Stabilized preparations containing recombinant stabilized galectin-9 protein

Abstract

The present invention relates to a stabilized formulation containing a recombinant stabilized galectin-9 protein. More specifically, the present invention provides a pharmaceutical formulation having improved stability due to the combination of a buffer and a stabilizer.

Description

[Technical Field]

[0001] The present invention relates to a stabilized preparation containing a recombinant stabilized galectin-9 protein, more specifically to a pharmaceutical preparation that can stably preserve the galectin-9 protein by combining a buffer and a stabilizer. [Background technology]

[0002] Galectin-9 is a β-galactoside lectin protein isolated from mouse embryonic kidney. Galectin-9 consists of two carbohydrate-recognition domains (CRDs) connected by a linker peptide region, with the N-terminal domain (NCRD) and C-terminal domain (CCRD) connected via a linker peptide. Galectin-9 is known as a ligand for HAVCR2 (Tim-3). It binds to Tim-3 to induce apoptosis of Tim-3-positive Th1 cells, suppressing excessive Th1 responses and thereby reducing autoimmune inflammation. Galectin-9 has also been observed in various cancers, including malignant melanoma, Hodgkin's lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, and colorectal cancer, and its mutants have been developed for therapeutic applications. In this regard, Patent Document 1 discloses a pharmaceutical composition containing a recombinant stabilized galectin-9 protein for the prevention or treatment of cancer. However, as disclosed, formulation development is required to enable technical application to the human body through administration to patients.

[0003] In general, the composition of a formulation, depending on the type and ratio of excipients used, significantly affects the efficacy and stability of the active ingredient when administered to the human body. In addition to pharmacokinetic factors such as duration of action, drug distribution, metabolism, and excretion after administration, formulation parameters must also be carefully considered, taking into account packaging conditions and container systems. For example, while poorly soluble drugs can be administered via capsules, administration may be challenging depending on the manufacturing method and composition ratio, requiring extensive formulation research. Furthermore, poor antibody stability can reduce antibody efficacy through denaturation, oxidation, aggregation, degradation, and other factors, which may in some cases lead to adverse effects such as toxicity. Therefore, various efforts are required to develop pharmaceutical formulations that are stable, effective, and convenient to use.

[0004] Therefore, the present inventors have conducted extensive research into formulations for stabilizing recombinant galectin-9 protein, and have identified formulations with improved stability, thereby completing the present invention. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korea Patent No. 10-2022-0068158 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a pharmaceutical preparation that allows stable storage of a recombinant stabilized galectin-9 protein. [Means for solving the problem]

[0007] To achieve the objectives of the present invention, the present invention provides a pharmaceutical formulation comprising: (1) a recombinant stabilized galectin-9 protein; (2) a buffering agent; and (3) a stabilizer.

[0008] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein is a protein having the amino acid sequence shown in SEQ ID NO:1.

[0009] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein is a protein having 90% or more homology with the amino acid sequence shown in SEQ ID NO:1.

[0010] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein comprises a deletion of the first amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO:1.

[0011] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein is at 1 to 20 mg / mL.

[0012] In one embodiment of the present invention, the buffer is at least one selected from the group consisting of citrate, phosphate, histidine, glycine, acetate, tartrate, aspartate, lactate, gluconate, glutamate, succinate, and combinations thereof.

[0013] In one embodiment of the present invention, the buffer has a pH of 4.5 to 7.

[0014] In one embodiment of the present invention, the concentration of the buffering agent is 5 to 20 mM.

[0015] In one embodiment of the invention, the stabilizer comprises: (i) one or more carbohydrates or sugars; and (ii) one or more amino acids or pharmaceutically acceptable salts thereof.

[0016] In one embodiment of the invention, the carbohydrate or sugar is selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, lactitol, sucrose, trehalose, mannose, maltose, lactose, xylose, ribose, glucose, raffinose, dextran, cyclodextrin, cellobiose, isomaltose, arabinose, glucosamine and fructose.

[0017] In one embodiment of the invention, said carbohydrate or sugar is selected from the group consisting of trehalose and mannitol.

[0018] In one embodiment of the invention, the amino acid is selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, and arginine.

[0019] In one embodiment of the present invention, the carbohydrate or sugar comprises one or more carbohydrates or sugars, each of which is 1 to 5 w / v %.

[0020] In one embodiment of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is present at a concentration of 1 to 20 mg / mL.

[0021] In one embodiment of the invention, the stabilizer further comprises a polysorbate.

[0022] In one embodiment of the present invention, the stabilizer further comprises 0.001 to 0.05 w / v % polysorbate 80.

[0023] In one embodiment of the invention, the pharmaceutical formulation is in liquid or lyophilized form.

[0024] The present invention also provides a pharmaceutical preparation for preventing or treating a bone disease, comprising a therapeutically effective amount of the pharmaceutical preparation.

[0025] Furthermore, the present invention provides a pharmaceutical preparation for preventing cancer or treating an autoimmune disease, comprising a therapeutically effective amount of the pharmaceutical preparation.

[0026] The present invention also provides use of the pharmaceutical preparation in the manufacture of a medicament for preventing or treating a bone disease.

[0027] Furthermore, the present invention provides the use of the pharmaceutical preparation in the manufacture of a medicament for the prevention or treatment of cancer or autoimmune disease.

[0028] The present invention also provides use of the pharmaceutical preparation for preventing or treating bone diseases.

[0029] Furthermore, the present invention provides use of the pharmaceutical preparation for the prevention or treatment of cancer or autoimmune diseases.

[0030] The present invention also provides a method for treating a bone disease, which comprises the step of administering a therapeutically effective amount of a pharmaceutical preparation to a subject.

