine, glycyl-glycine, and the like.

The buffering agent may be used in the form of a buffer solution that is commonly used, such as a phosphate buffer solution, a citrate buffer solution, and an acetate buffer solution.

The amount of buffer agent in the lyophilized formulation is, based on the concentration in the aqueous solution containing the hepatocyte growth factor, prior to lyophilization, for example, 1-100 mM. By adjusting the concentration within this range, a constant pH of the aqueous solution containing the hepatocyte growth factor obtained by dissolving the lyophilized formulation can be achieved.

[0026]

Hepatocyte growth factor has different solubility depending on pH, with the solubility minimum being near the isoelectric point. Therefore, the pH can be selected, while taking into account the isoelectric point, so that the hepatocyte growth factor can be dissolved without aggregation in an aqueous solution containing hepatocyte growth factor used to obtain a lyophilized composition, or in an aqueous solution containing hepatocyte growth factor obtained by dissolving the lyophilized composition.

In the present invention, the hepatocyte growth factor used in the examples in the present specification, for example, has a minimum solubility of 0.1-0.2 mg/mL near neutral pH, which is the isoelectric point, and has a solubility of 1.6 mg/mL or higher near pH 5. Therefore, the aqueous solution before lyophilization and the aqueous solution obtained by dissolving the lyophilized formulation have a pH of, for example, 4.5 to 6.5 and preferably 5.0 to 6.0.

In the process for preparing the hepatocyte growth factor lyophilized formulation of the present invention, a pH adjusting agent other than a buffering agent may be used to adjust the pH of the aqueous solution prior to lyophilization.

[0027]

The pH adjusting agent for use in the present invention is not particularly limited insofar as it is pharmaceutically acceptable, and examples thereof include hydrochloric acid, anhydrous citric acid, citric acid hydrate, sodium hydroxycitrate, glycine, succinic acid, acetic acid, glacial acetic acid, sodium hydroxyacetate , tartaric acid, sodium hydroxide, sodium bicarbonate, sodium hydroxycarbonate, dry sodium carbonate, monoethanolamine, triethanolamine, lactic acid, sodium lactate, phosphoric acid, disodium phosphate hydrate, anhydrous sodium monohydrogen phosphate, sodium dihydrogen phosphate, anhydrous sodium dihydrogen phosphate, crystalline sodium dihydrogen phosphate, trisodium phosphate , dipotassium phosphate, potassium dihydrogen phosphate, and the like.

[0028]

In order to prevent a decrease in the content of hepatocyte growth factor in the composition of the medicinal product to be administered due to the adsorption of hepatocyte growth factor on the glass or polymer of which the container is composed, after redissolving the lyophilized composition, the lyophilized composition may contain a surfactant.

The surfactant for use in the present invention is not particularly limited insofar as it is pharmaceutically acceptable, and examples thereof include nonionic surfactants, specifically including polysorbate 80, polysorbate 20, poloxamers, polyethylene glycol, HCO-40, HCO-60, etc. .P.

The amount of surfactant in the lyophilized formulation in an aqueous solution containing 1.0 mg/mL of hepatocyte growth factor obtained by dissolving the lyophilized formulation is, for example, 0.020 to 1.0 mg/mL, preferably 0.050 to 1.0 mg/ml, more preferably 0.075 to 0.5 mg/ml and even more preferably 0.075 to 0.2 mg/ml.

[0029]

The lyophilized composition of the present invention, while the total weight of the lyophilized composition by solid particles is 100.0 parts by weight, contains from 0.1 to 4.5 parts by weight of hepatocyte growth factor, from 20.0 to 95.0 parts by weight. weight of trehalose (hereinafter in this section in terms of an equivalent amount of trehalose anhydride), and from 1.5 to 60.0 parts by weight of the compound (3) (hereinafter in this document, in this section in terms of an equivalent amount of free form). In one embodiment, with the total weight of the lyophilized formulation on solids being 100.0 parts by weight, the hepatocyte growth factor is from 0.3 to 4.4 parts by weight, trehalose is from 30.0 to 94.0 parts by mass and the compound (3) is from 3.0 to 59.0 parts by mass. In another embodiment, hepatocyte growth factor is 0.3 to 4.4 parts by weight, trehalose is 30.0 to 94.0 parts by weight, compound (3) is 3.0 to 59.0 parts by weight. mass and polysorbate 80 is from 0.026 to 0.7 parts by mass.

[0030]

In the present invention, the lyophilized composition may further contain, if necessary, an additive such as a carrier, a tonicity adjusting agent, and an antioxidant.

[0031]

The process for preparing a lyophilized formulation of the present invention includes the steps of:

(I) before adding (1) hepatocyte growth factor pH adjusting aqueous solution containing at least (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, to a pH of 4.5-6.5, and obtaining an aqueous solution containing (1) hepatocyte growth factor, (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts; and

(II) lyophilizing the aqueous solution containing the hepatocyte growth factor obtained in step (I).

The lyophilized composition of the present invention can be obtained by the production method.

