Stabilized formulation comprising recombinant stable galectin 9 protein

Abstract

The invention relates to a stabilized preparation containing recombinant stable galectin 9 protein, in particular to a pharmaceutical preparation with improved stability by combining a buffer solution, a stabilizer and the like.

Description

Technical Field

[0001] The present invention relates to a stabilized preparation comprising a recombinant stabilized Galectin-9 protein, and to a pharmaceutical preparation capable of stably preserving the Galectin-9 protein by combining the preparation with a buffer and a stabilizer. Background Art

[0002] Galectin-9 is a beta-galactoside lectin protein isolated from mouse embryonic kidney. It consists of two carbohydrate recognition domains (CRDs) linked by a linker peptide region that connects the N-terminal carbohydrate recognition domain (NCRD) to the C-terminal carbohydrate recognition domain (CCRD). Galectin-9 is known to be a ligand for HAVCR2 (Tim-3), binding to Tim-3 and inducing apoptosis in Tim-3-positive Th1 cells, thereby suppressing autoimmune inflammation by inhibiting excessive Th1 responses. Furthermore, galectin-9 has been observed in various cancers, including melanoma, Hodgkin's disease, liver cancer, pancreatic cancer, gastric cancer, and colorectal cancer. Research is ongoing on its use as a therapeutic agent, including the preparation of variants. In this regard, Korean Patent Publication No. 10-2022-0068158 discloses a pharmaceutical composition comprising a recombinant stabilized galectin-9 protein for use in preventing or treating cancer. However, even based on this disclosure, formulation design is still required for human applications such as administration to patients.

[0003] Generally speaking, factors such as the type and ratio of additives in the formulation process will have a significant impact on the effectiveness and stability of the drug applied to the human body, and not only are there differences in the duration after administration, drug distribution, metabolism, excretion, etc., but also need to be carefully considered according to factors such as packaging status and packaging container. For example, for poorly soluble drugs, although they can be taken in the form of capsules, there may be problems with taking them depending on their preparation method or ratio, so various studies need to be conducted on the formulation. In addition, when the stability of the antibody is low, it may lose its efficacy due to denaturation, oxidation, aggregation, decomposition, etc., and may even cause risks such as toxicity in some cases. Therefore, in order to prepare stable, effective and easy-to-use pharmaceutical preparations, many efforts are needed.

[0004] To this end, the present inventors conducted extensive research on formulations to improve the stability of recombinant galectin-9 protein, ultimately developing a formulation with high stability, thereby completing the present invention.

[0005] Patent Literature

[0006] (Patent Document 0001) Korean Patent Publication No. 10-2022-0068158 Summary of the Invention

[0007] Technical issues

[0008] The present invention aims to provide a pharmaceutical preparation that can stably preserve recombinant stable galectin-9 protein.

[0009] Technical Solution

[0010] To achieve the above objectives, the present invention provides a pharmaceutical preparation comprising: (1) a recombinant stabilized galectin-9 protein; (2) a buffer; and (3) a stabilizer.

[0011] In one aspect of the present invention, the recombinant stabilized Galectin-9 protein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0012] In one aspect of the present invention, the recombinant stabilized galectin-9 protein has a homology greater than or equal to 90% with the amino acid sequence shown in SEQ ID NO: 1.

[0013] In one aspect 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.

[0014] In one aspect of the present invention, the concentration of the recombinant stabilized Galectin-9 protein is 1 mg / ml to 20 mg / ml.

[0015] In one aspect of the present invention, the buffering agent is at least one selected from the group consisting of citrate, phosphate, histidine, glycine, acetate, tartrate, aspartate, lactate, gluconate, glutamate, succinate, and combinations thereof.

[0016] In one aspect of the invention, the buffer has a pH of 4.5 to 7.

[0017] In one aspect of the invention, the concentration of the buffer is 5 mM to 20 mM.

[0018] In one aspect of the present invention, the stabilizer consists of (i) one or more carbohydrates or sugars; and (ii) one or more amino acids or pharmaceutically acceptable salts thereof.

[0019] In one aspect of the present 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.

[0020] In one aspect of the present invention, the carbohydrate or sugar is selected from the group consisting of trehalose and mannitol.

[0021] In one aspect 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.

[0022] In one aspect of the present invention, the carbohydrate or sugar includes one or more carbohydrates or sugars, and each is 1 w / v% to 5 w / v%.

[0023] In one aspect of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is in the range of 1 mg / ml to 20 mg / ml.

[0024] In one aspect of the present invention, the stabilizer further comprises polysorbate.

[0025] In one aspect of the present invention, the stabilizer further comprises 0.001 w / v% to 0.05 w / v% of polysorbate 80.

[0026] In one aspect of the present invention, the pharmaceutical preparation is a liquid dosage form or a lyophilized dosage form.

[0027] Furthermore, the present invention provides a pharmaceutical preparation for preventing or treating bone diseases, comprising a therapeutically effective amount of the pharmaceutical preparation.

[0028] In addition, the present invention provides a pharmaceutical preparation for preventing cancer or treating autoimmune diseases, which comprises a therapeutically effective amount of the pharmaceutical preparation.

[0029] In addition, the present invention provides use of a pharmaceutical preparation in preparing a medicament for preventing or treating bone diseases.

[0030] In addition, the present invention provides a use of a pharmaceutical preparation in preparing a medicament for preventing or treating cancer or autoimmune diseases.

[0031] Furthermore, the present invention provides use of a pharmaceutical preparation in preventing or treating bone diseases.

[0032] In addition, the present invention provides use of a pharmaceutical preparation in preventing or treating cancer or autoimmune diseases.

[0033] Furthermore, the present invention provides a method for treating bone diseases, comprising administering a therapeutically effective amount of a pharmaceutical preparation to a subject.

[0034] Furthermore, the present invention provides a method for treating cancer or autoimmune disease, comprising administering a therapeutically effective amount of a pharmaceutical preparation to a subject.

[0035] Effects of the Invention

[0036] The present invention relates to a pharmaceutical preparation comprising a recombinant galectin-9 protein, which has the advantage that a highly stable preparation can be provided by using a buffer and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a graph showing the results of a comparative experiment between a histidine buffer and a PBS buffer.

[0038] Figure 2 Graph showing comparative experimental results of acetate buffer and PBS buffer.

[0039] Figure 3 Graph showing the results of a comparative experiment between histidine acetate buffer and sodium acetate buffer.

[0040] Figures 4a to 4c This is a graph showing the results of a potency test using a histidine acetate buffer according to pH values.

[0041] Figure 5 3-3 is a graph showing the properties of one embodiment (Experimental Example 3-3) according to the present invention.

[0042] Figure 6 It is a graph showing the properties of one embodiment (Experimental Example 3-6) according to the present invention.

[0043] Figure 7 It is a diagram showing the results of SDS PAGE analysis according to one embodiment of the present invention (Experimental Examples 3-6).

