Pharmaceutical Formulation, Bottle, Syringe, and Use of the Pharmaceutical Formulation

Abstract

The present invention relates to a stabilized formulation comprising a recombinant stabilized galectin 9 protein and, more specifically, to a pharmaceutical formulation of which stability increases as the formulation is mixed and combined with a buffer, a stabilizer, or the like.

Description

44 Pharmaceutical Formulation, Bottle, Syringe, and Use of the Pharmaceutical Formulation FIELD OF THE INVENTION

[001] The present invention relates to a stabilized pharmaceutical formulation comprising a stabilized recombinant galectin-9 protein. More specifically, it relates to a pharmaceutical formulation in which the galectin-9 protein is stably preserved by combination with a buffer and a stabilizer. FUNDAMENTALS OF THE TECHNIQUE

[002] Galectin-9 is a beta-galactoside lectin protein isolated from the embryonic mouse kidney. It consists of two carbohydrate recognition domains (CRDs) and a linker peptide region connecting them, where the N-terminal carbohydrate recognition domain (NCRD) and the C-terminal carbohydrate recognition domain (CCRD) are linked via the linker peptide. Galectin-9 is known as one of the ligands of HAVCR2 (Tim-3) and binds to Tim-3 to induce apoptosis of Tim-3-positive Th1 cells and suppress excessive Th1 responses, thus alleviating autoimmune inflammation. Furthermore, galectin-9 has been observed in several cancer types, including malignant melanoma, Hodgkin's lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, and colorectal cancer, and efforts have been made to develop variants for therapeutic applications.In this regard, Korean Patent Publication No. 10-2022-0068158 describes a pharmaceutical composition comprising a stabilized recombinant galectin-9 protein for the prevention or treatment of cancer. However, even as described, formulation development is necessary to enable technical application in the human body through administration to patients.

[003] In general, pharmaceutical formulation significantly affects the efficacy and stability of an active ingredient when applied to the body. Petition 870250089978, dated 02 / 10 / 2025, page 6 / 104 / 44 human, depending on the type and proportion of excipients used. In addition to pharmacokinetic factors, such as duration of action, drug distribution, metabolism, and excretion after administration, formulation parameters must also be carefully considered in light of packaging conditions and container systems. For example, in the case of poorly soluble drugs, administration can be done through capsule formulations; however, depending on the manufacturing method or the proportion of the composition, such formulations may present difficulties in administration, requiring extensive formulation research. Furthermore, if the stability of an antibody is low, its efficacy may be compromised due to denaturation, oxidation, aggregation, or degradation, and in some cases, these phenomena can lead to adverse effects such as toxicity.Consequently, various efforts are needed to develop pharmaceutical formulations that are stable, effective, and convenient to use.

[004] Consequently, the present inventors concluded the present invention by conducting extensive studies on formulations for the stabilization of recombinant galectin-9 protein and identifying a formulation that exhibits enhanced stability. Prior Art Documents Patent Documents

[005] (Patent document 0001) Korean patent publication no. 10-2022-0068158 DETAILED DESCRIPTION OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[006] The present invention aims to provide a pharmaceutical formulation capable of stably preserving a stabilized recombinant galectin-9 protein. WAYS TO SOLVE THE PROBLEM Petition 870250089978, dated 02 / 10 / 2025, page 7 / 104 / 44

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

[008] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein may comprise an amino acid sequence represented by SEQ ID NO: 1.

[009] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein may comprise a protein with at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 1.

[0010] In one embodiment of the present invention, the recombinant stabilized galectin-9 protein may comprise a protein in which the first N-terminal amino acid of the amino acid sequence of SEQ ID NO: 1 is deleted.

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

[0012] In one embodiment of the present invention, the buffer may be one or more selected from the group consisting of citrate, phosphate, histidine, glycine, acetate, tartrate, aspartate, lactate, gluconate, glutamate and succinate.

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

[0014] In one embodiment of the present invention, the buffer may be present at a concentration of 5 to 20 mM.

[0015] In one embodiment of the present invention, the stabilizer may comprise: (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 present invention, the carbohydrate or sugar may be selected from the group consisting of mannitol, sorbitol, Petition 870250089978, dated 02 / 10 / 2025, page 8 / 104 / 44 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 present invention, the carbohydrate or sugar can be selected from the group consisting of trehalose and mannitol.

[0018] In one embodiment of the present invention, the amino acid may be 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 in an amount of 1 to 5% w / v.

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

[0021] In one embodiment of the present invention, the stabilizer may further comprise polysorbate.

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

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

[0024] In addition, the present invention provides a pharmaceutical formulation comprising a therapeutically effective amount thereof, for use in the prevention or treatment of a bone disease.

[0025] In addition, the present invention provides a pharmaceutical formulation comprising a therapeutically effective amount thereof, for use in the prevention or treatment of cancer or an autoimmune disease. Petition 870250089978, dated 02 / 10 / 2025, page 9 / 104 / 44

[0026] In addition, the present invention provides a use in the manufacture of a pharmaceutical formulation for the prevention or treatment of a bone disease.

[0027] In addition, the present invention provides a use in the manufacture of a pharmaceutical formulation for the prevention or treatment of cancer or an autoimmune disease.

[0028] In addition, the present invention provides the use of a pharmaceutical formulation for the prevention or treatment of a bone disease.

[0029] In addition, the present invention provides the use of a pharmaceutical formulation for the prevention or treatment of cancer or an autoimmune disease.

[0030] Furthermore, the present invention provides a method for treating a bone disease, comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation.

[0031] Furthermore, the present invention provides a method for treating cancer or an autoimmune disease, comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation. EFFECTS AND ADVANTAGES OF THE INVENTION

[0032] The present invention relates to a pharmaceutical formulation comprising a recombinant galectin-9 protein and has the advantage of providing a formulation with improved stability through the use of a buffer or similar. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a diagram showing the comparative experimental results between histidine buffer and PBS buffer.

[0034] Figure 2 is a diagram showing the comparative experimental results between acetate buffer and PBS buffer.

[0035] Figure 3 is a diagram showing the results Petition 870250089978, dated 02 / 10 / 2025, page 10 / 104 / 44 comparative experimental studies between histidine acetate buffer and sodium acetate buffer.

[0036] Figure 4a is a diagram showing the results of the potency tests according to the pH of the histidine acetate buffer.

[0037] Figure 4b is a diagram showing the results of the potency tests according to the pH of the histidine acetate buffer.