[0032]

In step (I), the pH of an aqueous solution containing at least (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl )methylamine and their pharmaceutically acceptable salts are adjusted to pH 4.5-6.5 and preferably to pH 5.0-6.0.

Prior to pH adjustment, the aqueous solution may contain (2) trehalose but does not contain (1) hepatocyte growth factor. It is after adjusting the pH of an aqueous solution containing at least compound (3) to pH 4.5-6.5 that (1) hepatocyte growth factor is added to it, or it is added simultaneously or sequentially (1) hepatocyte growth factor and (2 ) trehalose.

A buffering agent and/or a pH adjusting agent can be used to adjust the pH. The reaction temperature during pH adjustment is not particularly limited.

An aqueous solution containing at least compound (3) can be obtained by dissolving compound (3) in a solvent.

The solvent for obtaining an aqueous solution containing at least the compound (3) is not specifically limited to being, for example, pharmaceutically acceptable water. Examples thereof include water for injection, sterilized purified water, and the like.

[0033]

(1) Hepatocyte growth factor and (2) trehalose can be added as is or in the form of an aqueous solution. The solvent for obtaining an aqueous solution may be the same as or different from the solvent for obtaining an aqueous solution containing at least the compound (3). The temperature at the time of adding (1) hepatocyte growth factor and (2) trehalose is not particularly limited.

[0034]

In step (I), a surfactant, other additives (carrier, tonicity agent, antioxidant, etc.) and the like can be added as needed.

[0035]

In step (II), the aqueous solution containing the hepatocyte growth factor obtained in step (I) is lyophilized.

The aqueous solution containing the hepatocyte growth factor may be lyophilized according to the conventional method after pouring the aqueous solution containing the hepatocyte growth factor into a vial or ampoule.

An aqueous solution containing a hepatocyte growth factor sterilized by filtration through a filter or the like is preferable. and lyophilized.

Examples of the lyophilization method include a method including three single steps, namely, a freezing step for cooling and freezing below the nominal pressure, a primary drying step for sublimating and drying free water that is not entrained in solutes under reduced pressure, and a secondary drying step for removing adsorbed water and crystal water associated with solutes (for reference, Pharm. Tech. Japan, vol. 8, no. 1, p. 75-87, 1992, etc. may be referred to).

The temperature during the freezing step is, for example, 40° C. or less. The temperature during the primary drying step is, for example, -20°C to 10°C. The temperature during the secondary drying step is, for example, 20°C to 30°C. The reduced pressure is, for example, 10 Pa or less.

[0036]

The term "precipitate" as used herein refers to a porous solid in a lyophilized formulation. The characteristics of the lyophilized composition can be assessed by visual observation of the porous structure or color of the precipitate, or the solubility of the lyophilized composition in water, or visual observation of the aqueous solution thus obtained.

The lyophilized formulation of the present invention generally accepts a white precipitate and rapidly dissolves in water to provide a clear and colorless aqueous solution.

On the other hand, the aqueous solution obtained by dissolving the conventional lyophilized formulation may be cloudy, probably due to the formation of insoluble aggregates, which may be derivatives of hepatocyte growth factor and the like. Thus, the characteristics of the aqueous solution obtained by dissolving the lyophilized formulation may indicate the formation of insoluble aggregates. The fact that the aqueous solution is clear after storage of the lyophilized formulation indicates that the formation of insoluble aggregates is reduced.

EXAMPLES

[0037]

The present invention is more specifically described hereinafter by way of examples, which do not limit the present invention.

[0038]

The hepatocyte growth factor used in the present examples was obtained by whole gene synthesis based on the amino acid sequence information disclosed in patent publication JP-A-H11-056382, inserting the gene into a vector, expressing the protein in CHO cells, and isolating and purifying squirrel.

[0039]

(Examples 1-25 and Comparative Examples 1-7)

The lyophilized formulations from the Examples and Comparative Examples were prepared using the method described herein below. Tables 1-4 show the amounts (mg) of components per ml of each of the aqueous solutions containing hepatocyte growth factor. The values in the tables were rounded to one decimal place. Aqueous solutions containing hepatocyte growth factor were prepared in volumes ranging from 100 ml to 250 ml.

[0040]

Table 1

Examples Comparative examples one 2 3 four one 2 3 four 5 6 Hepatocyte growth factor 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.1 Trehalose dihydrate 100.0 100.0 100.0 100.0 - - - - - - Xylitol - - - - 100.0 - - - - - sucrose - - - - - 100.0 - - - 10.0 Sorbitol - - - - - - 100.0 - - - Mannitol - - - - - - - 100.0 - - L-arginine 17.4 - - - 17.4 17.4 17.4 17.4 17.4 - L-histidine - 15.5 - - - - - - - - L-lysine - - 14.6 - - - - - - - Meglumine - - - 19.5 - - - - - - L-alanine - - - - - - - - - 5.0 Sodium chloride - - - - - - - - 9.0 18.0 Citric acid monohydrate 2.1 Polysorbate 80 0.1 0.3