[0044] Figures 8a to 8d : is a graph showing the results of SEC HPLC analysis according to one example (Experimental Example 3-6) of the present invention.

[0045] Figures 9a to 9d : is a graph showing the results of IEX HPLC analysis according to one embodiment of the present invention (Experimental Examples 3-6).

[0046] Figures 10a to 10b It is a graph showing the potency results according to one embodiment of the present invention (Experimental Examples 3-6). DETAILED DESCRIPTION

[0047] Best Mode for Carrying Out the Invention

[0048] The following detailed description is provided through embodiments of the present invention so that those skilled in the art can easily implement the present invention. The embodiments of the present invention are used to further fully describe the present invention to those skilled in the art. Therefore, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0049] Throughout the specification of the present invention, when it is described that a part “includes” a certain constituent element, it means further including another constituent element, rather than excluding another constituent element, unless explicitly stated otherwise.

[0050] The present invention relates to a pharmaceutical preparation comprising (1) a recombinant stabilized galectin-9 protein; (2) a buffer; and (3) a stabilizer.

[0051] In the present invention, the recombinant stabilized Galectin-9 protein maintains the sugar chain recognition activity of wild-type Galectin-9 while having a more stable molecular structure against proteases.

[0052] Specifically, the recombinant stabilized galectin-9 protein is a protein produced by modifying the connecting region of two CRDs (Carbohydrate Recognition Domains) and CCRD (C-terminal domain) of wild-type galectin-9 (which has NCRD-linker-CCRD). More specifically, the recombinant stable galectin-9 protein may consist of the amino acid sequence shown in SEQ ID NO: , wherein all peptides in the linker region of the amino acid sequence are completely deleted, the amino acid sequence (SEQ ID NO: 3) at positions 1 to 10 in the CCRD (SEQ ID NO: 2) is deleted, and Ala (Alanine; A) at position 13 is substituted with Pro (Proline; P). The recombinant stable galectin-9 protein may include an amino acid sequence having a sequence homology to the amino acid sequence shown in SEQ ID NO: 1 of greater than or equal to 75%, preferably greater than or equal to 80%, further preferably greater than or equal to 90%, and most preferably greater than or equal to 95%, and may further include an amino acid sequence tailored for a specific purpose (targeting sequence, tag, labeling residue, improving half-life or peptide stability).

[0053] In addition, the recombinant proteins of the present invention can be obtained by various other methods known in the art. For example, they can be produced by polynucleotide recombination and protein expression systems, or synthesized in vitro by chemical synthesis methods such as peptide synthesis, or produced by cell-free protein synthesis methods.

[0054] As used herein, the term "polynucleotide" refers to a polymer of linked nucleotides that functions to transmit genetic information. For the purposes of the present invention, it encodes the recombinant protein of SEQ ID NO: 1 and may include sequences that share greater than or equal to 75%, preferably greater than or equal to 85%, more preferably greater than or equal to 90%, and most preferably greater than or equal to 95% sequence homology with the polynucleotide sequence encoding the recombinant protein.

[0055] The term "homology" as used in the present invention is intended to indicate the degree of similarity to a wild-type amino acid sequence or polynucleotide sequence. Such homology can be compared using comparison programs known in the art, and the homology of two or more sequences can be calculated as a percentage (%).

[0056] The term "prevention" used in the present invention refers to all actions to inhibit or delay the occurrence of a disease by administering a composition.

[0057] The term "treatment" used in the present invention refers to all actions that improve or benefit the symptoms of the disease by administering a composition.

[0058] The term "pharmaceutical preparation" used in the present invention refers to a finished product prepared by filling, lyophilizing and / or splitting a stock solution comprising a composition.

[0059] In one aspect of the present invention, the recombinant stabilized Galectin-9 protein has the amino acid sequence shown in SEQ ID NO: 1.

[0060] In one aspect of the present invention, the recombinant stabilized galectin-9 protein has an amino acid sequence identity of greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or greater than or equal to 99% to the amino acid sequence of SEQ ID NO: 1.

[0061] In one aspect 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.

[0062] In one aspect of the present invention, the concentration of the recombinant stable galectin-9 protein in the pharmaceutical preparation may be 0.01 mg / ml to 400 mg / ml. Specifically, the concentration of the recombinant stable galectin-9 protein may be 0.01 mg / ml to 250 mg / ml, more specifically, 0.01 mg / ml to 150 mg / ml, 0.01 mg / ml to 120 mg / ml, 0.01 mg / ml to 100 mg / ml, 0.1 mg / ml to 80 mg / ml, 0.1 mg / ml to 60 mg / ml, or 0.1 mg / ml to 40 mg / ml. In a specific aspect of the invention, the concentration of the recombinant stabilized Galectin-9 protein is 0.1 mg / ml to 20 mg / ml, 0.1 mg / ml to 18 mg / ml, 0.1 mg / ml to 16 mg / ml, 0.1 mg / ml to 15 mg / ml, 1 mg / ml to 15 mg / ml, 2 mg / ml to 15 mg / ml, 3 mg / ml to 15 mg / ml, 4 mg / ml to 15 mg / ml, or 5 mg / ml to 15 mg / ml. In a more specific aspect of the invention, the concentration of the recombinant stabilized Galectin-9 protein is 5.5 mg / ml to 15 mg / ml, 6 mg / ml to 15 mg / ml, 6.5 mg / ml to 15 mg / ml, 7 mg / ml to 15 mg / ml, 7.5 mg / ml to 15 mg / ml, 7.5 mg / ml to 14.5 mg / ml, 7.5 mg / ml to 14 mg / ml, 7.5 mg / ml to 13.5 mg / ml, 7.5 mg / ml to 13 mg / ml, or 7.5 mg / ml to 12.5 mg / ml.

[0063] In one aspect of the present invention, the buffer is at least one selected from the group consisting of the following substances: citrate, phosphate, histidine, glycine, acetate, tartrate, aspartate, lactate, gluconate, glutamate, succinate, and combinations thereof. In a specific aspect of the present invention, the buffer is at least one selected from the group consisting of the following substances: phosphate, histidine, acetate, and combinations thereof. In a more specific aspect of the present invention, the buffer is histidine acetate. In the present invention, including in terms of stability and pH regulation, the inclusion of histidine can show beneficial effects.

[0064] In one aspect of the invention, the buffering agent does not comprise a sodium salt.

[0065] In one aspect of the present invention, the pH of the buffer is 4 to 7. Specifically, the pH of the buffer may be 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 aspect of the present invention, the pH of the buffer may be 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 a more specific aspect of the present invention, the pH of the buffer is 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.