[0038] Figure 4c is a diagram showing the results of the potency tests according to the pH of the histidine acetate buffer.

[0039] Figure 5 is a diagram showing the appearance according to an embodiment of the present invention (Experimental Example 3-3).

[0040] Figure 6 is a diagram showing the appearance according to an embodiment of the present invention (Experimental Example 3-6).

[0041] Figure 7 is a diagram showing the result of the SDS-PAGE analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0042] Figure 8a is a diagram showing the results of the SEC-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0043] Figure 8b is a diagram showing the results of the SEC-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0044] Figure 8c is a diagram showing the results of the SEC-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0045] Figure 8d is a diagram showing the results of the SEC-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0046] Figure 9a is a diagram showing the results of IEX-HPLC analysis according to an embodiment of the present invention (Example Petition 870250089978, dated 02 / 10 / 2025, p. 11 / 104 / 44 Experimental 3-6).

[0047] Figure 9b is a diagram showing the results of the IEX-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0048] Figure 9c is a diagram showing the results of the IEX-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0049] Figure 9d is a diagram showing the results of the IEX-HPLC analysis according to an embodiment of the present invention (Experimental Example 3-6).

[0050] Figure 10a is a diagram showing the power results according to an embodiment of the present invention (Experimental Example 3-6).

[0051] Figure 10b is a diagram showing the power results according to an embodiment of the present invention (Experimental Example 3-6). BEST WAY TO CARRY OUT THE INVENTION

[0052] Embodiments of the present invention will be described in detail below, by way of examples, so that those skilled in the art may implement the invention without undue difficulty. The embodiments of the present invention are provided to explain the invention more fully to those with common knowledge of the art. Therefore, the embodiments of the present invention may be modified in various other ways, and the scope of the present invention is not intended to be limited to the embodiments described below.

[0053] It will be understood that, throughout this specification, when a component is said to “comprise” a particular element, this does not exclude the presence of other elements unless otherwise specified, but allows for the inclusion of additional elements. Petition 870250089978, dated 02 / 10 / 2025, p. 12 / 104 / 44

[0054] The present invention relates to a pharmaceutical formulation comprising: (1) a stabilized recombinant galectin-9 protein; (2) a buffer; and (3) a stabilizer.

[0055] In the present invention, recombinant stabilized galectin-9 protein retains the carbohydrate recognition activity of wild-type galectin-9, while possessing a more stable molecular structure against proteases.

[0056] Specifically, recombinant stabilized galectin-9 protein is a recombinant protein obtained by modifying the linking region that connects the two CRDs (Carbohydrate Recognition Domains) of wild-type galectin-9, which has an NCRD-linker-CCRD structure, and the CCRD (C-terminal Carbohydrate Recognition Domain). More specifically, recombinant stabilized galectin-9 protein may comprise an amino acid sequence represented by SEQ ID NO: 1, in which the peptide of the linking region is completely deleted; an amino acid sequence (SEQ ID NO: 3) corresponding to positions 1 to 10 of the CCRD (SEQ ID NO: 2) is deleted; and alanine (Ala; A) at position 13 is replaced by proline (Pro; P).The recombinant stabilized galectin-9 protein may comprise an amino acid sequence with at least 75%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 1. Furthermore, the recombinant stabilized galectin-9 protein may also comprise amino acid sequences introduced for specific purposes, such as targeting sequences, markers, labeled residues, or sequences designed to increase the half-life or stability of the peptide.

[0057] Furthermore, the recombinant protein of the present invention can be obtained by several methods well known in the art. For example, Petition 870250089978, dated 02 / 10 / 2025, page 13 / 104 / 44 it can be produced using polynucleotide recombination and a protein expression system, synthesized in vitro by chemical synthesis, such as peptide synthesis, or prepared by a cell-free protein synthesis method.

[0058] As used herein, the term “polynucleotide” refers to a polymer of nucleotides that functions to transmit genetic information. For the purposes of the present invention, the polynucleotide encodes the recombinant protein of SEQ ID NO: 1, and may include a nucleotide sequence with at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the polynucleotide sequence encoding said recombinant protein.

[0059] As used herein, the term “sequence identity” is intended to indicate the degree of similarity to the wild-type amino acid sequence or to the polynucleotide sequence. This sequence identity comparison can be performed using well-known comparison programs in the art, and the identity between two or more sequences can be calculated as a percentage (%).

[0060] As used herein, the term “prevention” refers to any act of inhibiting or delaying the onset of a disease through the administration of the composition.

[0061] As used herein, the term “treatment” refers to any act of relieving or favorably modifying the symptoms of a disease through the administration of the composition.

[0062] As used herein, the term “pharmaceutical formulation” refers to a final dosage form that is prepared by filling, lyophilizing and / or reconstituting a bulk solution comprising the composition.

[0063] In one aspect of the present invention, the galectin-9 protein Petition 870250089978, dated 02 / 10 / 2025, p. 14 / 104 / 44 stabilized recombinant protein is a protein comprising the amino acid sequence represented by SEQ ID NO: 1.

[0064] In one aspect of the present invention, the recombinant stabilized galectin-9 protein is a protein comprising an amino acid sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 1.

[0065] In one aspect of the present invention, the recombinant stabilized galectin-9 protein comprises a deletion of the first N-terminal amino acid residue of the amino acid sequence represented by SEQ ID NO: 1.

[0066] In one aspect of the present invention, recombinant stabilized galectin-9 protein may be present in the pharmaceutical composition in an amount ranging from 0.01 to 400 mg / ml. Specifically, recombinant stabilized galectin-9 protein may be present 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 more specific aspect of the present invention, the recombinant stabilized galectin-9 protein may be present 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 further specific aspect of the present invention, recombinant stabilized galectin-9 protein is present 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.

[0067] In one aspect 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. Petition 870250089978, dated 02 / 10 / 2025, page 15 / 104 / 44 combinations thereof. In a specific aspect 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 aspect of the present invention, the buffer is histidine acetate. The inclusion of histidine in the present invention may provide beneficial effects in terms of stability and pH control.

[0068] In one aspect of the present invention, the buffer does not include a sodium salt.

[0069] In one aspect of the present invention, the buffer has a pH in the range of 4 to 7. Specifically, the buffer may have a pH of 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 buffer may have a pH of 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 buffer has a pH of 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.