[0031] Furthermore, the present invention provides a method for treating cancer or an autoimmune disease, comprising the step of administering a therapeutically effective amount of the pharmaceutical preparation to a subject. [Effects of the Invention]

[0032] The present invention relates to a pharmaceutical preparation containing a recombinant galectin-9 protein, and has the advantage of being able to provide a preparation with improved stability by using a buffer or the like. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 shows the results of a comparative experiment using histidine buffer and PBS buffer. [Figure 2] FIG. 1 shows the results of a comparative experiment using acetate buffer and PBS buffer. [Figure 3] FIG. 1 shows the results of a comparative experiment using histidine acetate buffer and sodium acetate buffer. [Figure 4A]FIG. 1 shows the results of a potency test as a function of the pH of histidine acetate buffer. [Figure 4B] FIG. 1 shows the results of a potency test as a function of the pH of histidine acetate buffer. [Figure 4C] FIG. 1 shows the results of a potency test as a function of the pH of histidine acetate buffer. [Figure 5] FIG. 10 is a diagram showing the properties of an example (Experimental Example 3-3) of the present invention. [Figure 6] FIG. 10 is a diagram showing the properties of an example (Experimental Example 3-6) of the present invention. [Figure 7] FIG. 1 shows the results of SDS-PAGE analysis according to one example of the present invention (Experimental Example 3-6). [Figure 8A] FIG. 1 shows the results of SEC-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 8B] FIG. 1 shows the results of SEC-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 8C] FIG. 1 shows the results of SEC-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 8D] FIG. 1 shows the results of SEC-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 9A] FIG. 1 shows the results of IEX-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 9B] FIG. 1 shows the results of IEX-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 9C] FIG. 1 shows the results of IEX-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 9D] FIG. 1 shows the results of IEX-HPLC analysis according to one example (Experimental Example 3-6) of the present invention. [Figure 10A] FIG. 1 is a diagram showing the results of a potency test according to one example (Experimental Example 3-6) of the present invention. [Figure 10B] FIG. 1 is a diagram showing the results of a potency test according to one example (Experimental Example 3-6) of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0034] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Therefore, the embodiments of the present invention may be modified in various ways, and the scope of the present invention is not limited to the embodiments described below.

[0035] Throughout the specification of the present invention, when a part is described as "comprising" certain components, this does not mean that other components are excluded, but that other components may additionally be included unless expressly stated otherwise.

[0036] The present invention relates to a pharmaceutical formulation comprising: (1) a recombinant stabilized galectin-9 protein; (2) a buffering agent; and (3) a stabilizer.

[0037] In the present invention, the recombinant stabilized galectin-9 protein has a molecular structure that is more stable against proteases while retaining the sugar chain recognition activity of wild-type galectin-9.

[0038] Specifically, the recombinant stabilized galectin-9 protein is a recombinant protein obtained by modifying the linker region connecting the two glycan-recognition domains (CRDs) of wild-type galectin-9 having an NCRD-linker-CCRD structure and the C-terminal glycan-recognition domain (CCRD). More specifically, the recombinant stabilized galectin-9 protein may comprise the amino acid sequence shown in SEQ ID NO: 1, in which the peptide in the linker region is completely deleted, the amino acid sequence at positions 1 to 10 of CCRD (SEQ ID NO: 2) (SEQ ID NO: 3) is deleted, and alanine (Ala) at position 13 is replaced with proline (Pro; P). The recombinant stabilized galectin-9 protein may comprise an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. Additionally, the recombinant stabilized galectin-9 protein may further comprise amino acid sequences engineered for specific purposes, such as targeting sequences, tags, labeling residues, or sequences designed to improve half-life or peptide stability.

[0039] The recombinant protein of the present invention can be obtained by various methods known to those skilled in the art, including, for example, production using polynucleotide recombination and protein expression systems, in vitro synthesis by chemical synthesis such as peptide synthesis, or cell-free protein synthesis.

[0040] As used herein, the term "polynucleotide" refers to a polymer of nucleotides that functions to transmit genetic information. For purposes of the present invention, the polynucleotide may include a sequence that encodes the recombinant protein of SEQ ID NO: 1 and has 75% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more sequence identity with the polynucleotide sequence encoding the recombinant protein.

[0041] As used herein, the term "homology" is intended to indicate the degree of similarity to a wild-type amino acid sequence or polynucleotide sequence. Such homology comparisons can be performed by those skilled in the art using well-known comparison programs, and the homology between two or more sequences can be calculated as a percentage (%).

[0042] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering a composition.

[0043] As used herein, the term "treatment" refers to any action of administering a composition to alleviate or improve the symptoms of the disease to a favorable state.

[0044] As used herein, the term "pharmaceutical formulation" refers to a final product produced by filling, lyophilizing, and / or reconstituting a stock solution containing a composition.

[0045] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein is a protein having the amino acid sequence shown in SEQ ID NO:1.

[0046] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein is a protein having an amino acid sequence that is 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the amino acid sequence set forth in SEQ ID NO:1.

[0047] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein comprises a deletion of the first amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO:1.

[0048] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein may be contained in the pharmaceutical formulation in an amount ranging from 0.01 to 400 mg / mL. Specifically, the recombinant stabilized galectin-9 protein may be contained in an amount of 0.01 to 250 mg / mL, more specifically, 0.01 to 150 mg / mL, 0.01 to 120 mg / mL, 0.01 to 100 mg / mL, 0.1 to 80 mg / mL, 0.1 to 60 mg / mL, or 0.1 to 40 mg / mL. In a specific embodiment of the present invention, the recombinant stabilized galectin-9 protein is contained in an amount of 0.1 to 20 mg / mL, 0.1 to 18 mg / mL, 0.1 to 16 mg / mL, 0.1 to 15 mg / mL, 1 to 15 mg / mL, 2 to 15 mg / mL, 3 to 15 mg / mL, 4 to 15 mg / mL, or 5 to 15 mg / mL. In a more specific embodiment of the present invention, the recombinant stabilized galectin-9 protein is contained in an amount of 5.5 to 15 mg / mL, 6 to 15 mg / mL, 6.5 to 15 mg / mL, 7 to 15 mg / mL, 7.5 to 15 mg / mL, 7.5 to 14.5 mg / mL, 7.5 to 14 mg / mL, 7.5 to 13.5 mg / mL, 7.5 to 13 mg / mL, or 7.5 to 12.5 mg / mL.

[0049] In one embodiment of the present invention, the buffer is one or more selected from the group consisting of citrate, phosphate, histidine, glycine, acetate, tartrate, aspartate, lactate, gluconate, glutamate, succinate, and combinations thereof. In a specific embodiment of the present invention, the buffer is one or more selected from the group consisting of phosphate, histidine, acetate, and combinations thereof. In a more specific embodiment of the present invention, the buffer is histidine acetate. Inclusion of histidine in the present invention may have beneficial effects in terms of stability and pH control.

[0050] In one embodiment of the invention, the buffer does not include sodium salts.