[0041]

table 2

Examples 5 6 7 eight 9 ten eleven Hepatocyte growth factor 1.1 1.1 1.1 1.1 1.1 1.1 1.1 Trehalose dihydrate 100.0 L-arginine 4.2 - - - - - - L-arginine hydrochloride 16.0 - - - - - - Monosodium glutamate - 18.7 - - - - - Sodium aspartate - - 17.7 - - - - Proline - - - 11.5 - - - Creatine - - - - 14.9 - - Creatinine - - - - - 11.3 - Tris(hydroxymethyl)methylamine - - - - - - 12.1 Citric acid monohydrate 2.1 Polysorbate 80 0.1

[0042]

Table 3

Examples 12 13 fourteen fifteen 16 17 eighteen Hepatocyte growth factor 0.5 1.0 3.1 5.1 1.1 1.0 1.0 Trehalose dihydrate 100.0 100.0 100.0 100.0 10.0 25.0 50.0 L-arginine 5.5 6.8 12.1 17.4 4.2 4.2 4.2 L-arginine hydrochloride 14.4 12.8 6.4 - 16.0 16.0 16.0 Citric acid monohydrate 2.1 Polysorbate 80 0.1

[0043]

Table 4

Examples Comparative Example 7 19 twenty 21 22 23 24 25 Hepatocyte growth factor 1.0 1.0 1.1 1.1 1.1 1.1 1.1 1.1 Trehalose dihydrate 150.0 200.0 250.0 300.0 100.0 100.0 100.0 - L-arginine 4.2 4.2 4.2 4.2 3.5 8.7 26.1 4.2 L-arginine hydrochloride 16.0 16.0 16.0 16.0 - - - 16.0 Citric acid monohydrate 2.1 Polysorbate 80 0.1

[0044]

<Get Mixing Buffer>

According to the volume of each aqueous solution containing hepatocyte growth factor and the concentration indicated in tables 1-4, components other than hepatocyte growth factor or polysorbate 80 were mixed and shaken with the required amount of water for injection (approximately 80% of the volume of the aqueous solution, containing hepatocyte growth factor). After visual confirmation of dissolution, the pH was adjusted to 5.5 with hydrochloric acid and/or sodium hydroxide and the volume was adjusted with water for injection to provide a mixing buffer.

<Preparation of an aqueous solution containing hepatocyte growth factor>

An aqueous solution containing hepatocyte growth factor was obtained using the following procedure.

(a) An aqueous solution containing hepatocyte growth factor was adjusted to obtain the concentration of hepatocyte growth factor as indicated in Table 1 by ultrafiltration using a mixing buffer. The concentration of hepatocyte growth factor was calculated according to the following formula (A) after determining the optical density at 280 nm on a spectrophotometer in the ultraviolet and visible spectral region (using a cell with a light-absorbing layer thickness of approximately 1 cm):

Protein concentration (mg/ml)=(A280/1.73)×D(A),

where

A280: optical density at 280 nm;

1.73: theoretical optical density of the test solution at 0.1% concentration, with a light-absorbing layer thickness of approximately 1 cm and at 280 nm; and

D: dilution factor for measurement.

(b) Polysorbate 80 was then added to reach the desired concentration as indicated in Table 1, followed by filtration through a sterilizing filter (product name: 0.22 µm MILLEX GV filter available from Millipore) to obtain an aqueous solution, containing hepatocyte growth factor.

<lyophilization step>

The aqueous solution containing the hepatocyte growth factor was divided into 1 ml into 5 ml glass vials, which were then partially closed with rubber caps. After lyophilization according to the plan shown in Table 5, each vial was completely closed with a rubber cap. The aluminum cap was then attached to a glass vial to form a lyophilized formulation.

Comparative Example 5 is a lyophilized formulation prepared according to the formulation disclosed in WO 2000/072873. Comparative Example 6 is a lyophilized formulation prepared according to the formulation disclosed in WO 2008/102849.

[0045]

Table 5

Freezing Maintain Freeze pressure reduction The heating Primary drying The heating Secondary drying Temperature(°C) Room temp. → -40 -40 -40 -40 -20 -twenty -20 20 twenty Pressure (Pa) normal pressure normal pressure Normal pressure → 10 ten ten ten ten Time (h) 1.5 four one 2 48 eight 48

[0046]

(Test example 1)

The resulting lyophilized formulations in Examples 1-11 and Comparative Examples 1-6 were tested for the characteristics of the lyophilized formulations, the solubility of the lyophilized formulations in water for injection, and the characteristics of the aqueous solutions obtained after dissolution, according to the following procedures.

<Method>

Each resulting lyophilized composition was visually observed (precipitation and color) on white and black backgrounds in the environment at 1000-3000 lux. Water for injection (1 ml) was then added to each lyophilized formulation, which was then held before visual observation of the solubility of the lyophilized formulation and the color of the resulting solution.

The term "rapidly soluble" shown in Table 6 refers to a formulation that completely dissolved within 1 minute, and the term "slightly soluble" refers to a formulation that did not completely dissolve even after 1 minute.