[0066] In one aspect of the invention, the concentration of the buffer in the pharmaceutical formulation may be 0.01 mM to 50 mM. Specifically, the concentration of the buffer may be 0.01 mM to 45 mM, more specifically 0.01 mM to 40 mM, 0.01 mM to 35 mM, 0.01 mM to 30 mM, or 0.01 mM to 25 mM. In a specific aspect of the invention, the concentration of the buffer is 0.1 mM to 25 mM, 0.1 mM to 24 mM, 0.1 mM to 23 mM, 0.1 mM to 22 mM, 0.1 mM to 21 mM, 1 mM to 21 mM, 2 mM to 21 mM, 3 mM to 21 mM, or 4 mM to 21 mM. In a more specific aspect of the invention, the concentration of the buffer is 4 mM to 20.5 mM, 4 mM to 20 mM, 4.5 mM to 20 mM, or 5 mM to 20 mM.

[0067] In one aspect of the present invention, the stabilizer consists of (i) one or more carbohydrates or sugars; and (ii) one or more amino acids or pharmaceutically acceptable salts thereof.

[0068] In the present invention, carbohydrates or sugars may include sugars, sugar alcohols, sugar acids and sugar derivatives, for example, glucose, fructose, sucrose, lactose, maltose, trehalose, glycerol, erythritol, arabitol, xylitol, mannitol, sorbitol, galactitol, fucitol, iditol, maltitol, lactitol, maltotriol, maltotetraol and the like, but are not limited thereto.

[0069] In the present invention, amino acids may include natural amino acids or non-natural amino acids, for example, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, etc., but are not limited thereto.

[0070] In one aspect of the present 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. In a specific aspect of the present invention, the carbohydrate or sugar is selected from the group consisting of mannitol and trehalose.

[0071] In one aspect of the present invention, the stabilizer includes one or more carbohydrates or sugars, and each is 0.01 w / v % to 10 w / v %. Specifically, the concentration of the one or more carbohydrates or sugars included in the stabilizer can be 0.01 w / v % to 9.5 w / v %, 0.01 w / v % to 9 w / v %, 0.01 w / v % to 8.5 w / v %, 0.01 w / v % to 8 w / v %, 0.01 w / v % to 7.5 w / v %, 0.5 w / v % to 7.5 w / v % or 1 w / v % to 7.5 w / v %. In a specific aspect of the invention, the concentration of one or more carbohydrates or sugars included in the stabilizer is 1 w / v% to 6 w / v%, 1 w / v% to 5.9 w / v%, 1 w / v% to 5.8 w / v%, 1 w / v% to 5.7 w / v%, 1 w / v% to 5.6 w / v%, 1 w / v% to 5.5 w / v%, 1 w / v% to 5.4 w / v%, 1 w / v% to 5.3 w / v%, 1 w / v% to 5.2 w / v%, 1 w / v% to 5.1 w / v% or 1 w / v% to 5 w / v%.

[0072] In a specific aspect of the present invention, the stabilizer may include two or more carbohydrates or sugars. Specifically, it includes two or more carbohydrates or sugars, wherein the concentration of one carbohydrate or sugar is 1 w / v% to 3 w / v%, 1.1 w / v% to 3 w / v%, 1.2 w / v% to 3 w / v%, 1.3 w / v% to 3 w / v%, 1.4 w / v% to 3 w / v%, 1.5 w / v% to 3 w / v%, 1.5 w / v% to 2.9 w / v%, 1.5 w / v% to 2.8 w / v%, 1.5 w / v% to 2.7 w / v%, 1.5 w / v% to 2.6 w / v% or 1.5 w / v% to 2.5 w / v%. The concentration of the other carbohydrate or sugar is 3 w / v% to 5 w / v%, 3.1 w / v% to 5 w / v%, 3.2 w / v% to 5 w / v%, 3.3 w / v% to 5 w / v%, 3.4 w / v% to 5 w / v%, 3.5 w / v% to 5 w / v%, 3.5 w / v% to 4.9 w / v%, 3.5 w / v% to 4.8 w / v%, 3.5 w / v% to 4.7 w / v%, 3.5 w / v% to 4.6 w / v% or 3.5 w / v% to 4.5 w / v%. In a more specific aspect of the present invention, the stabilizer comprises two or more carbohydrates or sugars, wherein the concentration of one carbohydrate or sugar is 1.5 w / v% to 2.5 w / v%, 1.55 w / v% to 2.5 w / v%, 1.6 w / v% to 2.5 w / v%, 1.65 w / v% to 2.5 w / v%, 1.7 w / v% to 2.5 w / v%, 1.75 w / v% to 2.5 w / v%, 1.75 w / v% to 2.45 w / v%, 1.75 w / v% to 2.4 w / v%, 1.75 w / v% to 2.35 w / v%, 1.75 w / v% to 2.35 w / v%, or 1.75 w / v% to 2.45 w / v%. the concentration of the other carbohydrate or sugar is 3.5 w / v% to 4.5 w / v%, 3.55 w / v% to 4.5 w / v%, 3.6 w / v% to 4.5 w / v%, 3.65 w / v% to 4.5 w / v%, 3.7 w / v% to 4.5 w / v%, 3.75 w / v% to 4.5 w / v%, 3.75 w / v% to 4.45 w / v%, 3.75 w / v% to 4.4 w / v%, 3.75 w / v% to 4.35 w / v%, 3.75 w / v% to 4.3 w / v% or 3.75 w / v% to 4.25 w / v%.

[0073] In one aspect of the present invention, the carbohydrate or sugar comprises trehalose and mannitol. In addition, in one aspect of the present invention, the carbohydrate or sugar comprises trehalose and mannitol, each at 1 w / v% to 10 w / v%.

[0074] In a specific aspect of the present invention, the concentration of mannitol is 1 w / v% to 9 w / v%, 1 w / v% to 8 w / v%, 1 w / v% to 7 w / v%, 1 w / v% to 6 w / v%, 1 w / v% to 5 w / v%, 1.5 w / v% to 5 w / v%, 2 w / v% to 5 w / v%, 2.5 w / v% to 5 w / v%, 3 w / v% to 5 w / v%, 3.1 w / v% to 5w / v%, 3.2w / v% to 5w / v%, 3.3w / v% to 5w / v%, 3.4w / v% to 5w / v%, 3.5w / v% to 5w / v%, 3.5w / v% to 4.9w / v%, 3.5w / v% to 4.8w / v%, 3.5w / v% to 4.7w / v%, 3.5w / v% to 4.6w / v% or 3.5w / v% to 4.5w / v%. More specifically, the concentration of mannitol is 3.5 w / v% to 4.5 w / v%, 3.55 w / v% to 4.5 w / v%, 3.6 w / v% to 4.5 w / v%, 3.65 w / v% to 4.5 w / v%, 3.7 w / v% to 4.5 w / v%, 3.75 w / v% to 4.5 w / v%, 3.75 w / v% to 4.45 w / v%, 3.75 w / v% to 4.4 w / v%, 3.75 w / v% to 4.35 w / v%, 3.75 w / v% to 4.3 w / v% or 3.75 w / v% to 4.25 w / v%.