[0051] In one embodiment of the present invention, the buffer has a pH in the range of 4 to 7. Specifically, the pH of the buffer is 4.5 to 7, more specifically, 4.6 to 7, 4.7 to 7, 4.8 to 7, 4.9 to 7, or 5 to 7. In a specific embodiment of the present invention, the pH of the buffer is 5.1 to 7, 5.2 to 7, 5.3 to 7, 5.4 to 7, 5.5 to 7, 5.5 to 6.9, 5.5 to 6.8, 5.5 to 6.7, 5.5 to 6.6, or 5.5 to 6.5. In an even more specific embodiment of the present invention, the pH of the buffer is 5.6 to 6.5, 5.65 to 6.5, 5.6 to 6.5, 5.65 to 6.5, 5.7 to 6.5, 5.75 to 6.5, 5.75 to 6.4, or 5.75 to 6.35.

[0052] In one embodiment of the present invention, the buffering agent is contained in the pharmaceutical formulation at a concentration of 0.01 to 50 mM. Specifically, the concentration of the buffering agent is 0.01 to 45 mM, more specifically, 0.01 to 40 mM, 0.01 to 35 mM, 0.01 to 30 mM, or 0.01 to 25 mM. In a specific embodiment of the present invention, the concentration of the buffering agent is 0.1 to 25 mM, 0.1 to 24 mM, 0.1 to 23 mM, 0.1 to 23 mM, 0.1 to 22 mM, 0.1 to 21 mM, 1 to 21 mM, 2 to 21 mM, 3 to 21 mM, or 4 to 21 mM. In a more specific embodiment of the present invention, the concentration of the buffering agent is 4 to 20.5 mM, 4 to 20 mM, 4.5 to 20 mM, or 5 to 20 mM.

[0053] In one embodiment of the invention, the stabilizer comprises: (i) one or more carbohydrates or sugars; and (ii) one or more amino acids or pharmaceutically acceptable salts thereof.

[0054] In the present invention, the term carbohydrate or sugar is intended to encompass monosaccharides, sugar alcohols, sugar acids, and sugar derivatives, including, but not limited to, glucose, fructose, sucrose, lactose, maltose, trehalose, glycerol, erythritol, arabitol, xylitol, mannitol, sorbitol, galactitol, fucitol, iditol, maltitol, lactitol, maltotriitol, maltotetriitol, and the like.

[0055] In the present invention, amino acids include natural amino acids and unnatural amino acids, including, but not limited to, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, and the like.

[0056] In one embodiment of the invention, the carbohydrate or sugar is selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, lactitol, sucrose, trehalose, mannose, maltose, lactose, xylose, ribose, glucose, raffinose, dextran, cyclodextrin, cellobiose, isomaltose, arabinose, glucosamine and fructose, hi a specific embodiment of the invention, the carbohydrate or sugar is selected from the group consisting of mannitol and trehalose.

[0057] In one embodiment of the present invention, the stabilizer contains one or more carbohydrates or sugars, each at 0.01 to 10 w / v %. Specifically, the one or more carbohydrates or sugars contained in the stabilizer are 0.01 to 9.5 w / v %, 0.01 to 9 w / v %, 0.01 to 8.5 w / v %, 0.01 to 8 w / v %, 0.01 to 7.5 w / v %, 0.5 to 7.5 w / v %, or 1 to 7.5 w / v %. In a specific embodiment of the present invention, the one or more carbohydrates or sugars contained in the stabilizer are 1 to 6 w / v%, 1 to 5.9 w / v%, 1 to 5.8 w / v%, 1 to 5.7 w / v%, 1 to 5.6 w / v%, 1 to 5.5 w / v%, 1 to 5.4 w / v%, 1 to 5.3 w / v%, 1 to 5.2 w / v%, 1 to 5.1 w / v%, or 1 to 5 w / v%.

[0058] In a specific embodiment of the present invention, the stabilizer may contain two or more carbohydrates or sugars. Specifically, when two or more carbohydrates or sugars are contained, one of the carbohydrates or sugars is present at 1 to 3 w / v%, 1.1 to 3 w / v%, 1.2 to 3 w / v%, 1.3 to 3 w / v%, 1.4 to 3 w / v%, 1.5 to 3 w / v%, 1.5 to 2.9 w / v%, 1.5 to 2.8 w / v%, 1.5 to 2.7 w / v%, 1.5 to 2.6 w / v%, or 1.5 to 2.5 w / v%. In addition, the concentration of another carbohydrate or sugar is 3 to 5 w / v%, 3.1 to 5 w / v%, 3.2 to 5 w / v%, 3.3 to 5 w / v%, 3.4 to 5 w / v%, 3.5 to 5 w / v%, 3.5 to 4.9 w / v%, 3.5 to 4.8 w / v%, 3.5 to 4.7 w / v%, 3.5 to 4.6 w / v%, or 3.5 to 4.5 w / v%. In a more specific embodiment of the present invention, the stabilizer comprises two or more carbohydrates or sugars, one of which is present at 1.5 to 2.5 w / v%, 1.55 to 2.5 w / v%, 1.6 to 2.5 w / v%, 1.65 to 2.5 w / v%, 1.7 to 2.5 w / v%, 1.75 to 2.5 w / v%, 1.75 to 2.45 w / v%, 1.75 to 2.4 w / v%, 1.75 to 2.35 w / v%, 1.75 to 2.3 w / v%, or 1.75 to 2.25 w / v%. Another carbohydrate or sugar is 3.5 to 4.5 w / v%, 3.55 to 4.5 w / v%, 3.6 to 4.5 w / v%, 3.65 to 4.5 w / v%, 3.7 to 4.5 w / v%, 3.75 to 4.5 w / v%, 3.75 to 4.45 w / v%, 3.75 to 4.4 w / v%, 3.75 to 4.35 w / v%, 3.75 to 4.3 w / v%, or 3.75 to 4.25 w / v%.

[0059] In one embodiment of the present invention, the carbohydrate or sugar comprises trehalose and mannitol. Also, in one embodiment of the present invention, the carbohydrate or sugar comprises trehalose and mannitol, each of which is present at 1 to 10 w / v %.