[0047]

<Results>

The results for the samples from Examples 1-4 and Comparative Examples 1-6 are shown in Table 6. In Examples 1-4, white precipitates formed and acceptable solubility was demonstrated. Comparative Example 1 containing xylitol and Comparative Example 3 containing sorbitol did not form precipitates and white or colorless molten solids were observed. Comparative Example 5, which did not include the saccharide, was a white solid with no precipitation commonly seen with lyophilized formulations. Examples 5-11 also produced white precipitates similar to Examples 1-4 and showed acceptable solubility.

[0048]

Table 6

Sediment formation Sediment color Solubility Mortar color Example 1 Good White Quick Dissolve Transparent Example 2 Good White Quick Dissolve Transparent Example 3 Good White Quick Dissolve Transparent Example 4 Good White Quick Dissolve Transparent Comparative Example 1 Not Colorless sparingly soluble Transparent Comparative Example 2 Good White Quick Dissolve Transparent Comparative Example 3 Not White sparingly soluble Transparent Comparative Example 4 Good White Quick Dissolve Transparent Comparative Example 5 Not White Quick Dissolve Transparent Comparative Example 6 Good White Quick Dissolve Transparent

[0049]

The resulting lyophilized formulations were stored in a thermostatic chamber at 25°C, 40°C or 60°C for 2 weeks or 1 month, followed by the following tests.

[0050]

(Test example 2)

The amount of soluble aggregates in Examples 1-25 and Comparative Examples 1-7 was evaluated as follows.

<Method>

Water for injection (1 ml) was added to each lyophilized formulation of Examples and Comparative Examples to dissolve under ice cooling. The solution was shaken moderately to obtain a homogeneous solution, which was then held for 5 minutes. The resulting solution was dispensed into 250 µl microinserts, which are located in HPLC vials, to obtain samples.

Each sample was measured by size exclusion chromatography under the following analytical conditions to determine the amount of soluble aggregates before and after storage, and the amount of soluble aggregates was calculated using the percentage area method.

Detector: UV 280 nm;

Column: guard column (trade name: TSKgel G3000SWXL, available from Tosoh Corporation);

Column temperature: 30°C;

Mobile phase: prepared by dissolving 39 g of sodium dihydrogen phosphate dihydrate, 87.5 g of sodium chloride and 5.0 g of sodium lauryl sulfate in 5000 ml of water and adjusting the pH of the solution to 7.5 with 2 mol/l sodium hydroxide solution;

Sample sample: 50 µl; and

Consumption: 1 ml/min.

[0051]

<Results>

The results of measuring the amount of soluble aggregates in the samples in the initial state (before storage) and after storage at 25°C, 40°C or 60°C for 2 weeks or 1 month are shown in tables 7-9. The results of measuring the amount of soluble aggregates in the samples of Examples 1-4 and Comparative Examples 1-6 in the initial state and after storage at 60°C for 1 month are shown in FIG. 3. In addition, the correlation between the content of trehalose in freeze-dried formulations and the amount of soluble aggregates is shown in table 10,

[0052]

According to Table 7, the amount of soluble aggregates in Comparative Examples 1-6 increased with increasing storage time and storage temperature. The number of soluble aggregates increased significantly after storage at 60°C for 1 month.

In addition, according to tables 7 and 8, such a significant change in the number of soluble aggregates as in comparative examples was not observed for examples 1-11 under the tested storage conditions. Thus, the formation of soluble aggregates was reduced in examples 1-11 even under storage conditions of 60°C.

[0053]

According to tables 9 and 10, the number of soluble aggregates increased significantly in comparative example 7, which did not include trehalose.

In addition, a slight change in the amount of soluble aggregates was observed in examples 12-15 containing different amounts of hepatocyte growth factor, and examples 23-25 containing different amounts of arginine.

[0054]

Table 7

Number of aggregates (%) Initial value Storage 2 weeks Storage 1 month Storage at 25°C Storage at 40°C Storage at 60°C Storage at 25°C Storage at 40°C Storage at 60°C Example 1 0.3 0.2 0.2 0.2 0.2 0.2 0.3 Example 2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Example 3 0.3 0.4 0.4 0.4 0.3 0.3 0.4 Example 4 0.3 0.3 0.3 0.3 0.3 0.3 0.8 Comparative Example 1 0.3 0.6 1.1 26.4 0.8 1.6 44.1 Comparative Example 2 0.3 0.3 0.3 0.6 0.3 0.3 1.4 Comparative Example 3 0.3 0.3 0.7 11.5 0.4 0.9 21.5 Comparative Example 4 0.3 0.3 0.3 1.9 0.3 0.4 5.6 Comparative Example 5 0.3 0.3 0.4 1.9 0.4 0.4 3.1 Comparative Example 6 0.4 0.8 1.1 6.8 1.0 1.4 18.4

[0055]

Table 8

Number of aggregates (%) Initial value Storage at 60°C/2 weeks Example 5 0.2 0.2 Example 6 0.2 0.3 Example 7 0.3 0.3 Example 8 0.2 0.2 Example 9 0.2 0.3 Example 10 0.2 0.2 Example 11 0.2 0.3