[0075] In a specific aspect of the invention, the concentration of trehalose is 1 w / v% to 3 w / v%, 1.1 w / v% to 3 w / v%, 1.2 w / v% to 3 w / v%, 1.3 w / v% to 3 w / v%, 1.4 w / v% to 3 w / v%, 1.5 w / v% to 3 w / v%, 1.5 w / v% to 2.9 w / v%, 1.5 w / v% to 2.8 w / v%, 1.5 w / v% to 2.7 w / v%, 1.5 w / v% to 2.6 w / v% or 1.5 w / v% to 2.5 w / v%. More specifically, the concentration of trehalose is 1.5 w / v% to 2.5 w / v%, 1.55 w / v% to 2.5 w / v%, 1.6 w / v% to 2.5 w / v%, 1.65 w / v% to 2.5 w / v%, 1.7 w / v% to 2.5 w / v%, 1.75 w / v% to 2.5 w / v%, 1.75 w / v% to 2.45 w / v%, 1.75 w / v% to 2.4 w / v%, 1.75 w / v% to 2.35 w / v%, 1.75 w / v% to 2.3 w / v% or 1.75 w / v% to 2.25 w / v%.

[0076] In one aspect of the present 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 aspect of the present invention, the amino acid is glycine.

[0077] In one aspect of the present invention, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.01 mg / ml to 20 mg / ml. Specifically, the concentration of the amino acid or its pharmaceutically acceptable salt is 0.01 mg / ml to 19 mg / ml, 0.01 mg / ml to 18 mg / ml, 0.01 mg / ml to 17 mg / ml, 0.01 mg / ml to 16 mg / ml, 0.01 mg / ml to 15 mg / ml, 0.01 mg / ml to 14 mg / ml, 0.01 mg / ml to 13 mg / ml, 0.01 mg / ml to 12 mg / ml, 0.01 mg / ml to 11 mg / ml, 0.01 mg / ml to 10 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.2 mg / ml to 10 mg / ml, 0.3 mg / ml to 10 mg / ml, 0.4 mg / ml to 10 mg / ml, 0.5 mg / ml to 10 mg / ml, 0.6 mg / ml to 10 mg / ml, 0.7 mg / ml to 10 mg / ml, 0.8 mg / ml to 10 mg / ml, 0.9 mg / ml to 10 mg / ml or 1 mg / ml to 10 mg / ml. In a specific aspect of the invention, the amino acid or pharmaceutically acceptable salt thereof is present at a concentration of 1 mg / ml to 10 mg / ml, 1.5 mg / ml to 10 mg / ml, 2 mg / ml to 10 mg / ml, 2.5 mg / ml to 10 mg / ml, 2.5 mg / ml to 9.5 mg / ml, 2.5 mg / ml to 9 mg / ml, 2.5 mg / ml to 8.5 mg / ml, 2.5 mg / ml to 8 mg / ml or 2.5 mg / ml to 7.5 mg / ml. In a more specific aspect of the invention, the concentration of the amino acid or pharmaceutically acceptable salt thereof is 2.6 mg / ml to 7.5 mg / ml, 2.7 mg / ml to 7.5 mg / ml, 2.8 mg / ml to 7.5 mg / ml, 2.9 mg / ml to 7.5 mg / ml, 3 mg / ml to 7.5 mg / ml, 3 mg / ml to 7 mg / ml, 3 mg / ml to 6.5 mg / ml, 3 mg / ml to 6.4 mg / ml, 3 mg / ml to 6.3 mg / ml, 3 mg / ml to 6.2 mg / ml, 3 mg / ml to 6.1 mg / ml or 3 mg / ml to 6 mg / ml.

[0078] In one aspect of the present invention, the stabilizer further comprises a nonionic surfactant. For example, the nonionic surfactant includes Pluronic, polysorbate, poloxamer, etc., but is not limited thereto.

[0079] In one aspect of the present invention, the stabilizer further comprises polysorbate, which is polysorbate 20, polysorbate 80, or a combination thereof.

[0080] In one aspect of the present invention, the stabilizer further comprises 0.001 w / v% w / v% to 0.05 w / v% of polysorbate 20, polysorbate 80, or a combination thereof.

[0081] In a specific aspect of the invention, the concentration of polysorbate 20 or polysorbate 80 is 0.001 w / v% to 0.02 w / v%, 0.002 w / v% to 0.02 w / v%, 0.003 w / v% to 0.02 w / v%, 0.004 w / v% to 0.02 w / v%, 0.005 w / v% to 0.02 w / v%, 0.005 w / v% to 0.019 w / v%, 0.005 w / v% to 0.018 w / v%, 0.005 w / v% to 0.017 w / v%, 0.005 w / v% to 0.016 w / v% or 0.005 w / v% to 0.015 w / v%. More specifically, the concentration of polysorbate 20 or polysorbate 80 is 0.006w / v% to 0.015w / v%, 0.0065w / v% to 0.015w / v%, 0.007w / v% to 0.015w / v%, 0.0075w / v% to 0.015w / v%, 0.0075w / v% to 0.014w / v%, 0.006w / v% to 0.013w / v% or 0.006w / v% to 0.0125w / v%.

[0082] In one aspect of the present invention, the potency of the pharmaceutical preparation is 60% w / v% to 130% of the standard potency. Specifically, the pharmaceutical preparation is 70% w / v% to 120% of the standard potency.

[0083] In addition, in one aspect of the present invention, the purity (SEL-HPLC) of the pharmaceutical preparation is greater than or equal to 90%. Specifically, the purity is greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, and more specifically, the purity is greater than or equal to 95%.

[0084] In one aspect of the present invention, the pharmaceutical preparation is a liquid dosage form or a lyophilized dosage form.

[0085] In a specific aspect of the present invention, the pharmaceutical preparation is a lyophilized dosage form. Compared to a liquid dosage form, the pharmaceutical preparation of the present invention exhibits high stability in a lyophilized dosage form.

[0086] The specific form of the pharmaceutical preparation of the present invention may vary depending on the product form, and it can be present in a filled state in a soft tube, cartridge, syringe, polymer vial, glass vial, etc., but is not limited thereto. In addition, the present invention can also be filled by conventional methods known to those skilled in the art.

[0087] In one aspect of the present invention, the present invention relates to a vial filled with the pharmaceutical formulation.

[0088] In one aspect of the present invention, the present invention relates to a syringe filled with the pharmaceutical preparation.