[0060] In a specific embodiment of the present invention, the mannitol is 1 to 9 w / v%, 1 to 8 w / v%, 1 to 7 w / v%, 1 to 6 w / v%, 1 to 5 w / v%, 1.5 to 5 w / v%, 2 to 5 w / v%, 2.5 to 5 w / v%, 3 to 5 w / v%, 3.1 to 5 w / v%, 3.2 to 5 w / v%, 3.3 to 5 w / v%, 3.4 to 5 w / v%, 3.5 to 5 w / v%, 3.5 to 4.9 w / v%, 3.5 to 4.8 w / v%, 3.5 to 4.7 w / v%, 3.5 to 4.6 w / v%, or 3.5 to 4.5 w / v%. More specifically, the mannitol is 3.5 to 4.5 w / v%, 3.55 to 4.5 w / v%, 3.6 to 4.5 w / v%, 3.65 to 4.5 w / v%, 3.7 to 4.5 w / v%, 3.75 to 4.5 w / v%, 3.75 to 4.45 w / v%, 3.75 to 4.4 w / v%, 3.75 to 4.35 w / v%, 3.75 to 4.3 w / v%, or 3.75 to 4.25 w / v%.

[0061] In a specific embodiment of the present invention, the trehalose is 1 to 3 w / v%, 1.1 to 3 w / v%, 1.2 to 3 w / v%, 1.3 to 3 w / v%, 1.4 to 3 w / v%, 1.5 to 3 w / v%, 1.5 to 2.9 w / v%, 1.5 to 2.8 w / v%, 1.5 to 2.7 w / v%, 1.5 to 2.6 w / v%, or 1.5 to 2.5 w / v%. More specifically, the trehalose is 1.5 to 2.5 w / v%, 1.55 to 2.5 w / v%, 1.6 to 2.5 w / v%, 1.65 to 2.5 w / v%, 1.7 to 2.5 w / v%, 1.75 to 2.5 w / v%, 1.75 to 2.45 w / v%, 1.75 to 2.4 w / v%, 1.75 to 2.35 w / v%, 1.75 to 2.3 w / v%, or 1.75 to 2.25 w / v%.

[0062] In one embodiment of the invention, the amino acid is selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, and arginine. In a specific embodiment of the invention, the amino acid is glycine.

[0063] In one embodiment of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is present at a concentration of 0.01 to 20 mg / mL. Specifically, the amino acid or a pharmaceutically acceptable salt thereof is 0.01 to 19 mg / mL, 0.01 to 18 mg / mL, 0.01 to 17 mg / mL, 0.01 to 16 mg / mL, 0.01 to 15 mg / mL, 0.01 to 14 mg / mL, 0.01 to 13 mg / mL, 0.01 to 12 mg / mL, 0.01 to 11 mg / mL, 0.01 to 10 mg / mL, 0.1 to 10 mg / mL, 0.2 to 10 mg / mL, 0.3 to 10 mg / mL, 0.4 to 10 mg / mL, 0.5 to 10 mg / mL, 0.6 to 10 mg / mL, 0.7 to 10 mg / mL, 0.8 to 10 mg / mL, 0.9 to 10 mg / mL, or 1 to 10 mg / mL. In a specific embodiment of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is 1 to 10 mg / mL, 1.5 to 10 mg / mL, 2 to 10 mg / mL, 2.5 to 10 mg / mL, 2.5 to 9.5 mg / mL, 2.5 to 9 mg / mL, 2.5 to 8.5 mg / mL, 2.5 to 8 mg / mL, or 2.5 to 7.5 mg / mL. In a more specific embodiment of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is 2.6 to 7.5 mg / mL, 2.7 to 7.5 mg / mL, 2.8 to 7.5 mg / mL, 2.9 to 7.5 mg / mL, 3 to 7.5 mg / mL, 3 to 7 mg / mL, 3 to 6.5 mg / mL, 3 to 6.4 mg / mL, 3 to 6.3 mg / mL, 3 to 6.2 mg / mL, 3 to 6.1 mg / mL, or 3 to 6 mg / mL.

[0064] In one embodiment of the present invention, the stabilizer further comprises a non-ionic surfactant, such as, but not limited to, Pluronic®, polysorbate, poloxamer, etc.

[0065] In one embodiment of the invention, the stabilizing agent further comprises a polysorbate, wherein the polysorbate is polysorbate 20, polysorbate 80, or a combination thereof.

[0066] In one embodiment of the present invention, the stabilizer further comprises 0.001 to 0.05 w / v % of polysorbate 20, polysorbate 80, or a combination thereof.

[0067] In a specific embodiment of the present invention, the concentration of polysorbate 20 or polysorbate 80 is 0.001 to 0.02 w / v%, 0.002 to 0.02 w / v%, 0.003 to 0.02 w / v%, 0.004 to 0.02 w / v%, 0.005 to 0.02 w / v%, 0.005 to 0.019 w / v%, 0.005 to 0.018 w / v%, 0.005 to 0.017 w / v%, 0.005 to 0.016 w / v%, or 0.005 to 0.015 w / v%. More specifically, the concentration of the polysorbate 20 or 80 is 0.006 to 0.015 w / v%, 0.0065 to 0.015 w / v%, 0.007 to 0.015 w / v%, 0.0075 to 0.015 w / v%, 0.0075 to 0.014 w / v%, 0.006 to 0.013 w / v%, or 0.006 to 0.0125 w / v%.

[0068] In one embodiment of the present invention, the pharmaceutical preparation has a reference potency of 60 to 130%. Specifically, the pharmaceutical preparation has a reference potency of 70 to 120%.

[0069] In one embodiment of the present invention, the purity of the pharmaceutical formulation (measured by SEL-HPLC) is 90% or more, specifically 91% or more, 92% or more, 93% or more, or 94% or more, and more specifically 95% or more.

[0070] In one embodiment of the invention, the pharmaceutical formulation is in liquid or lyophilized form.

[0071] In a specific embodiment of the present invention, the pharmaceutical formulation is in a lyophilized dosage form. The pharmaceutical formulation of the present invention exhibits increased stability in a lyophilized dosage form compared to a liquid form.

[0072] The pharmaceutical formulations of the present invention may take a variety of forms depending on the specific product configuration, and may be provided in a filled form, such as, but not limited to, a tube, a cartridge, a syringe, a polymeric vial, a glass vial, etc. Additionally, the present invention may be filled or otherwise processed by methods known to those skilled in the art.

[0073] In one embodiment of the present invention, the invention relates to a vial filled with the pharmaceutical formulation.

[0074] In one embodiment of the present invention, the invention relates to a syringe filled with the pharmaceutical formulation.