[0070] In one aspect of the present invention, the buffer in the pharmaceutical formulation may be present at a concentration of 0.01 to 50 mM. Specifically, the buffer may be present at a concentration of 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 aspect of the present invention, the buffer may be present at a concentration of 0.1 to 25 mM, 0.1 to 24 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 aspect of the present invention, the buffer is present in a concentration of 4 to 20.5 mM, 4 to 20 mM, 4.5 to 20 mM, or 5 to 20 mM.

[0071] In one aspect of the present invention, the stabilizer comprises (i) at least one carbohydrate or sugar; and (ii) at least one amino acid or pharmaceutically acceptable salt thereof. According to Petition 870250089978, dated 02 / 10 / 2025, page 16 / 104 / 44 used in the present invention, the term carbohydrate or sugar is intended to encompass monosaccharides, sugar alcohols, sugar acids and sugar derivatives. Examples include, but are not limited to, glucose, fructose, sucrose, lactose, maltose, trehalose, glycerol, erythritol, arabitol, xylitol, mannitol, sorbitol, galactitol, fucitol, iditol, maltitol, lactitol, maltotriitol and maltotetraitol.

[0072] As used in the present invention, amino acid may include natural amino acids or non-natural amino acids. Examples include, but are not limited to, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, and tryptophan.

[0073] 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.

[0074] In one aspect of the present invention, the stabilizer comprises at least one carbohydrate or sugar, each present at 0.01 to 10% w / v. Specifically, the at least one carbohydrate or sugar included in the stabilizer may be present at 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 aspect of the present invention, at least one carbohydrate or sugar included in the stabilizer is present at 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.

[0075] In a specific aspect of the present invention, the stabilizer may comprise two or more carbohydrates or sugars. Specifically, when two or more carbohydrates or sugars are included, one of the carbohydrates or sugars may be present at 1 to 3% w / v, 1.1 to 3% Petition 870250089978, dated 02 / 10 / 2025, p. 17 / 104 / 44 p / v, 1.2 to 3% p / v, 1.3 to 3% p / v, 1.4 to 3% p / v, 1.5 to 3% p / v, 1.5 to 2.9% p / v, 1.5 to 2.8% p / v, 1.5 to 2.7% p / v, 1.5 to 2.6% p / v or 1.5 to 2.5% p / v. In addition, another carbohydrate or sugar may be present in 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 aspect of the present invention, the stabilizer comprises two or more carbohydrates or sugars, wherein one of the carbohydrates or sugars 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, and the other carbohydrate or sugar is present at 3.5 to 4.5% w / v, 3.55 to 4.5% w / v, 3.6 to or 3.75 to 4.25 w / v%.

[0076] In one aspect of the present invention, the carbohydrate or sugar comprises trehalose and mannitol. In one aspect of the present invention, the carbohydrate or sugar comprises trehalose and mannitol, each in an amount of 1 to 10% w / v.

[0077] In a specific aspect of the present invention, mannitol is present in an amount of 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%. More specifically, mannitol is present in amounts of 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%.

[0078] In a specific aspect of the present invention, trehalose is present in an amount of 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% Petition 870250089978, dated 02 / 10 / 2025, p. 18 / 104 / 44 p / v, 1.5 to 2.6% p / v or 1.5 to 2.5% p / v. More specifically, trehalose is present in amounts of 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%.

[0079] 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 one aspect of the present invention, the amino acid is glycine.

[0080] In one aspect of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is present in an amount of 0.01 to 20 mg / ml. Specifically, the amino acid or a pharmaceutically acceptable salt thereof is present in an amount of 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 aspect of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is present in an amount of 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 aspect of the present invention, the amino acid or a pharmaceutically acceptable salt thereof is present in an amount of 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.

[0081] In one aspect of the present invention, the stabilizer further comprises a nonionic surfactant. The nonionic surfactant may include, for example, poloxamers, polysorbates and pluronic acids, but is not Petition 870250089978, dated 02 / 10 / 2025, page 19 / 104 / 44 limited to them.

[0082] In one aspect of the present invention, the stabilizer further comprises polysorbate. The polysorbate may be polysorbate 20, polysorbate 80, or a combination thereof.

[0083] In one aspect of the present invention, the stabilizer further comprises polysorbate 20, polysorbate 80 or a combination thereof in an amount of 0.001 to 0.05% w / v.

[0084] In a specific aspect of the present invention, polysorbate 20 or polysorbate 80 is present in an amount of 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, polysorbate 20 or 80 is present in amounts of 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%.

[0085] In one aspect of the present invention, the pharmaceutical formulation has a potency of 60 to 130% relative to the reference potency. Specifically, the pharmaceutical composition has a potency of 70 to 120% relative to the reference potency.

[0086] In one aspect of the present invention, the pharmaceutical formulation has a purity (measured by SEL-HPLC) of 90% or more. Specifically, the purity is 91% or greater, 92% or greater, 93% or greater, or 94% or greater, and most specifically, the purity is 95% or greater.

[0087] In one aspect of the present invention, the pharmaceutical formulation is in liquid or lyophilized form.

[0088] In a specific aspect of the present invention, the pharmaceutical formulation is in a lyophilized form. The pharmaceutical formulation of the present invention exhibits greater stability in lyophilized form compared to the liquid form. Petition 870250089978, dated 02 / 10 / 2025, p. 20 / 104 / 44

[0089] The pharmaceutical formulation of the present invention may assume various forms depending on the specific configuration of the product and may be supplied in a pre-filled form, such as in a tube, cartridge, syringe, polymer vial or glass vial, but is not limited to these. Furthermore, the present invention may be pre-filled or otherwise processed by methods known to those with common knowledge in the field.

[0090] In one aspect of the present invention, the present invention relates to a bottle in which the pharmaceutical formulation is filled.

[0091] In one aspect of the present invention, the present invention relates to a syringe in which the pharmaceutical formulation is filled.