[0089] In one aspect of the present invention, the present invention can be prepared by the following method: after preparing a stock solution including 1 / 2 weight part of protein and excipients based on 1 weight part of the finished product filled with the pharmaceutical preparation, the prepared stock solution is filled in a vial with 2 times the capacity. Specifically, a finished product comprising 1 mg / mL to 20 mg / mL of recombinant stable galectin-9 protein, 1 w / v% to 10 w / v% of mannitol, 1 w / v% to 10 w / v% of trehalose, 1 mg / mL to 20 mg / mL of amino acids, and 0.001 w / v% to 0.05 w / v% of polysorbate can be prepared by filling 2 mL of the stock solution in each vial and then lyophilizing the product. Each mL of the stock solution comprises 0.5 mg / mL to 10 mg / mL of recombinant stable galectin-9 protein, 0.5 w / v% to 5 w / v% of mannitol, 0.5 w / v% to 5 w / v% of trehalose, 0.5 mg / mL to 10 mg / mL of amino acids, and 0.0005 w / v% to 0.025 w / v% of polysorbate. More specifically, a finished product comprising 10 mg / mL to 12 mg / mL of recombinant stable galectin-9 protein, 4 w / v% mannitol, 2 w / v% trehalose, 5 mg / mL of amino acids, and 0.01 w / v% of polysorbate can be prepared by filling 2 mL of the stock solution in each vial and then lyophilizing. Each 1 mL of the stock solution comprises 5 mg / mL to 6 mg / mL of recombinant stable galectin-9 protein, 2 w / v% mannitol, 1 w / v% trehalose, 2.5 mg / mL of amino acids, and 0.005 w / v% of polysorbate, but is not limited thereto.

[0090] Furthermore, the present invention relates to a pharmaceutical preparation for preventing or treating bone diseases, comprising a therapeutically effective amount of the pharmaceutical preparation.

[0091] In the present invention, the bone disease may be at least one selected from the group consisting of the following diseases: osteoporosis, bone defect, osteomalacia, osteopenia, osteogenesis imperfecta, bone formation disorder, primary bone tumor, degenerative bone disease, osteoarthritis, rheumatoid arthritis, fracture, osteonecrosis, periodontal disease, inflammatory alveolar bone resorption disease, inflammatory bone resorption disease and Paget's disease, but is not limited thereto.

[0092] In addition, the present invention also relates to a pharmaceutical preparation for preventing cancer or treating autoimmune diseases, which comprises a therapeutically effective amount of the pharmaceutical preparation.

[0093] In the present invention, the cancer may be at least one selected from the group consisting of colorectal cancer, lung cancer, thyroid cancer, gastric cancer, breast cancer, brain cancer, osteosarcoma, glioblastoma, melanoma, glioma, glioblastoma, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, gallbladder cancer, bile duct cancer, esophageal cancer, colon cancer, rectal cancer, pancreatic cancer, blood cancer, leukemia, myeloid leukemia, lymphoma, head and neck cancer, skin cancer and liver cancer; but not limited thereto.

[0094] In the present invention, the autoimmune disease may be at least one selected from the group consisting of systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, photosensitive skin disease, autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, malignant Anemia, autoimmune atrophic gastritis, Addison's disease, early menopause, male infertility, childhood diabetes, Gutt's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, lenticular uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary sclerosis, chronic active hepatitis (Hbs negative), latent cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatomyositis and discoid lupus erythematosus; but not limited to these.

[0095] In addition, as non-limiting examples, autoimmune diseases may 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 antibody 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 and neuronal nerve disorders, Barlow's disease, and other autoimmune diseases. disease), Behçet's disease, pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Cheger-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome Syndrome, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Gutpacher syndrome, granulomatosis with polyangiitis (GPA)with polyangiitis), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, pediatric arthritis, pediatric diabetes mellitus, Kawasaki syndrome, Ayton-Lambert syndrome, leukemia Cytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), systemic lupus erythematosus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), erosive corneal ulcer, Mucha-Haberman disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (streptococcal infection), Infection-related pediatric autoimmune neuropsychiatric disorders), brain tumor-induced cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), facial hemitrophy, Parsonnage-Turner syndrome, intermediate uveitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis (perivenousencephalomyelitis), pernicious anemia, POEMS syndrome, polyarteritis nodosa, polyglandular autoimmune syndrome type I, II, and III, polymyalgia rheumatica, polymyositis, Post-myocardial infarction syndrome, postpericardiotomy syndrome, progestogen dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, simple red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless syndrome, retroperitoneal fibrosis, rheumatoid fever, rheumatoid arthritis, granulomatous disease, Simi's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-man syndrome person syndrome), subacute bacterial endocarditis (SBE), Susac 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 dermatitis, vitiligo, Waldenstrom's macroglobulinemia (WM), and Wegener's granulomatosis (granulomatosis with polyangiitis, GPA).

[0096] Furthermore, the present invention relates to use of a pharmaceutical preparation in preparing a medicament for preventing or treating bone diseases.

[0097] In addition, the present invention relates to use of a pharmaceutical preparation in preparing a medicament for preventing or treating cancer or autoimmune diseases.

[0098] Furthermore, the present invention relates to use of a pharmaceutical preparation for preventing or treating bone diseases.

[0099] Furthermore, the present invention relates to use of a pharmaceutical preparation for preventing or treating cancer or autoimmune diseases.

[0100] Furthermore, the present invention relates to a method for treating bone diseases, comprising administering a therapeutically effective amount of a pharmaceutical formulation to a subject.

[0101] In addition, the present invention also relates to a method for treating cancer or autoimmune disease, which comprises administering a therapeutically effective amount of a pharmaceutical preparation to a subject.

[0102] Specific ways of implementing the invention

[0103] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0104] However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

[0105] <Preparation Example 1> Preparation of recombinant stable galectin-9 protein (sGal-9)

[0106] After preparing an expression vector, the expression vector containing a gene encoding a recombinant stable galectin-9 protein having the amino acid sequence of SEQ ID NO: 1 was introduced into Escherichia coli (E. coli) via heat shock. The E. coli was cultured in LB medium containing 50 μg / mL kanamycin, and arabinose was added when the absorbance at 600 nm reached 0.7 to induce expression of the recombinant protein. The cells induced to express the recombinant protein were then lysed and filtered, and the target protein was captured using cation exchange methods and affinity columns, resulting in high-purity recombinant stable galectin-9 protein with high yield.

[0107] <Preparation Example 2> Preparation of stabilized preparation

[0108] Preparations were prepared using the recombinant stabilized galectin-9 protein prepared in Preparation Example 1 (hereinafter referred to as "recombinant galectin-9"). The compositions of each preparation are shown in the following experimental examples. In one embodiment, the purified solution was subjected to ultrafiltration (UF) / diafiltration (DF) using 5 mM histidine acetate (HisAce) pH 6.0 to a concentration of 11 mg / mL to 15 mg / mL. Subsequently, mannitol (2 w / v%), trehalose (1 w / v%), and glycine (2.5 mg / mL) were added for dilution to a protein concentration of 5 mg / mL to 7 mg / mL. The solution was then filtered through a 0.2 μm filter and finally filled into 2.0 mL / vial, followed by freeze-drying.

[0109] <Experimental Example 1> Buffer Evaluation Method

[0110] <Experimental Example 1-1> Appearance

[0111] The appearance of all samples was inspected for transparency, color, and visible particles using a YB-2 light box against a black and white background according to the standard operating procedure (SOP) method (PD-DPD-LAB-001-07).