[0075] In one embodiment of the present invention, the present invention relates to a method for producing vials filled with a pharmaceutical formulation by preparing a stock solution containing a protein and excipients in an amount of 1 / 2 part by weight per part by weight of the final pharmaceutical formulation, and filling the prepared stock solution into vials at a volume twice the volume of the final pharmaceutical formulation. Specifically, a final formulation containing 1 to 20 mg / mL of recombinant stabilized galectin-9 protein, 1 to 10 w / v% mannitol, 0.5 to 5 w / v% trehalose, 0.5 to 10 mg / mL amino acids, and 0.0005 to 0.025 w / v% polysorbate can be produced by filling 2 mL of the stock solution into vials per vial and lyophilizing the solution. More specifically, 1 mL of a stock solution containing 5-6 mg / mL of recombinant stabilized galectin-9 protein, 2 w / v% mannitol, 1 w / v% trehalose, 2.5 mg / mL of amino acids, and 0.005 w / v% polysorbate is filled into vials at 2 mL per vial and lyophilized to produce a final formulation containing 10-12 mg / mL of recombinant stabilized galectin-9 protein, 4 w / v% mannitol, 2 w / v% trehalose, 5 mg / mL of amino acids, and 0.01 w / v% polysorbate; however, the present invention is not limited to this.

[0076] The present invention also relates to a pharmaceutical preparation for preventing or treating bone diseases, which contains a therapeutically effective amount of the pharmaceutical preparation.

[0077] In the present invention, the bone disease may be one or more selected from the group consisting of osteoporosis, bone defect, osteomalacia, osteopenia, osteogenesis imperfecta, osteodystrophy, primary bone tumor, degenerative bone disease, osteoarthritis, rheumatoid arthritis, bone fracture, osteonecrosis, periodontal disease, inflammatory alveolar bone resorption disease, inflammatory bone resorption, and Paget's disease, but is not limited to these.

[0078] The present invention also relates to a pharmaceutical preparation for preventing cancer or treating an autoimmune disease, comprising a therapeutically effective amount of the pharmaceutical preparation.

[0079] In the present invention, the cancer may be one or more selected from the group consisting of colon cancer, lung cancer, thyroid cancer, gastric cancer, breast cancer, brain tumor, osteosarcoma, glioblastoma, melanoma, glioma, glioblastoma, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, gallbladder cancer, biliary tract cancer, esophageal cancer, colon cancer, colorectal cancer, pancreatic cancer, blood cancer, leukemia, myeloid leukemia, lymphoma, head and neck cancer, skin cancer, and liver cancer, but is not limited to these.

[0080] In the present invention, the autoimmune disease includes systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, discoid lupus erythematosus, photosensitive dermatosis, autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, and premature diabetes. The cause may be, but is not limited to, one or more selected from the group consisting of: urinary tract disease (type 1 diabetes), Goodpasture's syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary sclerosis, active chronic hepatitis Hbs-ve, idiopathic liver cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatomyositis, and discoid LE.

[0081] Examples of autoimmune diseases include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplant rejection, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune diabetic retinopathy, Autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathy, Baro's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, decompensated myelopathy, dyslipidemia ... Myelogenous neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans' syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gravidarum, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), Mooren's ulcer, Mukka-Habermann disease,Multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devik's disease), neutropenia, ocular pemphigoid, optic neuritis, relapsing rheumatoid arthritis, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Paley-Romberg syndrome, Parsonage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polyglandular autoimmune syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progestational dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis , pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff-person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular dermatosis, vitiligo, Waldenström's macroglobulinemia (WM), and Wegener's granulomatosis (granulomatosis with polyangiitis (GPA)).

[0082] The present invention also relates to use of the pharmaceutical preparation in the manufacture of a pharmaceutical preparation for preventing or treating bone diseases.

[0083] The present invention also relates to the use of the pharmaceutical preparation in the manufacture of a medicament for the prevention or treatment of cancer or autoimmune diseases.

[0084] The present invention also relates to the use of the pharmaceutical preparation for preventing or treating bone diseases.

[0085] The present invention also relates to the use of the pharmaceutical preparation for the prevention or treatment of cancer or autoimmune diseases.

[0086] The present invention also relates to a method for treating bone diseases, which comprises the step of administering a therapeutically effective amount of a pharmaceutical preparation to a subject.

[0087] The present invention also relates to a method for treating cancer or an autoimmune disease, which comprises the step of administering a therapeutically effective amount of a pharmaceutical preparation to a subject.

[0088] The present invention will be described in detail below with reference to examples and experimental examples.

[0089] However, it should be understood that the following examples and experimental examples are merely illustrative and are not intended to limit the scope of the present invention.

[0090] <Production Example 1> Production of recombinant stabilized galectin-9 protein (sGal-9) An expression vector containing a gene encoding a recombinant stabilized galectin-9 protein having the amino acid sequence of SEQ ID NO: 1 was constructed and introduced into Escherichia coli (E. coli) by the heat shock method. Expression of the recombinant protein was induced by culturing E. coli in LB medium containing 50 μg / mL kanamycin and adding arabinose when the absorbance at 600 nm reached 0.7. The cells in which recombinant protein expression was induced were then lysed and filtered. The target protein was then captured using cation exchange chromatography and affinity chromatography, yielding highly pure and highly yielded recombinant stabilized galectin-9 protein.

[0091] <Production Example 2> Production of stabilized formulation Preparations were produced using the recombinant stabilized galectin-9 protein (hereinafter referred to as recombinant galectin-9) obtained in Preparation Example 1. The composition of each preparation is as described in the following experimental example. For example, the purified solution was subjected to ultrafiltration (UF) / dilution filtration (DF) using a 5 mM histidine acetate (HisAce) buffer solution at pH 6.0 to adjust the protein concentration to 11-15 mg / mL. Subsequently, mannitol (2 w / v%), trehalose (1 w / v%), and glycine (2.5 mg / mL) were added, and the solution was diluted to a final protein concentration of 5-7 mg / mL. The resulting solution was filtered through a 0.2 μm filter, filled into 2.0 mL vials, and lyophilized.

[0092] <Experimental Example 1> Evaluation method of buffer solution <Experimental Example 1-1> Appearance The appearance of all samples was evaluated according to standard operating procedure (SOP) method PD-DPD-LAB-001-07, which included evaluation of clarity, color, and visible particles, under a black and white background using a YB-2 light box.

[0093] <Experimental Example 1-2> pH The pH was measured using a pH meter equipped with a glass electrode. Measurements were taken at least twice and the average value was recorded.