[0056]

Table 9

Number of aggregates (%) Initial value Storage 1 month Storage at 25°C Storage at 40°C Storage at 60°C Example 12 0.3 0.2 0.2 0.2 Example 13 0.2 0.2 0.2 0.2 Example 14 0.2 0.3 0.2 0.2 Example 15 0.2 0.2 0.3 0.3 Example 16 0.2 0.2 0.2 0.6 Example 17 0.2 0.2 0.2 0.4 Example 18 0.2 0.2 0.3 0.2 Example 19 0.2 0.3 0.2 0.3 Example 20 0.2 0.2 0.2 0.2 Example 21 0.2 0.2 0.2 0.2 Example 22 0.2 0.2 0.2 0.2 Example 23 0.2 0.2 0.3 0.3 Example 24 0.2 0.3 0.2 0.3 Example 25 0.3 0.2 0.2 0.2 Comparative Example 7 0.2 0.2 0.2 1.5

[0057]

Table 10

Conc. trehalose (mg/ml) Number of aggregates (%) Initial value Storage 1 month Storage at 25°C Storage at 40°C Storage at 60°C Comparative Example 7 0 0.2 0.2 0.2 1.5 Example 16 ten 0.2 0.2 0.2 0.6 Example 17 25 0.2 0.2 0.2 0.4 Example 18 fifty 0.2 0.2 0.3 0.2 Example 13 100 0.2 0.2 0.2 0.2 Example 19 150 0.2 0.3 0.2 0.3 Example 20 200 0.2 0.2 0.2 0.2 Example 21 250 0.2 0.2 0.2 0.2 Example 22 300 0.2 0.2 0.2 0.2

[0058]

(Test example 3)

The amount of impurities in Examples 1-25 and Comparative Examples 1-7 was evaluated as follows.

<Method>

The samples used were those in HPLC vials obtained to evaluate the amount of soluble aggregates.

Each sample was measured by reverse phase chromatography under the following analytical conditions to determine the amount of impurities before and after storage, and the amount of impurities was calculated by the percentage area method.

Detector: UV 215 nm;

Column: reverse phase column (trade name: Inertsil WP300-C8, available from GL Sciences Inc.) attached to a GL Cart guard column;

Column temperature: 40°C;

Mobile phase A: water/trifluoroacetic acid (1000:1);

Mobile phase B: acetonitrile/trifluoroacetic acid (1000:0.85);

Sample sample: 25 µl;

Consumption: 1 ml/min.; and

Gradient: Volume ratio of mobile phase B to total mobile phase A and mobile phase B: 15% (baseline) - 70% (27.5 min.) - 70% (30 min.) - 15% (30.01 min.) .) - 15% (35 min.).

[0059]

<Results>

The results of measuring the amount of impurities in the samples from examples 1-11 and comparative examples 1-6 in the initial state (before storage) and after storage at 25°C, 40°C or 60°C for 2 weeks or 1 month are shown in tables 11 -12. The results of measuring the total amount of impurities in the samples of Examples 1-4 and Comparative Examples 1-6 in the initial state and after storage at 60°C for 1 month are shown in FIG. four.

[0060]

According to table 11, the total amount of impurities in examples 1-4 practically did not change under storage conditions at 25°C and 40°C. According to Tables 11 and 12, the increase in the total amount of impurities in Examples 1 to 11 was no more than 2-fold relative to the original value even under storage conditions of 60°C, while the increase in the amount in the compositions of all comparative examples was almost 10-fold or more . Under storage conditions of 60°C, it was found that any of ImpA, ImpB and other imp increased in the compositions of all comparative examples. Thus, the formation of impurities was reduced in examples 1-11 even under storage conditions of 60°C.

In examples 12-25, a slight change in the total amount of impurities was observed under any storage conditions.

[0061]

Table 11

Total impurities (%) Initial value Storage 2 weeks Storage 1 month Storage at 25°C Storage at 40°C Storage at 60°C Storage at 25°C Storage at 40°C Storage at 60°C Example 1 0.18 0.19 0.19 0.36 0.20 0.20 0.32 Example 2 0.31 0.33 0.34 0.55 0.33 0.35 0.55 Example 3 0.20 0.28 0.21 0.41 0.61 0.23 0.32 Example 4 0.19 0.20 0.21 0.42 0.20 0.20 0.47 Comparative Example 1 0.23 0.51 0.74 2.34 0.38 0.50 4.31 Comparative Example 2 0.20 0.19 0.68 0.95 0.19 0.21 2.25 Comparative Example 3 0.19 0.21 0.31 1.42 0.28 0.48 2.40 Comparative Example 4 0.19 0.20 0.48 0.89 0.29 0.31 1.71 Comparative Example 5 0.22 0.35 0.43 1.02 0.23 0.49 2.69 Comparative Example 6 0.91 1.58 1.75 2.86 1.84 2.11 3.56

[0062]

Table 12

Total impurities (%) Initial value Storage at 60°C/2 weeks Example 5 0.21 0.38 Example 6 0.20 0.41 Example 7 0.21 0.34 Example 8 0.21 0.36 Example 9 0.20 0.38 Example 10 0.21 0.39 Example 11 0.20 0.39

[0063]

(Test example 4)

The ratio of isoforms in examples 1-25 and comparative examples 1-7 was evaluated as follows.