[0092] In one aspect of the present invention, the present invention may relate to a process for preparing a bottle filled with a final pharmaceutical formulation, wherein a bulk solution comprising a protein and an excipient is prepared in an amount of 1 / 2 by weight based on 1 part by weight of the final pharmaceutical formulation, and the prepared bulk solution is placed in a bottle with twice the volume of the final pharmaceutical formulation. Specifically, 1 mL of a bulk solution comprising 0.5 to 10 mg / mL of stabilized recombinant Galectin-9 protein, 0.5 to 5% w / v mannitol, 0.5 to 5% w / v trehalose, 0.5 to 10 mg / mL of an amino acid, and 0.0005 to 0.025% w / v polysorbate can be placed in a vial at 2 mL per vial and lyophilized to prepare a final formulation comprising 1 to 20 mg / mL of stabilized recombinant Galectin-9 protein, 1 to 10% w / v mannitol, 1 to 10% w / v trehalose, 1 to 20 mg / mL of an amino acid, and 0.001 to 0.05% w / v polysorbate.More specifically, 1 mL of a bulk solution comprising 5 to 6 mg / mL of stabilized recombinant Galectin-9 protein, 2% w / v mannitol, 1% w / v trehalose, 2.5 mg / mL of an amino acid, and 0.005% w / v polysorbate can be placed in a vial at 2 mL per vial and lyophilized to prepare a final formulation comprising 10 to 12 mg / mL of Galectin-9 protein. Petition 870250089978, dated 02 / 10 / 2025, page 21 / 104 / 44 recombinant stabilized, 4% w / v mannitol, 2% w / v trehalose, 5 mg / mL of an amino acid and 0.01% w / v polysorbate; however, the present invention is not limited to this.

[0093] The present invention also relates to a pharmaceutical formulation for the prevention or treatment of a bone disease, comprising a therapeutically effective amount of the pharmaceutical formulation.

[0094] In the present invention, bone disease may be at least one selected from the group consisting of osteoporosis, bone defects, osteomalacia, osteopenia, osteogenesis imperfecta, bone formation disorders, primary bone tumors, degenerative bone diseases, osteoarthritis, rheumatoid arthritis, bone fractures, osteonecrosis, periodontal disease, inflammatory alveolar bone resorption, inflammatory bone resorption and Paget's disease, but is not limited to them.

[0095] Furthermore, the present invention relates to a pharmaceutical formulation for the prevention of cancer or the treatment of autoimmune diseases, comprising a therapeutically effective amount of the pharmaceutical formulation.

[0096] In the present invention, cancer may be one or more selected from the group consisting of colorectal cancer, lung cancer, thyroid cancer, gastric cancer, breast cancer, brain cancer, osteosarcoma, astrocytoma, melanoma, neuroglioma, glioblastoma, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, renal cancer, gallbladder cancer, biliary tract cancer, esophageal cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, hematologic cancer, leukemia, myeloid leukemia, lymphoma, head and neck cancer, skin cancer and liver cancer, but is not limited to these.

[0097] In the present invention, the autoimmune disease may be one or more selected from the group consisting of systemic lupus erythematosus Petition 870250089978, dated 02 / 10 / 2025, page 22 / 104 / 44 (SLE), insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, photosensitive dermatoses, autoimmune arthritis, myasthenia gravis, thyroiditis, experimental autoimmune uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigus foliaceus, sympathetic ophthalmia, phacoanaphylactic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary sclerosis, chronic active hepatitis. (Hbs-negative), latent cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis / dermatomyositis and discoid LE, but is not limited to these.

[0098] In addition, examples of autoimmune diseases include, but are not limited to, 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 neuropathies, Balo's disease, Behçet's disease, pemphigus foliaceus, cardiomyopathy, Castleman's disease, infantile lipodystrophy, Chagas disease, chronic fatigue syndrome.Chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucosal pemphigoid, Crohn's disease, syndrome of, Petition 870250089978, dated 02 / 10 / 2025, page 23 / 104 / 44 Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus erythematosus, Dressler's syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, gestational herpes, hypogammaglobulinemia, hypergammaglobulinemia, thrombocytopenic purpura idiopathic (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoprotein abnormalities, inclusion body myositis,Inflammatory bowel disease, insulin-dependent diabetes mellitus (type I), interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Eaton-Lambert syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (SLE), Lyme disease, Ménière's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing polychondritis, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, pars planitis (intermediate uveitis), pemphigus,peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndrome type, Petition 870250089978, dated 02 / 10 / 2025, page 24 / 104 / 44 Types I, II, or III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone 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, Sheehan's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular related autoimmunity, stiff-person syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, purpura thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCT), uveitis, vasculitis,Vesiculobullous dermatoses, vitiligo, Waldenstrom's macroglobulinemia (WM), and Wegener's granulomatosis (granulomatosis with polyangiitis (GPA)).

[0099] Furthermore, the present invention relates to its use in the manufacture of a pharmaceutical formulation for the prevention or treatment of a bone disease.

[00100] Furthermore, the present invention relates to its use in the manufacture of a pharmaceutical formulation for the prevention or treatment of cancer or an autoimmune disease.

[00101] Furthermore, the present invention relates to the use of a pharmaceutical formulation for the prevention or treatment of a bone disease.

[00102] Furthermore, the present invention relates to the use of a pharmaceutical formulation for the prevention or treatment of cancer or an autoimmune disease.

[00103] In addition, the present invention relates to a method for Petition 870250089978, dated 02 / 10 / 2025, page 25 / 104 / 44 to treat a bone disease, comprising administering to a subject a therapeutically effective quantity of a pharmaceutical formulation.

[00104] Furthermore, the present invention relates to a method for treating cancer or an autoimmune disease, comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation.

[00105] Furthermore, the present invention relates to the use of a pharmaceutical formulation for the prevention or treatment of a bone disease.

[00106] Furthermore, the present invention relates to the use of a pharmaceutical formulation for the prevention or treatment of cancer or an autoimmune disease.

[00107] Furthermore, the present invention relates to a method for treating a bone disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation.

[00108] Furthermore, the present invention relates to a method for treating cancer or an autoimmune disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation. METHOD OF CARRYING OUT THE INVENTION

[00109] The present invention will now be described in more detail with reference to the following Examples and Experimental Examples.