[0094] <Experimental Example 1-3> Protein concentration Protein concentrations were measured using a ThermoUV spectrophotometer. Measurements were performed according to SOP method (PD-DPD-EQU-088-01). For all assays, the extinction coefficient was 1.0 AU × mL × mg. -1 ×cm -1 Each sample (2.5 μL) was measured at least twice, and the average value was recorded.

[0095] <Experimental Example 1-4> Size Exclusion Chromatography (SEC-HPLC) SEC-HPLC was performed according to the following procedure.

[0096] If the sample concentration exceeded 1.0 mg / mL, the sample was diluted to 1.0 mg / mL with the mobile phase before SEC analysis. 50 μg of sample was injected into the HPLC and analyzed. SEC-HPLC analysis was performed using a Hitachi Chromaster CM5000 system equipped with an Agilent TSKgel2000SWxl (7.8 mm × 300 mm, 5 μm) and a UV detector (detection wavelength: 280 nm). The column temperature was maintained at 25°C, and the loading volume was 50 μL. The mobile phase consisted of 300 mM sodium chloride, 10% isopropyl alcohol, and 50 mM phosphate buffer (pH 6.8 ± 0.1), with a flow rate of 0.4 mL / min.

[0097] <Experimental Example 1-5> Protein electrophoresis (SDS-PAGE) SDS-PAGE analysis was performed using a heat block (DAIHAN MaXtable® 10H), an electrophoresis apparatus (Bio-Rad BR 165-8033FC), and a rocker (Daihan RK-1D). The test solution was diluted 3:1 with 4x sample buffer and heated at 98°C for 5 minutes. A 12% acrylamide gel was prepared, and protein standards and samples were loaded into separate wells. Electrophoresis was performed at 100V for 90 minutes. The gel containing the separated proteins was stained in CBBR-250 solution on a rocker for 30 minutes and then destained in destaining solution on a rocker for 30 minutes. After background destaining was complete, a distinct protein band around 31 kD was identified and analyzed.

[0098] <Experimental Example 1-6> Potency test Preparation of cell lines A 1 mL vial of Molt-4T cells was thawed at 37°C for 1–2 min. The thawed cells were transferred to 10 mL of medium (RPMI 1640 supplemented with 10% FBS and 1% penicillin / streptomycin) and centrifuged at 1,300 rpm for 3 min at room temperature (24°C). After removing the supernatant, the cell pellet was resuspended in fresh culture medium to a final concentration of 2–4 × 10. 5 The cell suspension was added to a 25cm 2The cells were transferred to a culture flask and cultured at 37°C in a 5% CO2 incubator. 6 When the cell density reached 2–4 × 10 cells / mL, the medium was replaced and 5 The doubling time and cell density were then checked and adjusted as needed.

[0099] Potency Test The titer test was performed using Molt4-T cells with a doubling time of 24 hours and a viability of 95% over 2 weeks. 4 The cells were diluted with fresh medium to 100 cells / mL. Next, 90 μL of the cell suspension was dispensed into each well of a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 hours. The test solution was serially diluted with PBS(-) to achieve concentrations of 300, 100, 30, 10, 3, 1, and 0 μg / mL. Ten μL of each dilution was added to each well to achieve final concentrations in the medium of 30, 10, 3, 0.3, 0.1, and 0 μg / mL, respectively. After sample addition, the cells were further incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, 10 μL of WST-1 detection solution was added to each well and incubated at 37°C in a 5% CO2 incubator for 2 hours. The optical density (OD) at 450 nm was then measured using a microplate reader. The change in OD and LD were correlated. 50 was calculated using a four-parameter logistic fit model.

[0100] <Experimental Example 1-7> Osmolality Osmotic concentration was measured using an osmometer. The accuracy of the osmometer was verified before and after the test using a Clinitrol 290 mOsm / kg standard solution. Each sample was analyzed using 20 μL of sample.

[0101] <Experimental Example 2> Liquid preparation <Experimental Example 2-1> Comparative analysis of buffer systems (1) sGal-9 protein stability was evaluated under stress conditions (37°C) using a pH / buffer system. In each experiment, the sGal-9 concentration was set at 2 mg / mL. The appearance of each sample was evaluated over a 4-week period according to the appearance analysis described in Experimental Example 1. The buffer conditions used in the experiment are shown in Table 1 below.

[0102] [Table 1] The results of the experiment are shown in Figures 1 and 2 and Table 2.

[0103] A comparison of histidine buffer and PBS buffer is shown in Figure 1. As shown in Figure 1, precipitation was observed in PBS buffer, confirming that the protein in histidine buffer was more stable than in PBS buffer.

[0104] Table 2 and Figure 2 show a comparison between acetate buffer and PBS buffer. As shown in Table 2 and Figure 2, there was a difference in particle count measured at 25°C 3 days after filtration with PBS, confirming that acetate buffer performed better than PBS buffer.

[0105] (#1: 20 mM PBS, 50 mM NaCl, pH 7.3, 0.26 mg / mL; #2: acetate buffer, pH 5.0, 0.62 mg / mL; #3: acetate buffer, pH 5.3, 0.62 mg / mL; #4: #2 after filtration; #5: #3 after filtration)

[0106] [Table 2] (*CL (= clear liquid) / P (= particles) / P(n) (= number of particles) / PF (= no particles))

[0107] <Experimental Example 2-2> Comparative analysis of buffer solutions (2) (sodium salt) The stability of sGal-9 protein was evaluated using the same method as in Experimental Example 2-1 above. The experiment was conducted over a two-week period, and the appearance, UV, pH, SEC-HPLC, SDS-PAGE, and titer were evaluated according to the procedures described in Experimental Example 1. The buffer conditions used are shown in Table 3 below.

[0108] [Table 3] Appearance, UV, pH, SEC-HPLC, SDS-PAGE, and titer were measured at TO. Appearance, UV, pH-SEC-HPLC, and SDS-PAGE analyses were performed at week 1. Appearance, UV, pH, SEC-HPLC, SDS-PAGE, and titer were measured again at week 2.

[0109] The results of the appearance analysis are shown in Table 4 below and in FIG.

[0110] [Table 4] (* C (colorless) / SY (slightly yellow) / Y (yellow) / CL (clear liquid) / SO (slightly milky white liquid) / O (milky white liquid) / FP (no visible particles) / PO (visible particles, <5) / PO+ (a few visible particles) / PO++ (many visible particles))

[0111] The results of the SEC-HPLC experiments are shown in Tables 5 and 6.

[0112] [Table 5]

[0113] [Table 6] The results of the potency tests are shown in Table 7.