<Method>

The samples used were those in HPLC vials obtained to estimate the number of aggregates.

Each sample was measured by ion exchange chromatography under the following analytical conditions to determine the number of isoforms before and after storage, and the isoform ratio was calculated by the area percentage method.

Detector: UV 215 nm;

Column: trade name: Agilent Bio SCX NP5 PK, available from Agilent Technologies;

Column temperature: 30°C;

Mobile phase A: 50 mmol/l Tris-HCl buffer (pH 9.0);

Mobile phase B: 50 mmol/l Tris-HCl buffer (pH 9.0), 1 mol/l NaCl

Sample sample: 50 µl;

Consumption: 0.5 ml/min; and

Gradient: volume ratio of mobile phase B to total mobile phase A and mobile phase B: 20% (initial value) - 60% (50 min.) - 80% (51 min.) - 80% (60 min.) - 20 % (60.01 min.) - 20% (80 min.).

[0064]

<Results>

The results of measuring the change in the number of isoforms in terms of the ratio of peak B to the sum of peak A and peak B in samples in the initial state (before storage) and after storage at 25°C, 40°C or 60°C for 2 weeks or 1 month are shown in tables 13-15. The results of measuring the change in the ratio of isoforms in terms of the ratio of peak B in the samples of Examples 1-4 and Comparative Examples 1-6 in the initial state and after storage at 60°C for 1 month are shown in FIG. 5. In addition, the correlation between trehalose content in lyophilized formulations and peak B ratio is shown in FIG. 6, while the correlation between arginine content and peak B ratio is shown in FIG. 7.

[0065]

According to Tables 13 and 14, a slight change in peak B isoform ratio was observed in Examples 1-11 and Comparative 5 under the storage conditions tested, while peak B isoform ratio changed significantly in the other Comparatives under storage conditions of 60°C. Thus, the change in the ratio of isoforms was reduced in examples 1-11 even under storage conditions of 60°C. In Comparative Example 1, a significant change was observed after storage, namely, peak A isoform was excluded so that the detectable isoform was only peak B and a peak that was not the isoform that was observed.

[0066]

According to FIG. 6, it was found that the peak B isoform ratio decreased with increasing trehalose content in the formulations. Similarly, a decrease in peak B isoform ratio was also observed for the formulation of Comparative Example 7, which did not include trehalose.

According to FIG. 7, it was found that the peak B isoform ratio decreased with decreasing arginine content in the formulations under storage conditions at 60° C. for 1 month.

In addition, there was no change in the ratio of isoforms depending on the change in the content of hepatocyte growth factor in the formulations.

[0067]

Table 13

Peak B ratio (%) Initial value Storage 2 weeks Storage 1 month Storage at 25°C Storage at 40°C Storage at 60°C Storage at 25°C Storage at 40°C Storage at 60°C Example 1 90.59 90.10 90.89 89.81 90.87 90.87 90.68 Example 2 90.62 90.60 90.89 90.24 90.96 90.96 90.51 Example 3 91.51 90.22 90.79 91.09 91.04 91.21 90.70 Example 4 90.69 90.84 90.58 90.59 91.08 91.05 90.70 Comparative Example 1 90.28 90.74 89.79 85.37 89.94 90.06 100.00 Comparative Example 2 90.82 90.89 90.62 90.07 90.71 90.78 88.93 Comparative Example 3 90.53 90.99 90.84 88.44 90.75 90.52 84.56 Comparative Example 4 90.75 91.43 90.57 89.80 90.38 90.89 86.08 Comparative Example 5 91.22 90.72 90.40 90.37 90.27 90.86 90.35 Comparative Example 6 91.17 90.40 90.54 90.24 90.66 91.45 83.05

[0068]

Table 14

Peak B ratio (%) Initial value Storage at 60°C/2 weeks Example 5 89.95 89.76 Example 6 89.89 89.89 Example 7 89.57 89.82 Example 8 90.00 89.35 Example 9 90.23 89.73 Example 10 89.70 89.96 Example 11 90.04 90.12

[0069]

Table 15

Peak B ratio (%) Initial value Storage 1 month Storage at 25°C Storage at 40°C Storage at 60°C Example 12 90.09 89.94 90.38 89.66 Example 13 90.18 89.97 90.35 89.88 Example 14 89.71 90.20 90.26 89.76 Example 15 89.71 90.00 90.20 89.86 Example 16 90.59 90.44 90.79 90.55 Example 17 90.28 90.38 90.54 90.45 Example 18 90.15 90.19 90.15 90.49 Example 19 89.87 89.97 90.09 90.07 Example 20 89.76 90.02 89.90 89.54 Example 21 89.68 89.63 89.76 89.83 Example 22 89.51 89.72 89.61 89.29 Example 23 90.02 90.26 89.98 88.68 Example 24 90.35 89.95 90.17 89.34 Example 25 90.12 90.30 90.34 89.93 Comparative Example 7 90.05 90.37 90.65 89.77

[0070]

(Test example 5)

The amount of insoluble aggregates in Examples 1-25 and Comparative Examples 1-7 was evaluated as follows.