[00110] It should be understood, however, that the following Examples and Experimental Examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. <Exemplo de Preparação 1> Production of Stabilized Recombinant Galectin-9 Protein (sGal-9)

[00111] An expression vector comprising a gene encoding a recombinant Galectin-9 protein stabilized with the sequence of Petition 870250089978, dated 02 / 10 / 2025, page 26 / 104 / 44 amino acids of SEQ ID NO: 1 were constructed and introduced into Escherichia coli (E. coli) by the heat shock method. The expression of the recombinant protein was induced by culturing E. coli in LB medium containing 50 μg / mL of kanamycin and adding arabinose when the optical density at 600 nm reached 0.7. Subsequently, the cells in which the expression of the recombinant protein was induced were lysed and filtered. The target protein was then captured using cation exchange and affinity chromatography, thus obtaining the stabilized recombinant Galectin-9 protein with high purity and high yield. <Exemplo de Preparação 2> Preparation of the Stabilized Formulation

[00112] A formulation was prepared using the stabilized recombinant Galectin-9 protein (hereinafter referred to as recombinant Galectin-9) obtained in Preparation Example 1. The composition of each formulation is described in the following Experimental Examples. In one example, the purified solution was subjected to ultrafiltration (UF) and diafiltration (DF) using 5 mM histidine acetate buffer (HisAce) at pH 6.0 to adjust the protein concentration to 11-15 mg / mL. Then, mannitol (2 w / v%), trehalose (1 w / v%) and glycine (2.5 mg / mL) were added and diluted to produce a final protein concentration of 5-7 mg / mL. The resulting solution was filtered through a 0.2 μm filter and placed in flasks at 2.0 mL / flask, followed by lyophilization (freeze-drying). <Exemplo Experimental 1> Assessment method for tampon<Exemplo Experimental 1-1> Appearance

[00113] The appearance of all samples was evaluated according to the standard operating procedure (SOP) method PD-DPD-LAB-001-07. The evaluation included assessing clarity, color, and visible particles, which was performed using a YB-2 light box under black and white backgrounds. <Exemplo Experimental 1-2> pH

[00114] The pH was measured using a pH meter equipped with a Petition 870250089978, dated 02 / 10 / 2025, page 27 / 104 / 44 glass electrode. The measurement was performed at least twice, and the average value was recorded. <Exemplo Experimental 1-3> Protein Concentration

[00115] Protein concentration was measured using a Thermo UV spectrophotometer. The measurement was performed according to the SOP method (PD-DPD-EQU-088-01). For all assessments, the absorbance coefficient was set as 1.0 AU χ mL χ mg1χ cm⁻¹, and each sample (2.5 μL) was measured at least twice. The average value was recorded. <Exemplo Experimental 1-4>Size Exclusion Chromatography (SECHPLC)

[00116] Size exclusion chromatography (SEC-HPLC) was performed according to the following procedure.

[00117] If the sample concentration was greater than 1.0 mg / mL, the sample was diluted with mobile phase to 1.0 mg / mL before analysis. A 50 μg portion of the sample was injected into the HPLC system for analysis. SEC-HPLC analysis was conducted using a Hitachi Chromaster CM5000 system equipped with a UV detector (detection wavelength: 280 nm) and an Agilent TSKgel 2000SWxl column (7.8 mm χ 300 mm, 5 μm). 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. <Exemplo Experimental 1-5> Protein Electrophoresis (SDS-PAGE)

[00118] SDS-PAGE analysis was performed using a heat block (DAIHAN MaXtable™10H), an electrophoresis system (Bio-Rad BR 1658033FC), and a shaker (Daihan RK-1D). Test samples were diluted 3:1 with 4x sample buffer and heated to 98°C for 5 minutes. A 12% acrylamide gel was prepared, and protein standards and test samples were loaded into separate wells. Electrophoresis was Petition 870250089978, dated 02 / 10 / 2025, page 28 / 104 / 44 performed at 100 V for 90 minutes. The gel with separated proteins was stained in CBB R-250 solution on a shaker for 30 minutes, followed by decolorization in a decolorizing solution on a shaker for 30 minutes. After completion of background decolorization, a distinct protein band was confirmed near 31 kDa for analysis. <Exemplo Experimental 1-6> Power Test Cell preparation

[00119] A 1 mL vial of Molt-4 T cells was thawed at 37°C for 1-2 minutes. The thawed cells were transferred to 10 mL of culture medium (RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin) and centrifuged at 1,300 rpm for 3 minutes 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 χ¹⁰⁵ cells / mL. The cell suspension was transferred to a 25 cm² culture flask and incubated at 37°C in a 5% CO₂ incubator solution. When the cell density reached 1 x 10⁶ cells / mL, the culture medium was replaced and the cell concentration was adjusted to maintain 2-4 x 10⁵ cells / mL. Subsequently, the doubling time and cell density were monitored and maintained appropriately. Power test

[00120] The potency assay was conducted using Molt-4 T cells exhibiting a doubling time of 24 hours and a viability of 95% over a two-week period. Cells were diluted with fresh culture medium to a density of 5.6 χ¹⁰⁴ cells / mL. Then, 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 sample was serially diluted in PBS(-) to concentrations of 300, 100, 30, 10, 3, 1, and 0 μg / mL. From each dilution, 10 μL were added to Petition 870250089978, dated 02 / 10 / 2025, page 29 / 104 / 44 wells so that the final concentrations in the medium were 30, 10, 3, 0.3, 0.1 and 0 μg / mL, respectively. After sample addition, the cells were incubated for a further 24 hours at 37°C in a 5% CO2 incubator solution. After incubation, 10 μL of WST-1 detection solution were added to each well and the plate was incubated for 2 hours at 37°C in a 5% CO2 incubator atmosphere. The optical density (OD) at 450 nm was then measured using a microplate reader. Changes in OD and LD50 values ​​were calculated using a four-parameter logistic fitting model. <Exemplo Experimental 1-7> Osmolality

[00121] Osmolality was measured using an osmometer. The accuracy of the osmometer was checked before and after the test using a Clinitrol 290 mOsm / kg reference solution. Each sample was analyzed using a 20 μL aliquot. <Exemplo Experimental 2>Liquid Formulation<Exemplo Experimental 2-1> Comparative Analysis of Buffer Systems (1)

[00122] The stability of the sGal-9 protein was evaluated under stress conditions (37°C) using various pH / buffer systems. In each experiment, the concentration of sGal-9 was fixed at 2 mg / mL. The appearance of each sample was evaluated over a period of 4 weeks according to the appearance analysis described in Experimental Example 1. The buffer conditions used in the experiment are shown in Table 1 below. Table 1] Surfactant buffer F01 20 mM Histidine (His) pH 6.0 0.02% PS80 (Polysorbate 80) F02 20 mM PBS (phosphate buffer) pH 7.4 F03 20 mM Acetate pH 5.0

[00123] The experimental results are shown in Figures 1 and 2 and TABLE 2.

[00124] Figure 1 shows a comparison between a buffer of Petition 870250089978, dated 02 / 10 / 2025, page 30 / 104 / 44 histidine and a PBS buffer. As shown in Figure 1, precipitation was observed in the PBS buffer, confirming that the protein in the histidine buffer was more stable than in the PBS buffer.