[0112] <Experimental Example 1-2> pH

[0113] The pH value was measured at least twice using a pH meter equipped with a glass electrode, and the average pH value was recorded.

[0114] <Experimental Example 1-3> Protein concentration

[0115] Protein concentration was determined using a Thermo UV spectrophotometer. The assay was performed according to the standard operating procedure (SOP) method (PD-DPD-EQU-088-01), with an absorbance of 1.0 AU × mL × mg for each evaluation. -1 ×cm -1 The sample volume is 2.5 μL, the detection times are no less than two times, and the average value is taken and recorded.

[0116] <Experimental Example 1-4> Size Exclusion Chromatography (SEC-HPLC)

[0117] SEC-HPLC was performed according to the following steps.

[0118] When the sample concentration was greater than or equal to 1.0 mg / mL, the sample was diluted to 1.0 mg / mL using mobile phase prior to SEC analysis, and 50 μg of sample was then injected into the HPLC system for analysis. SEC-HPLC was performed using a Hitachi Chromaster CM5000 system, which included an Agilent TSKgel 2000SWxl column (7.8 mm × 300 mm, 5 μm) and a UV detector (detection wavelength: 280 nm). Analysis conditions were a column temperature of 25°C, an injection volume of 50 μL, a mobile phase of 300 mM sodium chloride, 10% isopropanol, and 50 mM phosphate buffer (pH 6.8 ± 0.1), and a solvent flow rate of 0.4 mL / min.

[0119] <Experimental Example 1-5> Protein electrophoresis (SDS-PAGE)

[0120] SDS-PAGE was performed using a heater (DAIHAN MaXtable TM 10H), electrophoresis system (Bio-Rad BR 165-8033FC) and rocker (Daihan RK-1D) for analysis. The sample solution was diluted with 4 times loading buffer at a ratio of 3:1 and heated at 98 ° C for 5 minutes. The protein standard (Protein Standard) and the sample were added to a 12% acrylamide gel respectively and electrophoresed at 100V for 90 minutes. The gel after protein separation was stained with CBB (Coomassie Brilliant Blue) R-250 solution in a rocker (Rocker) for 30 minutes. Subsequently, destaining was performed with a destaining solution in a rocker for 30 minutes. After the background destaining was completed, the analysis was performed by confirming the dark band near 31kD.

[0121] <Experimental Example 1-6> Potency Experiment

[0122] Prepare cell lines

[0123] Thaw a 1 mL vial of Molt 4T cells at 37°C for 1 to 2 minutes. Add the cells to 10 mL of RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and centrifuge at room temperature (24°C) at 1,300 rpm for 3 minutes. Remove the medium and add fresh medium to disperse the cells and adjust the cell concentration to 24 × 10 5 cells / mL, seeded in 25cm 2 Culture flasks were cultured in a 37°C, 5% CO2 incubator. 6 When the number of cells / mL reached 24×10 5Afterwards, the doubling time and density of the cells were confirmed and maintained.

[0124] Potency experiment

[0125] The titer experiment was performed using Molt 4T cells that had been cultured for 2 weeks, with a doubling time of 24 hours and a density of 95%. The cells were diluted to 5.6×10 4 Cells / mL were plated in a 96-well culture plate, 90 μL was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator for 24 hours. The sample solution was diluted with calcium- and magnesium-free phosphate buffered saline (PBS(-)) to 300 μg / mL, 100 μg / mL, 30 μg / mL, 10 μg / mL, 3 μg / mL, 1 μg / mL, and 0 μg / mL, so that the final concentrations in the culture medium were 30 μg / mL, 10 μg / mL, 3 μg / mL, 0.3 μg / mL, 0.1 μg / mL, and 0 μg / mL, and 10 μL of the diluted sample was added to each well. Subsequently, after culturing in an incubator at 37°C and 5% CO2 for 24 hours, 10 μL of WST-1 detection reagent was added to each well and reacted in an incubator at 37°C and 5% CO2 for 2 hours. The optical density (OD) value at 450 nm was then measured using a microplate reader, and the OD value change and half-maximal inhibitory concentration (LD50) were calculated using a four-parameter logistic fit model. 50 ).

[0126] <Experimental Example 1-7> Osmolality

[0127] Osmolality was measured using an osmometer. Clinitrol 290 mOsm / kg standard solution was used before and after testing to confirm the accuracy of the osmometer. 20 μL of each sample was analyzed.

[0128] <Experimental Example 2> Liquid Preparation

[0129] <Experimental Example 2-1> Comparative Analysis of Buffer Solutions (1)

[0130] The stability of the sGal-9 protein was evaluated using a pH / buffer system under stress conditions (37°C). The sGal-9 concentration in each experiment was 2 mg / mL. The experiment was conducted over a 4-week period, using the same appearance analysis as in Experimental Example 1. The buffer conditions are shown in Table 1.

[0131] Table 1

[0132]

[0133] The experimental results are as follows Figure 1 、 Figure 2 and as shown in Table 2.

[0134] Figure 1 This is the comparison result between histidine buffer and PBS buffer. Figure 1 As shown, precipitation was observed when PBS buffer was used, and the protein stability was higher in histidine buffer compared to PBS buffer.

[0135] Table 2 and Figure 2 The comparison results of acetate buffer and PBS buffer are shown in Table 2 and Figure 2 As shown, there is a difference in the particle count measured at 25°C 3 days after PBS filtration, confirming that acetate buffer is superior to PBS buffer. (#1: 20mM PBS, 50mM NaCl, pH 7.3, 0.26mg / mL; #2: Acetate buffer, pH 5.0, 0.62mg / mL; #3: Acetate buffer, pH 5.3, 0.62mg / mL; #4: After filtration from #2; #5: After filtration from #3)

[0136] Table 2

[0137]

[0138] (*CL (=clear liquid); P (=particles); P(n) (=number of particles); PF (=no particles))

[0139] <Experimental Example 2-2> Comparative Analysis of Buffer Solutions (2) (Sodium Salt)

[0140] The stability of the sGal-9 protein was evaluated using the same methods as in Experimental Example 2-1. Appearance, UV absorption, pH, SEC-HPLC, SDS-PAGE, and potency were analyzed as in Experimental Example 1. The experiment lasted for 2 weeks. The buffer conditions are shown in Table 3.

[0141] Table 3

[0142]

[0143] Appearance, ultraviolet absorption (UV), pH, SEC-HPLC, SDS-PAGE and potency experiments were performed at T0; appearance, UV, pH, SEC-HPLC and SDS-PAGE experiments were performed 1 week later; appearance, UV, pH, SEC-HPLC, SDS-PAGE and potency experiments were performed again 2 weeks later.

[0144] The appearance test results are shown in Table 4 and Figure 3 shown.

[0145] Table 4

[0146]

[0147] (*C (=colorless); SY (=light yellow; Y (=yellow); CL (=clear liquid); SO (=slightly opalescent liquid); O (=opalescent liquid); FP (=no visible particles); PO (=visible particles <5); PO+ (=few visible particles); PO++ (=many visible particles))

[0148] The SEC-HPLC test results are shown in Tables 5 and 6.