<Method>

Water for injection (1 ml) was added to each lyophilized composition, which was kept and then dissolved in water for injection, or each placebo aqueous solution to obtain a sample. The amount of insoluble microparticles before and after storage in each sample was measured on a particle flow imaging analyzer and the amount of insoluble microparticles was calculated according to the following formula:

Number of microparticles (number/vial)=P×1000×V/(n×v),

where

P: number of detected particles;

V: volume of sample solution (ml);

n: number of measurements; and

v: sample volume per measurement.

[0071]

<Results>

The results of measuring the amount of insoluble aggregates in the samples from examples 1-11 and comparative examples 1-6 in the initial state (before storage) and after storage at 60°C for 2 weeks or 1 month are shown in tables 16 and 17.

[0072]

According to Table 16, the amount of insoluble aggregates in Comparative Examples 1-6 increased after storage at 60°C for 1 month. In addition, according to tables 16 and 17, such a significant change in the amount of soluble aggregates, as in comparative examples, was not observed for examples 1-11. Thus, the formation of insoluble aggregates was reduced in examples 1-11 even under storage conditions at 60°C for 1 month. A slight change in the number of insoluble aggregates was observed for examples 12-25.

[0073]

Table 16

Number of insoluble microparticles (quantity/vial) < 10 µm ≥ 10 µm ≥ 25 µm Initial value Storage at 60°C/1-month Initial value Storage at 60°C/1-month Initial value Storage at 60°C/1-month negative control 6786 - 143 - 0 - Example 1 13929 9286 333 190 0 0 Example 2 7857 3929 143 95 0 48 Example 3 7143 12143 286 333 95 48 Example 4 19643 6429 381 0 95 0 Comparative Example 1 18571 390000 333 87524 190 49857 Comparative Example 2 15357 7857 286 1095 48 333 Comparative Example 3 30000 7392857 381 1476 0 48 Comparative Example 4 28929 334643 238 70286 0 24476 Comparative Example 5 11071 66786 143 143 48 0 Comparative Example 6 3214 5153214 238 197238 48 18524

[0074]

Table 17

Number of insoluble microparticles (quantity/vial) < 10 µm ≥ 10 µm ≥ 25 µm Initial value Storage at 60°C/2 weeks Initial value Storage at 60°C/2 weeks Initial value Storage at 60°C/2 weeks Example 5 714 357 0 0 0 0 Example 6 714 357 48 48 0 0 Example 7 2143 0 95 0 48 0 Example 8 1071 357 0 0 0 0 Example 9 714 1786 95 48 95 0 Example 10 1429 357 0 190 0 48 Example 11 357 357 48 0 0 0

[0075]

(Test example 6)

The biological activity in Example 1, Comparative Example 5 and Comparative Example 6 was evaluated as follows.

<Method>

Mink lung epithelial cells (Mv.1.Lu cells) were cultured, cell viability was confirmed, and then a 1×10 ⁵ /ml cell solution was prepared. A solution of transformed growth factor β-1 (TGFβ-1) was added to each well of a 96-well assay plate and a sample solution was added to each well containing hepatocyte growth factor at a concentration of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 or 128 ng/ml. Then a solution of cells 1×10 ⁵ /ml was added and the plate was incubated in a 5% carbon dioxide incubator at 37°C for 69 hours. Cell count kit (10 µl) available from Dojindo Molecular Technologies, Inc. was added to the plate, which was then incubated for 3 hours, and the optical density was measured at 450 nm.

The obtained absorbance value was plotted on the vertical axis with respect to the HGF concentration in the samples (horizontal axis) and the EC50 of the samples was calculated using analytical software (SoftMax Pro available from Molecular Devices) using parallel line analysis and 4D logic analysis. Relative titer (%) was calculated by dividing the EC50 before storage by the EC50 after storage.

[0076]

<Results>

The relative EC50 titer of the samples of Example 1 and Comparative Examples 5 and 6 after storage at 60°C for 1 month is shown in Table 18.

[0077]

According to Table 18, a decrease in relative titer was observed for Comparative Examples 5 and 6 after storage at 60° C. for 1 month.

In addition, no decrease in relative titer was observed for Example 1, indicating that the biological activity was maintained even under storage conditions at 60° C. for 1 month.

[0078]

Table 18

Example 1 Comp. Example 5 Comp. Example 6 Relative titer (%) 101 80 45

INDUSTRIAL APPLICABILITY

[0079]

The industrial applicability of the lyophilized composition of the present invention is that high quality hepatocyte growth factor formulations can be obtained in medical practice.

EXPANDED SEQUENCE LISTING

[0080]

SEQ ID NO: 1 is the amino acid sequence of human hepatocyte growth factor.