[00125] TABLE 2 and Figure 2 show a comparison between an acetate buffer and a PBS buffer. As shown in TABLE 2 and Figure 2, a difference in the number of particles was observed when measured at 25°C three days after filtration with PBS, indicating that the acetate buffer performed better than the PBS buffer.

[00126] (#1: 20 mM PBS, 50 mM NaCl, pH 7.3, 0.26 mg / mL; #2: in acetate buffer, pH 5.0, 0.62 mg / mL; #3: in acetate buffer, pH 5.3, 0.62 mg / mL; #4: #2 after filtration; #5: #3 after filtration) Table 2] Appearance Identification T0 25 C / After 3 days #1 SO, P+ SO, P+ #2 CL, P CL, P #3 CL, P CL, P #4 CL, PF CL, P(2) #5 CL, PF CL, P(>10) (* CL(=clear liquid) / P(=particle) / P(n)(=number of particles) / PF(=no particles))<Exemplo Experimental 2-2> Comparative Analysis of Buffer Systems (2) (Sodium Salts)

[00127] The stability of the sGal-9 protein was evaluated using the same method described in<Exemplo Experimental 2-1> The experiment was conducted over a two-week period and included assessments of appearance, UV absorbance, pH, SEC-HPLC, SDS-PAGE, and potency according to the procedures described in Experimental Example 1. The buffer conditions used are shown in Table 3 below. Table 3] Surfactant buffer F01 Histidine acetate 20 mM (His Ace) pH 6.0 0.04% PS80 (Polysorbate 80) F02 Sodium acetate 20 mM (Na Ace) pH 7.4 Petition 870250089978, dated 02 / 10 / 2025, page 31 / 104 / 44

[00128] Appearance, UV absorbance, pH, SEC-HPLC, SDS-PAGE and potency tests were performed at T0. In week 1, appearance, UV absorbance, pH, SEC-HPLC and SDS-PAGE analyses were performed. In week 2, appearance, UV absorbance, pH, SEC-HPLC, SDS-PAGE and potency tests were performed again.

[00129] The results of the appearance analysis are shown in Table 4 and Figure 3 below. Table 4] Identification Appearance T0 40 C 25 C 1w 2w 1w 2w F01 C, CL, PO, P++ O, P++ C, SO, P+ C, CL, P++ F02 C, CL, PF SO, PF SO, PF C, CL, PF C, CL, PF (* C=colorless / SY=slightly yellow / Y=yellow / CL=transparent liquid / SO=slightly opalescent liquid / O=opalescent liquid / FP=free of visible particles / PO=observed particles, <5) / PO+=some visible particles / PO++=many visible particles)

[00130] The results of the SEC-HPLC analysis are shown in Tables 5 and 6. [Table 5] Identification Buffer T0 25°C, 1 sem. 40°C, 1 sem. Main HMW LMW Main HMW LMW Main HMW LMW F1 His Ace 6.0 89.5 10.5 ND 78.6 21.4 ND 42.4 44.3 13.3 F2 Na Acetate 5.0 73.0 27.0 ND 51.8 46.5 ND 0 75.2 24.8 [Table 6] Identification Buffer 25°C, 2 sem. 40C,2w Principal HMW LMW Principal HMW LMW F1 His Ace 6.0 78.4 21.6 ND 0 76.8 23.2 F2 Na Acetate 5.0 43.4 54.7 1.9 0 68.8 31.2

[00131] The results of the power test are shown in Table 7 Petition 870250089978, dated 02 / 10 / 2025, page 32 / 104 / 44 Table 7] Identification Buffer Concentration (mg / mL) T0 40oC, 2 weeks 25oC, 2 weeks Potency (pg / mL) Max. (OD45 0OD630) Min. (OD45 0OD630) Potency (pg / mL) Max. (OD45 0OD630) Min. (OD45 0OD630) Potency (pg / mL) Max. (OD45 0OD630) Min. (OD45 0OD630) F1 His Ace 6.0 4.2 1.76 0.25 0.13 NA 0.27 0.27 2.32 0.26 0.14 F2 Na Acetate 5.0 4.5 2.56 0.25 0.13 NA 0.27 0.27 6.46 0.27 0.14 Standard SG 0.263 3.65 0.24 0.13 5.69 0.27 0.15 4.59 0.27 0.14

[00132] As shown in Table 4 and Figure 3, histidine acetate exhibited whitish particles or a high number of visible particles at 25°C and 40°C, while no visible particles were observed in the sodium acetate formulation. However, in terms of purity and potency, the protein in the histidine acetate buffer proved to be more stable than that in the sodium acetate buffer.

[00133] Furthermore, as shown in Tables 5 to 7, it was confirmed that the sodium salt form had a negative effect on the stability of the sGal-9 protein, indicating that the sodium salt form is unsuitable for use in the formulation. <Exemplo Experimental 2-3> Comparative Analysis of Buffer Solutions (3) (pH / Excipient)

[00134] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The evaluation was conducted over a four-week period using appearance, UV absorbance, pH, protein concentration (100 μL), SEC-HPLC, osmolality, and potency assay, as described in Experimental Example 1. Buffer conditions are shown in Table 8 below. Petition 870250089978, dated 02 / 10 / 2025, page 33 / 104 / 44 Table 8] Identification Buffer pH Concentration Sucrose Glycine PS (polysorbate 80) mg / mL % w / v mg / mL % w / v F01 Histidine acetate 20 mM (His Ace) pH 5.0 2 8 2 0.04 F02 Histidine acetate 20 mM (His Ace) pH 6.0

[00135] At T0, tests were conducted for appearance, UV absorbance, pH, protein concentration (100 μL), SEC-HPLC, and osmolality. After one week, tests were performed for UV absorbance, pH, protein concentration (100 μL), SEC-HPLC, and potency at 40 °C. After four weeks, tests were performed for UV absorbance, pH, protein concentration (100 pL), SEC-HPLC, and potency at 25 °C.