[0114] [Table 7] As shown in Table 4 and Figure 3, histidine acetate showed milky white particles or numerous visible particles at both 25°C and 40°C, whereas no visible particles were observed in the sodium acetate formulation. However, in terms of purity and potency, proteins in histidine acetate buffer were found to be more stable than proteins in sodium acetate buffer.

[0115] Furthermore, as shown in Tables 5 to 7, it was confirmed that the sodium salt form had an adverse effect on the stability of the sGal-9 protein, indicating that the sodium salt form is unsuitable for use in formulations.

[0116] <Experimental Example 2-3> Comparative analysis of buffer solutions (3) (pH / excipients) The stability of sGal-9 protein was evaluated in the same manner as in Example 2-1 above. Evaluation was performed over a 4-week period using the same methods as in Example 1, including appearance, UV, pH, protein concentration (100 μL), SEC-HPLC, permeate concentration, and titer tests. The buffer conditions are shown in Table 8 below.

[0117] [Table 8] At TO, appearance, UV, pH, protein concentration (100 μL), SEC-HPLC, and permeate concentration were examined. After one week at 40°C, UV absorbance, pH, protein concentration (100 μL), SEC-HPLC, and titer were examined. After four weeks at 25°C, UV absorbance, pH, protein concentration (100 μL), SEC-HPLC, and titer were examined.

[0118] The results of the appearance, pH, concentration and permeate concentration experiments are shown in Tables 9 and 10 below.

[0119] [Table 9] (*C (= colorless) / CL (= transparent liquid) / PF (= particle-free))

[0120] [Table 10] The results of the SEC-HPLC analysis are shown in Table 11.

[0121] [Table 11] The results of the potency test are shown in Tables 12 to 14 and Figures 4A to 4C.

[0122] (B201-20210301-T0=F1 / B201-20210302-T0=F2)

[0123] Results of the titer test at TO [Table 12]

[0124] Titer test results after 1 week of incubation at 40°C [Table 13]

[0125] Titer test results after 4 weeks of incubation at 25°C [Table 14] The experimental results demonstrated that histidine buffer (pH 6.0) exhibited superior performance compared to histidine buffer (pH 5.0), and that sGal-9 protein was stable when 8% sucrose, 0.04% polysorbate 80, and 2 mg / mL glycine were used as excipients.

[0126] <Experimental Example 2-4> Comparative analysis of buffer solutions (4) (pH) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. Experiments were performed using a range of pH and excipients, and the buffer conditions were as shown in Table 15 below.

[0127] [Table 15] The results of the SEC-HPLC analysis, protein concentration and potency tests are shown in Table 16 below.

[0128] [Table 16] The results of the experiment confirmed that histidine acetate buffer at pH 6.0 exhibited superior performance compared to buffers at other pH values, and no significant effects of other excipients such as polysorbate 20 (PS20) or glycine were observed.

[0129] <Experimental Example 2-5> Comparative analysis of buffer solutions (5) (concentration) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The experiment was carried out based on the concentration change, and the buffer conditions are shown in Table 17 below.

[0130] [Table 17] The results of the SEC-HPLC analysis, protein concentration and potency tests are shown in Table 18 below.

[0131] [Table 18] The results of the experiment confirmed that histidine acetate buffer had higher protein stability at concentrations of 10 mM and 20 mM compared to 30 mM, and that stability improved at lower concentrations in the order of 10 mM > 20 mM > 30 mM.

[0132] <Experimental Example 2-6> Comparative analysis of buffer solutions (6) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The buffer conditions are shown in Table 19 below.

[0133] [Table 19] The SEC-HPLC analysis, protein concentration and potency test results are shown in Table 20 below.

[0134] [Table 20] The results of the experiment confirmed that histidine acetate buffer was most effective at 10 mM, and that the use of sucrose resulted in higher stability.

[0135] <Experimental Example 3> Freeze-dried preparation Based on the results of Experimental Example 2, excipients and formulations were compared while maintaining the buffer and pH at 10 mM histidine acetate (pH 6.0). Lyophilized formulations were prepared by mixing sGal-9 protein with each excipient, filling 10 mL vials to contain 10 mg of sGal-9 per vial, and then lyophilizing. Analysis was performed by dissolving the lyophilized samples in 1.0 mL of deionized water at each time point.

[0136] <Experimental Example 3-1> Comparative analysis of excipients (1) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The excipient conditions are shown in Table 21 below.

[0137] [Table 21] The results of the SEC-HPLC analysis, protein concentration and titer tests are shown in Table 22 below.

[0138] [Table 22] The results of the experiment confirmed that the purity of the sample decreased after 2 weeks (T2W) under harsh conditions (37°C). It was also confirmed that the addition of PS80 (0.01%) decreased the stability.

[0139] <Experimental Example 3-2> Comparative analysis of excipients (2) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The excipient conditions are shown in Table 23 below.

[0140] [Table 23] The results of the SEC-HPLC analysis, protein concentration and potency tests are shown in Tables 24 and 25 below.

[0141] [Table 24]

[0142] [Table 25] The results of the experiment confirmed that no changes were observed in the experimental parameters of SEC-HPLC, UV, and PAGE after 24 hours, and that the formulation was stable when mannitol was added.

[0143] <Experimental Example 3-3> Comparative analysis of excipients (3) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The excipient conditions are shown in Table 26 below.

[0144] [Table 26] The results of the SEC-HPLC analysis, protein concentration and potency tests are shown in Tables 27 and 28 below.

[0145] [Table 27]

[0146] [Table 28] The results of the above experiment confirmed that the formulation was stable when trehalose was included.

[0147] <Experimental Example 3-4> Comparative analysis of excipients (4) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The excipient conditions are shown in Table 29 below.

[0148] [Table 29] The results of the SEC-HPLC analysis, protein concentration and titer tests are shown in Tables 30 to 32 below.

[0149] [Table 30]

[0150] [Table 31]

[0151] [Table 32] The results of the above experiments confirmed that there were no significant differences in stability between the excipient candidates.

[0152] <Experimental Example 3-5> Comparative analysis of excipients (5) The stability of sGal-9 protein was evaluated in the same manner as in Experimental Example 2-1. The excipient conditions are shown in Table 33 below.

[0153] [Table 33] The results of the SEC-HPLC analysis, protein concentration and titer tests are shown in Tables 34 to 37 below.