[0149] Table 5

[0150]

[0151]

[0152] Table 6

[0153]

[0154] The results of the titer experiment are shown in Table 7.

[0155] Table 7

[0156]

[0157] As shown in Table 4 and Figure 3 As shown in the figure, milky white particles or a large number of particles were observed when histidine acetate was used at 25°C and 40°C, while no visible particles were observed when sodium acetate was used. However, in terms of purity and titer, the protein using histidine acetate buffer showed higher stability than that using sodium acetate buffer.

[0158] As shown in Table 4 and Figure 3 As shown in the figure, milky white particles or a large number of particles were observed when histidine acetate was used at 25°C and 40°C, while no visible particles were observed when sodium acetate was used. However, in terms of purity and titer, the protein using histidine acetate buffer showed higher stability than that using sodium acetate buffer.

[0159] <Experimental Example 2-3> Comparative Analysis of Buffer Solutions (3) (pH / Excipients)

[0160] The stability of sGal-9 protein was evaluated according to the same method as in Experimental Example 2-1. According to the method of Experimental Example 1, appearance, ultraviolet absorption (UV), pH, protein concentration (100 μL), SEC-HPLC, osmotic pressure concentration and titer experiments were performed, and the experiment lasted for 4 weeks. The buffer conditions are shown in Table 8. At T0, appearance, UV, pH, protein concentration (100 μL), SEC-HPLC and osmotic pressure concentration experiments were performed; after 1 week, UV, pH, protein concentration (100 μL), SEC-HPLC and titer experiments were performed at 40 ° C; after 4 weeks, UV, pH, protein concentration (100 μL), SEC-HPLC and titer experiments were performed again at 25 ° C.

[0161] Table 8

[0162]

[0163] Appearance, ultraviolet absorption (UV), pH, protein concentration (100 μL), SEC-HPLC and osmotic pressure concentration experiments were performed at T0; UV, pH, protein concentration (100 μL), SEC-HPLC and potency experiments were performed at 40°C one week later; UV, pH, protein concentration (100 μL), SEC-HPLC and potency experiments were performed again at 25°C four weeks later.

[0164] The test results of appearance, pH, concentration and osmotic pressure concentration are shown in Tables 9 and 10 below.

[0165] Table 9

[0166]

[0167] (*C (=colorless); CL (=clear liquid); PF (=particle-free))

[0168] Table 10

[0169]

[0170] The SEC-HPLC test results are shown in Table 11.

[0171] Table 11

[0172]

[0173] The results of the titer experiments are shown in Tables 12 to 14 and Figures 4A to 4C.

[0174] (B201-20210301-T0=F1 / B201-20210302-T0=F2)

[0175] Table 12 Titer experiment results at T0

[0176]

[0177] Table 13 Potency test results at 40°C after 1 week

[0178]

[0179] Table 14 Potency test results at 25°C after 4 weeks

[0180]

[0181] The above experimental results confirmed that histidine buffer at pH 6.0 was superior to histidine buffer at pH 5.0. In addition, sGal-9 protein showed good stability when 8% sucrose, 0.04% polysorbate 80, and 2 mg / mL glycine were used as excipients.

[0182] <Experimental Example 2-4> Comparative Analysis of Buffer Solutions (4) (pH)

[0183] The stability of the sGal-9 protein was evaluated using the same method as in Experimental Example 2-1. Experiments were conducted under different pH ranges and excipient conditions. The buffer conditions are shown in Table 15 below.

[0184] Table 15

[0185]

[0186] The results of SEC-HPLC, protein concentration and titer experiments are shown in Table 16 below.

[0187] Table 16

[0188]

[0189] The above experimental results confirm that histidine acetate buffer at pH 6.0 is superior to buffers at other pH conditions. In addition, no significant effect of other excipients such as polysorbate 20 (PS20) or glycine was observed.

[0190] <Experimental Example 2-5> Comparative Analysis of Buffer Solutions (5) (Concentration)

[0191] The stability of sGal-9 protein was evaluated using the same method as in Experimental Example 2-1 above. Experiments were conducted at different concentrations, and the buffer conditions are shown in Table 17 below.

[0192] Table 17

[0193]

[0194] -SEC-HPLC, protein concentration and titer experimental results are shown in Table 18 below.

[0195] Table 18

[0196]

[0197]

[0198] According to the above experimental results, the protein stability in histidine acetate buffer decreases in 10 mM, 20 mM and 30 mM, and the stability at 10 mM and 20 mM is better than that at 30 mM.

[0199] <Experimental Example 2-6> Comparative Analysis of Buffer Solutions (6)

[0200] The stability of the sGal-9 protein was evaluated using the same method as in Experimental Example 2-1. The buffer conditions are shown in Table 19 below.

[0201] Table 19

[0202]

[0203]

[0204] -SEC-HPLC, protein concentration and titer experimental results are shown in Table 20 below.

[0205] Table 20

[0206]

[0207] The above experimental results confirmed that the histidine acetate buffer was most effective at 10 mM and that the protein was more stable when sucrose was used.

[0208] <Experimental Example 3> Lyophilized Formulation

[0209] Based on the results of Experimental Example 2 above, the buffer and pH were maintained at 10 mM histidine acetate, pH 6.0, and excipients and dosage forms were compared. Lyophilized formulations were prepared by preparing sGal-9 protein with various excipients, filling 10 mL vials to a volume of 10 mg of sGal-9, and then lyophilizing. Lyophilized samples at each time point were dissolved in 1.0 mL of deionized water for analysis.

[0210] <Experimental Example 3-1> Comparative Analysis of Excipients (1)

[0211] 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.

[0212] Table 21

[0213]

[0214] -SEC-HPLC, protein concentration and titer experimental results are shown in Table 22 below.

[0215] Table 22

[0216]

[0217]

[0218] The above experimental results confirmed that the purity decreased under harsh conditions (37°C) in the second-week (T2W) analysis. In addition, the stability was confirmed to decrease when 0.01% polysorbate 80 (PS80) was included.

[0219] <Experimental Example 3-2> Comparative Analysis of Excipients (2)

[0220] 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.

[0221] Table 23

[0222]

[0223]

[0224] -SEC-HPLC, protein concentration and titer experimental results are shown in Tables 24 and 25.

[0225] Table 24

[0226]

[0227] Table 25

[0228]

[0229]

[0230] The above experimental results confirmed that all experimental data (SEC-HPLC, UV, PAGE) remained unchanged within 24 hours. In addition, it was confirmed that the solution exhibited good stability when mannitol was included.

[0231] <Experimental Example 3-3> Comparative Analysis of Excipients (3)

[0232] 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.

[0233] Table 26

[0234]

[0235] -SEC-HPLC, protein concentration and titer experimental results are shown in Tables 27 and 28 below.