[00136] The results of the appearance, pH, protein concentration and osmolality tests are shown in Tables 9 and 10 below. [Table 9] Identification Buffer pH Concentration (mg / ml) Appearance Osmolality (mOsm / kg) T0 40°C 25°C 1 week 4 weeks F1 20 mM His-Ace 5.0 2.0 C,CL,PF C,CL,PF C,CL,PF 296 F2 6.0 C,CL,PF C,CL,PF C,CL,PF 297 (* C=colorless / CL=clear liquid / PF=no particles) Table 10] Identification Buffer pH Concentration (mg / ml) pH Concentration T0 40°C 25°C T0 40°C 25°C 1 sem. 4 sem. 1w 4 sem. F1 20 mM His-Ace 5.0 2.0 5.0 5.0 5.0 2.0 2.1 2.0 F2 6.0 6.0 6.0 6.0 1.9 1.9 2.0

[00137] The results of the SEC-HPLC analysis are shown in the Table. 11. Petition 870250089978, dated 02 / 10 / 2025, page 34 / 104 / 44 Table 11] Identification Buffer pH Concentration (mg / ml) T0 40°C, 1 week 25°C, 4 weeks %Monomer (Main) Peak HM W LM W %Monomer (Main) Peak HM W LM W %Monomer (Main) Peak HM W LM W F1 20 mM HisAce 5.0 2 95.2 4.8 ND 3.2 95.8 1.1 69.2 30.8 ND F2 6.0 92.7 7.3 ND 89.2 10.8 ND 93.0 7.0 ND

[00138] The results of the power test are shown in Tables 14 and FIGS 4a to 4c.

[00139] (B201-20210301-T0 = F1 / B201-20210302-T0 = F2) [Table 12] Result of the power test at T0. Identification Concentration (mg / ml) Potency (Kg / mL) Max. (OD450-OD630) Min. (OD450-OD630) F1 2.0 4.11 0.37 0.13 F2 2.0 4.12 0.37 0.13 Standard SG 0.263 5.37 0.35 0.15 [Table 13] Results of the power test after 1 week at 40°C. Identification Concentration (mg / ml) Potency (Kg / mL) Max. (OD450-OD630) Min. (OD450-OD630) F1 2.1 NA 0.37 0.39 F2 2.0 6.63 0.37 0.16 Standard SG 0.263 5.44 0.37 0.16 [Table 14] Results of the power test after 4 weeks at 25°C. Identification Concentration (mg / ml) Potency (Kg / mL) Max. (OD450-OD630) Min. (OD450-OD630) F1 2.0 4.88 0.37 0.14 F2 2.0 4.12 0.37 0.14 Standard SG 0.263 4.37 0.36 0.17

[00140] Based on the experimental results above, it was confirmed that the histidine buffer (pH 6.0) showed superior performance compared to the histidine buffer (pH 5.0). Furthermore, it was confirmed that the sGal-9 protein showed stability when 8% sucrose, 0.04% of Petition 870250089978, dated 02 / 10 / 2025, page 35 / 104 / 44 polysorbate 80 and 2 mg / mL of glycine were used as excipients. <Exemplo Experimental 2-4> Comparative Analysis of Buffer Solutions (4) (pH)

[00141] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The experiment was conducted according to the pH range and excipient conditions, and the buffer conditions are shown in Table 15 below. Table 15] Identification Buffer pH Sucrose Glycine PS 20 (polysorbate 20) % w / v mg / ml % w / v C1 20 mM His-Ace 5.0 8 - - C2 6.0 8 - - C3 7.0 8 - - C4 6.0 8 - 0.02 C5 6.0 8 2 0.02

[00142] The results of the SEC-HPLC analysis, protein concentration and potency assay are shown in Table 16 below. [Table 16] C1 C2 C3 C4 C5 T0 Purity: Area SEC-HPLC (%) 92.5 92.2 89.6 85.4 85.4 Protein concentration (A280 / BF) 2.0 / 2.01 2.1 / 1.78 2.1 / 1.76 2.2 / 2.24 2.2 / 2.3 Potency (µg / ml) 6.39 5.03 T4D Purity: Area SEC-HPLC (%) 35.44 / 97.1 66.83 90.17 90.14 Protein concentration (A280) 2.3 / 2.09 2.2 / 1.8 2.9 / 1.96 2.4 / 1.96 2.6 / 1.82 Potency (µg / ml) > 31.5 4.56 5.90 3.26 5.51

[00143] Based on the experimental results above, it was confirmed that the histidine acetate buffer at pH 6.0 showed superior performance when compared to buffers at other pH values. Furthermore, it was confirmed that the effects of other excipients, such as polysorbate 20 (PS 20) or glycine, were not clearly observed. Petition 870250089978, dated 02 / 10 / 2025, page 36 / 104 / 44<Exemplo Experimental 2-5> Comparative Analysis of Buffer Solutions (5) (Concentration)

[00144] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The experiment was conducted based on variations in concentration, and the buffer conditions are shown in Table 17 below. [Table 17] Identification Buffer Mannitol Sucrose PS 20 (polysorbate 20) Glycine (w / v%) (w / v%) (w / v%) (mg / ml) C1 20 mM His-Ace pH 6.0 - 8 - 5 C2 - 8 0.02 5 C3 5 - - 5 C4 10 mM His-Ace pH 6.0 - 8 - 5 C5 - 8 0.02 5 C6 30 mM His-Ace pH 6.0 - 8 - 5 C7 - 8 0.02 5 C8 5 - - 5

[00145] The results of the SEC-HPLC analysis, protein concentration, and potency assay are shown in Table 18 below. [Table 18] Identification 20 mM His-Ace / 5 mg / ml Glycine 10 mM His-Ace / 5 mg / ml Glycine 30 mM His-Ace / 5 mg / ml Glycine 8% sucrose 8% sucrose 0.02% PS20 5% Mannitol 8% sucrose 8% sucrose 0.02% PS20 8% sucrose 8% sucrose 0.02% PS20 5% Mannitol C1 C2 C3 C4 C5 C6 C7 C8 T0 Purity (%) 96.74 86.79 96.41 96.17 95.55 96.13 96.70 91.73 A280 / BF (mg / ml) 2.52 / 2.45 2.44 / 2.42 1.84 / 1.84 1.80 / 1.85 1.77 / 1.91 2.46 / 2.34 2.45 / 2.35 1.79 / 1.89 Potency (ug / ml) 0.94 0.887 1.765 0.884 0.598 0.703 0.998 0.803 T2W Purity(%) 97.30 96.23 96.37 97.79 96.71 95.60 92.19 94.25 A280 / BF (mg / ml) 2.98 / 2.28 3.30 / 2.16 2.15 / 1.68 2.01 / 1.47 2.21 / 1.53 3.00 / 2.32 3.75 / 1.88 2.47 / 1.35 Potency (ug / ml) 3.00 4.34 3.71 2.74 3.94 2.98 5.88 4.12 Petition 870250089978, dated 02 / 10 / 2025, page 37 / 104 / 44 Purity(%) 95.80 93.53 95.08 97.91 97.08 94.10 88.69 95.70 A280 / BF 3.26 / 3.93 / 2.57 / 2.13 / 2.46 / 3.42 / 4.34 / 3.08 / T3W (mg / ml) 1.79 1.69 1.18 1.34 1.26 1.65 1.42 0.97 Potency 3.172 ± 5.778 ± 3.328 1.696 1.919 1.921 3.965 0.587 ± (ug / ml) 0.471 0.455 ±0.189 ±0.361 ±0.264 ±0.310 ±1.258 0.249