[0154] [Table 34]

[0155] [Table 35]

[0156] [Table 36]

[0157] [Table 37] The results of the above experiment confirmed that the presence or absence of PS80 did not significantly affect stability.

[0158] <Experimental Example 3-6> Comparative analysis of excipients (6) Lyophilized preparations were produced in the same manner as in the above <Production Examples 2 and 3>. The excipient conditions are shown in Table 38 below.

[0159] [Table 38] Furthermore, the stability of sGal-9 protein was evaluated using the same method as in Example 2-1. The purpose of the analyses, including SEC-HPLC, IEX-HPLC, SDS-PAGE, protein, and potency tests, was to compare the stability of sGal-9 protein in the 2% mannitol formulation and the 4% mannitol formulation. The test results are shown in Tables 39 and 40 below.

[0160] [Table 39]

[0161] [Table 40]

[0162] Therefore, it was confirmed that the sGal-9 protein of the present invention exhibits superior stability in a lyophilized formulation containing 10 mM histidine acetate buffer (pH 6.0), 4 w / v% mannitol, 2 w / v% trehalose, 0.01 w / v% polysorbate 80, and 5 mg / mL glycine per 10 mg of sGal-9 protein, compared to formulations using other excipients or buffer systems.

[0163] (Industrial Applicability) The present invention relates to pharmaceutical formulations containing recombinant galectin-9 protein, and can provide formulations with improved stability through the use of buffers and other excipients.

Claims

1. (1) Recombinant stabilized galectin-9 protein; (2) a buffer; and (3) stabilizers; 10. A pharmaceutical formulation comprising:

2. 2. The pharmaceutical preparation according to claim 1, wherein the recombinant stabilized galectin-9 protein is a protein having the amino acid sequence shown in SEQ ID NO:

1.

3. 2. The pharmaceutical preparation according to claim 1, wherein the recombinant stabilized galectin-9 protein is a protein having 90% or more homology with the amino acid sequence shown in SEQ ID NO:

1.

4. 2. The pharmaceutical formulation of claim 1, wherein the recombinant stabilized galectin-9 protein comprises a deletion of the first amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO:

1.

5. 2. The pharmaceutical formulation of claim 1, wherein the recombinant stabilized galectin-9 protein is 1 to 20 mg / mL.

6. 2. The pharmaceutical formulation according to claim 1, wherein the buffering agent is one or more selected from the group consisting of citrate, phosphate, histidine, glycine, acetate, tartrate, aspartate, lactate, gluconate, glutamate, succinate, and combinations thereof.

7. 2. The pharmaceutical formulation according to claim 1, wherein the buffering agent is one or more selected from the group consisting of phosphate, histidine, acetate, and combinations thereof.

8. 2. The pharmaceutical formulation of claim 1, wherein the buffering agent is histidine acetate.

9. 2. The pharmaceutical formulation according to claim 1, wherein the pH of the buffer is 4.5 to 7.

10. 2. The pharmaceutical formulation according to claim 1, wherein the pH of the buffer is 5.5 to 6.

5.

11. 2. The pharmaceutical preparation according to claim 1, wherein the concentration of the buffering agent is 5 to 20 mM.

12. 2. The pharmaceutical preparation according to claim 1, wherein the concentration of the buffering agent is 5 to 15 mM.

13. The stabilizer is (i) one or more carbohydrates or sugars; and (ii) one or more amino acids or pharmaceutically acceptable salts thereof; 2. The pharmaceutical formulation of claim 1, comprising:

14. 14. The pharmaceutical formulation of claim 13, wherein the carbohydrate or sugar is selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, lactitol, sucrose, trehalose, mannose, maltose, lactose, xylose, ribose, glucose, raffinose, dextran, cyclodextrin, cellobiose, isomaltose, arabinose, glucosamine, and fructose.

15. 14. The pharmaceutical formulation of claim 13, wherein the carbohydrate or sugar is selected from the group consisting of trehalose and mannitol.

16. 14. The pharmaceutical formulation of claim 13, wherein the amino acid is selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, and arginine.

17. 14. The pharmaceutical formulation of claim 13, wherein the amino acid is glycine.

18. 14. The pharmaceutical formulation according to claim 13, wherein the carbohydrate or sugar comprises one or more carbohydrates or sugars, each of which is present in an amount of 1 to 10 w / v %.

19. 14. The pharmaceutical formulation according to claim 13, wherein the carbohydrate or sugar comprises trehalose and mannitol, each of which is present in an amount of 1 to 5 w / v %.

20. 14. The pharmaceutical formulation according to claim 13, wherein the amino acid or the pharmaceutically acceptable salt thereof is present at a concentration of 1 to 20 mg / mL.

21. 14. The pharmaceutical formulation of claim 13, wherein the stabilizer further comprises a polysorbate.

22. 14. The pharmaceutical formulation of claim 13, wherein the stabilizer further comprises 0.001 to 0.05 w / v % of polysorbate 20, polysorbate 80, or a combination thereof.

23. 14. The pharmaceutical formulation of claim 13, wherein the pharmaceutical formulation is in a liquid or lyophilized dosage form.

24. A vial filled with the pharmaceutical formulation according to any one of claims 1 to 23.

25. A syringe filled with the pharmaceutical preparation according to any one of claims 1 to 23.

26. A pharmaceutical preparation for preventing or treating a bone disease, comprising a therapeutically effective amount of the pharmaceutical preparation according to any one of claims 1 to 23.

27. A pharmaceutical preparation for preventing cancer or treating an autoimmune disease, comprising a therapeutically effective amount of the pharmaceutical preparation according to any one of claims 1 to 23.

28. Use of the pharmaceutical preparation according to any one of claims 1 to 23 in the manufacture of a medicament for preventing or treating a bone disease.

29. Use of the pharmaceutical preparation according to any one of claims 1 to 23 in the manufacture of a medicament for the prevention or treatment of cancer or an autoimmune disease.

30. Use of the pharmaceutical preparation according to any one of claims 1 to 23 for the prevention or treatment of bone diseases.

31. Use of the pharmaceutical preparation according to any one of claims 1 to 23 for the prevention or treatment of cancer or autoimmune diseases.

32. A method for treating a bone disease, comprising the step of administering a therapeutically effective amount of the pharmaceutical preparation according to any one of claims 1 to 23 to a subject.

33. A method for treating cancer or autoimmune disease, comprising the step of administering a therapeutically effective amount of the pharmaceutical preparation according to any one of claims 1 to 23 to a subject.

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