[0236] Table 27

[0237]

[0238]

[0239] Table 28

[0240]

[0241] From the above experimental results, it was confirmed that good stability was exhibited when trehalose was included.

[0242] <Experimental Example 3-4> Comparative Analysis of Excipients (4)

[0243] 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.

[0244] Table 29

[0245]

[0246] -SEC-HPLC, protein concentration and titer experimental results are shown in Tables 30 to 32 below.

[0247] Table 30

[0248]

[0249] Table 31

[0250]

[0251]

[0252] Table 32

[0253]

[0254]

[0255] Based on the above experimental results, it was confirmed that there was no significant difference in the stability between the candidate excipients.

[0256] <Experimental Example 3-5> Comparative Analysis of Excipients (5)

[0257] 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.

[0258] Table 33

[0259]

[0260] -SEC-HPLC, protein concentration and titer experimental results are shown in Tables 34 to 37 below.

[0261] Table 34

[0262]

[0263]

[0264] Table 35

[0265]

[0266] Table 36

[0267]

[0268]

[0269] Table 37

[0270]

[0271] Based on the above experimental results, it was confirmed that the presence or absence of polysorbate 80 (PS80) had no significant effect on stability.

[0272] <Experimental Examples 3-6> Comparative Analysis of Excipients (6)

[0273] Lyophilized preparations were prepared in the same manner as in <Preparation Examples 2 and 3>. Excipient conditions are shown in Table 38 below.

[0274] Table 38

[0275]

[0276] The stability of the sGal-9 protein was evaluated using the same methods as in Experimental Example 2-1. The stability of the sGal-9 protein in the presence of 2% and 4% mannitol was compared using SEC-HPLC, IEX-HPLC, SDS-PAGE, and protein and potency assays. The results are shown in Tables 39 and 40 below.

[0277] Table 39

[0278]

[0279]

[0280] Table 40

[0281]

[0282] Therefore, compared to the use of other excipients or other buffers, the sGal-9 protein according to the present invention exhibited excellent stability in a lyophilized dosage form based on 10 mg of sGal-9 protein in 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.

[0283] Industrial Applicability

[0284] The present invention relates to a pharmaceutical preparation comprising a recombinant galectin-9 protein, which can provide a preparation with high stability by using a buffer and the like.

[0285] Sequence Listing Free Text

[0286] sequence:

[0287] Sequence Listing Number: 1

[0288] Length: 284

[0289] Sequence type: AA

[0290] Feature Position / Qualifier:

[0291] REGION, 1..284

[0292] >Note: sGal-9

[0293] Source, 1..284

[0294] Molecule type (mol_type): protein

[0295] >Source organism: synthetic construct

[0296] Amino acid residues:

[0297] MAFSGSQAPY LSPAVPFSGT IQGGLQDGLQ ITVNGTVLSS SGTRFA VNFQ TGFSGNDIAF 60

[0298] HFNPRFEDGG YVVCNTRONG SWGPEERKTH MPFQKGMPFD LCFLVQSSDF KVMVNGILFV 120

[0299] QYFHRVPFHR VDTISVNGSV QLSYISFQHP PYPMPFITTI LGGLYPSKSI LLSGTVLPSA 180

[0300] QRFHINLCSG NHIAFHLNPR FDENAVVRNT QIDNSWGSEE RSLPRKMPFV RGQSFSVWIL 240

[0301] CEAHCLKVAV DGQHLFEYYH RLRNLPTINR LEVGGDIQLT HVQT 284

[0302] Sequence Listing Number: 2

[0303] Length: 146

[0304] Sequence type: AA

[0305] Feature Position / Qualifier:

[0306] REGION, 1..146

[0307] >Note: CCRD

[0308] Source, 1..146

[0309] Molecule type (mol_type): protein

[0310] >Source organism: synthetic construct

[0311] Amino acid residues:

[0312] TPAIPPMMYP HPAYPMPFIT TILGGLYPSK SILLSGTVLP SAQRFHINLC SGNHIAFHLN 60

[0313] PRFDENAVVR NTQIDNSWGS EERSLPRKMP FVRGQSFSVW ILCEAHCLKV AVDGQHLFEY 120

[0314] YHRLRNLPTI NRLEVGGDIQLTHVQT 146

[0315] Sequence Listing Number: 3

[0316] Length: 10

[0317] Sequence type: AA

[0318] Feature Position / Qualifier:

[0319] REGION, 1..10

[0320] >Note: CCRD 1-10

[0321] Source, 1..10

[0322] Molecule type (mol_type): protein

[0323] >Source organism: synthetic construct

[0324] Amino acid residues:

[0325] TPAIPPMMYP 10.

Claims

1. A pharmaceutical preparation comprising: (1) Recombinant stable galectin-9 protein; (2) buffer; as well as (3) Stabilizer.

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

1.

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

1.

4. The pharmaceutical preparation according to 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. The pharmaceutical preparation according to claim 1, wherein The recombinant stable galectin-9 protein is 1 mg / ml to 20 mg / ml.

6. The pharmaceutical preparation according to claim 1, wherein 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.

7. The pharmaceutical preparation according to claim 1, wherein The buffer is at least one selected from the group consisting of phosphate, histidine, acetate, and combinations thereof.

8. The pharmaceutical preparation according to claim 1, wherein The buffer is histidine acetate.

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

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

5.

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

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

13. The pharmaceutical preparation according to claim 1, wherein The stabilizer consists of (i) one or more carbohydrates or sugars; and (ii) one or more amino acids or pharmaceutically acceptable salts thereof.

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

16. The pharmaceutical preparation according to 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. The pharmaceutical preparation according to claim 13, wherein The amino acid is glycine.

18. The pharmaceutical preparation according to claim 13, wherein The carbohydrate or sugar includes one or more carbohydrates or sugars, each in an amount of 1 w / v% to 10 w / v%.

19. The pharmaceutical preparation according to claim 13, wherein The carbohydrates or sugars include trehalose and mannitol, each in an amount of 1 w / v% to 5 w / v%.

20. The pharmaceutical preparation according to claim 13, wherein The amino acid or a pharmaceutically acceptable salt thereof is in an amount of 1 mg / ml to 20 mg / ml.

21. The pharmaceutical preparation according to claim 13, wherein The stabilizer further includes polysorbate.

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

23. The pharmaceutical preparation according to claim 13, wherein The pharmaceutical preparation is in liquid dosage form or lyophilized dosage form.

24. A vial filled with the pharmaceutical preparation 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 bone diseases, 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 autoimmune diseases, 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 preparation of a medicament for preventing or treating bone diseases.

29. Use of the pharmaceutical preparation according to any one of claims 1 to 23 in the preparation of a medicament for preventing or treating cancer or autoimmune diseases.

30. Use of a pharmaceutical formulation according to any one of claims 1 to 23 for preventing or treating bone diseases.

31. Use of the pharmaceutical preparation according to any one of claims 1 to 23 in preventing or treating cancer or autoimmune diseases.

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

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

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