--->

SEQUENCE LIST

<110> Eisai ARandDi Management Co., Ltd.

<120> HGF LYOPHILIZED COMPOSITION

<130> E0405AGP0001

<150> 2014-092888

<151> 2014-04-28

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 728

<212> PROTEIN

<213> Human

<400> 1

Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu

1 5 10 15

Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln

20 25 30

Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr

35 40 45

Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val

50 55 60

Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu

65 70 75 80

Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys

85 90 95

Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe

100 105 110

Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys

115 120 125

Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys

130 135 140

Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His

145 150 155 160

Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr

165 170 175

Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser

180 185 190

Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu

195 200 205

Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp

210 215 220

His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro

225 230 235 240

His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp

245 250 255

Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr

260 265 270

Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys

275 280 285

Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu

290 295 300

Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile

305 310 315 320

Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu

325 330 335

His Asp Met Thr Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn

340 345 350

Tyr Cys Arg Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr

355 360 365

Asp Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp

370 375 380

Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met

385 390 395 400

Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp

405 410 415

Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala

420 425 430

Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His

435 440 445

Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys

450 455 460

Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu

465 470 475 480

Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val

485 490 495

Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg

500 505 510

Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp

515 520 525

Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr

530 535 540

Glu Ala Trp Leu Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys

545 550 555 560

Cys Lys Gln Val Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly

565 570 575

Ser Asp Leu Val Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp

580 585 590

Phe Val Ser Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu

595 600 605

Lys Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn

610 615 620

Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu

625 630 635 640

Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu

645 650 655

Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp

660 665 670

Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu

675 680 685

Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly

690 695 700

Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile

705 710 715 720

Leu Thr Tyr Lys Val Pro Gln Ser

725

<---

Claims (22)

1. Stable freeze-dried composition containing: (1) hepatocyte growth factor; (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in which the mass ratio of hepatocyte growth factor and trehalose is from 1:4 to 1:460, and the mass ratio of hepatocyte growth factor and each of these one or more compounds is from 1:1 to 1:50 in the lyophilized composition. 2. Stable freeze-dried formulation according to claim 1, wherein the weight ratio of hepatocyte growth factor and trehalose is from 1:4 to 1:370, and the weight ratio of hepatocyte growth factor and each of said one or more compounds is from 1:1 to 1 :40 in lyophilized formulation. 3. Stable freeze-dried formulation according to claim 1, wherein the weight ratio of hepatocyte growth factor and trehalose is from 1:8 to 1:280, and the weight ratio of hepatocyte growth factor and each of said one or more compounds is from 1:2 to 1 :35 in lyophilized formulation. 4. A method for obtaining a lyophilized composition, including: (1) adjusting the pH of an aqueous solution containing one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts , up to pH 4.5-6.5; and (2) adding hepatocyte growth factor to the aqueous solution after the step of adjusting the pH of the aqueous solution (1) and adding trehalose to the aqueous solution before or after adjusting the pH in step (1); and (3) lyophilization of the aqueous solution obtained by step (2), in which the mass ratio of hepatocyte growth factor and trehalose is from 1:4 to 1:460, and the mass ratio of hepatocyte growth factor and each of these one or more compounds is from 1:1 to 1:50 in the lyophilized composition. 5. The method according to claim 4, wherein the weight ratio of hepatocyte growth factor and trehalose is from 1:4 to 1:370, and the weight ratio of hepatocyte growth factor and each of said one or more compounds is from 1:1 to 1:40 in lyophilized composition. 6. The method according to claim 4, wherein the mass ratio of hepatocyte growth factor and trehalose is from 1:8 to 1:280, and the mass ratio of hepatocyte growth factor and each of said one or more compounds is from 1:2 to 1:35 in lyophilized composition. 7. The method according to any one of paragraphs. 4-6, wherein trehalose is added to the aqueous solution after pH adjustment in step (1), simultaneously or sequentially with the addition of hepatocyte growth factor in step (2). 8. A method for stabilizing hepatocyte growth factor, including lyophilization of an aqueous solution to obtain a lyophilized composition containing: (1) hepatocyte growth factor, (2) trehalose and (3) one or more compounds selected from the group consisting of arginine, histidine, lysine, meglumine, glutamic acid, aspartic acid, proline, creatine, creatinine, tris(hydroxymethyl)methylamine and their pharmaceutically acceptable salts, in which the mass ratio of hepatocyte growth factor and trehalose is from 1:4 to 1:460, and the mass ratio of hepatocyte growth factor and each of these one or more compounds is from 1:1 to 1:50 in the lyophilized composition. 9. The method according to claim 8, wherein the mass ratio of hepatocyte growth factor and trehalose is from 1:4 to 1:370, and the mass ratio of hepatocyte growth factor and each of said one or more compounds is from 1:1 to 1:40 in lyophilized composition. 10. The method according to claim 8, wherein the mass ratio of hepatocyte growth factor and trehalose is from 1:8 to 1:280, and the mass ratio of hepatocyte growth factor and each of said one or more compounds is from 1:2 to 1:35 in lyophilized composition.

Images