[00146] Based on the experimental results above, it was confirmed that the histidine acetate buffer showed greater protein stability at concentrations of 10 mM and 20 mM compared to 30 mM, in the order of 10 mM > 20 mM > 30 mM, indicating greater stability at lower concentrations. <Exemplo Experimental 2-6> Comparative Analysis of Buffer Solutions (6)

[00147] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The buffer conditions are shown in Table 19 below. Table 19] Identification Buffer Mannitol Sucrose PS 20 (polysorbate 20) Glycine (w / v%) (w / v%) (w / v%) (mg / ml) C1 20 mM His-Ace pH 6.0 - 8 - 5 C2 - 8 0.02 5 C3 5 - - 5 C4 10 mM His-Ace pH 6.0 - 8 - 5 C5 - 8 0.02 5

[00148] The results of the SEC-HPLC analysis, protein concentration and potency assay are shown in Table 20 below. Table 20] __________________________________________________ Identification 20 mM His-Ace / 5 mg / ml Glycine 10 mM His-Ace / 5 mg / ml Glycine 8% sucrose 8% sucrose 0.02% PS20 5% Mannitol 8% sucrose 8% sucrose 0.02% PS20 C1 C2 C3 C4 C5 T0 Purity (%) 98.16 98.06 / 97.17 99.35 97.90 A280 / BF (mg / ml) 2.04 / 1.95 2.05 / 2.24 2.06 / 1.80 1.93 / 1.97 2.07 / 2.34 Potency (pg / ml) 7.642 ± 1.873 2.447 ± 0.559 3.113 ± 0.394 2.630 ± 0.473 1.248 ± 0.113 T2W Purity(%) 91.84 85.76 90.24 92.83 80.63 Petition 870250089978, dated 02 / 10 / 2025, page 38 / 104 / 44 A280 / BF (mg / ml) 2.49 / 2.07 3.53 / 1.67 2.53 / 1.64 2.23 / 1.60 2.82 / 1.61 Potency (ug / ml) 2.454 ± 0.455 5.551 ± 3.583 4.771 ± 1.287 5.084 ± 1.298 5.834 ± 1.624

[00149] Based on the experimental results above, it was confirmed that the histidine acetate buffer was more effective at 10 mM, and that greater stability was observed when sucrose was used. <Exemplo Experimental 3> Freeze-dried formulation

[00150] Based on the results obtained in Experimental Example 2, the buffer and pH were maintained at 10 mM histidine acetate (pH 6.0), and the excipients and formulations were compared. The lyophilized formulation was prepared by formulating the sGal-9 protein with each excipient, filling the formulation into 10 mL vials to contain 10 mg of sGal-9 per vial, and then subjecting it to lyophilization. Analyses were performed by reconstituting the lyophilized samples in 1.0 mL of deionized water at each time point. <Exemplo Experimental 3-1>Comparative Analysis of Excipients (1)

[00151] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The excipient conditions are shown in Table 21 below. Table 21] Identification Buffer pH Sucrose PS 80 (polysorbate 80) Glycine (w / v%) (w / v%) (mg / ml) C1 10 mM His-Ace 6.0 8 - 5 C2 6.0 8 0.01 5

[00152] The results of the SEC-HPLC analysis, protein concentration and potency assay are shown in Table 22 below. [Table 22] Identification 10 mM His-Ace pH 6.0 / 8% Sucrose, 5 mg / ml Glycine 10 mM His-Ace pH 6.0 / 8% Sucrose, 5 mg / ml Glycine / 0.01% PS80 25°C 37°C 25°C 37°C Before Purity (%) 97.94 98.23 Petition 870250089978, dated 02 / 10 / 2025, page 39 / 104 / 44 FD A280 (mg / ml) 2.26 (11.3 mg) 2.30 (11.5 mg) Potency (pg / ml) 1.18±0.07 1.71±0.16 T0 (After FD) Purity (%) 97.07 96.29 A280 (mg / ml) 8.52 9.42 Potency (pg / ml) 0.78±0.16 1.76±0.15 T2W (37C) T3W (25C) T3W(25OC) T2W(37C) T3W(25C) T2W(37C) Purity (%) 95.71 92.87 96.00 71.98 A280 (mg / ml) 8.26 8.90 9.05 9.46 Potency (pg / ml) 1.358±0.136 1.062±0.028 (Observations - 1.124, 1.346±0.217)

[00153] Based on the experimental results above, it was confirmed that purity decreased under stressful conditions (37°C) at the 2-week analysis point (T2W). Furthermore, it was confirmed that the inclusion of PS80 (0.01%) resulted in reduced stability. <Exemplo Experimental 3-2> Comparative Analysis of Excipients (2)

[00154] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The excipient conditions are shown in Table 23 below. [Table 23] Identification Buffer Sucrose Mannitol Trehalose Glycine PS 80 (w / v%) (w / v%) (w / v%) (mg / ml) (w / v%) C1 10 mM His-Ace (pH 6.0) 8 - - 5 0.01 C2 - 5 - 5 0.01 C3 - 4 2 5 0.01 C4 2 4 - 5 0.01

[00155] The results of the SEC-HPLC analysis, protein concentration, and potency assay are shown in Tables 24 and 25 below. [Table 24] Identification C1 C2 C3 C4 STD Before FD Purity (%) 96.81 99.47 96.43 96.82 97.19 Petition 870250089978, dated 02 / 10 / 2025, page 40 / 104 / 44 A280 (mg / ml) 3.23 3.21 3.21 3.26 1.85 T0 Purity (%) 97.82 97.63 97.82 97.71 96.53 A280 (mg / ml) 9.55 9.72 9.82 9.69 T0 (5C, 1 day) Purity (%) 97.52 97.4 97.54 97.56 97.69 A280 (mg / ml) 9.41 9.54 9.83 9.82 Table 25] C1 C2 C3 C4 STD Room temperature 37 C Room temperature 37 C Room temperature 37 C Room temperature 37 C Before FD Purity (%) 96.81 99.47 96.43 96.82 97.19 A280 (mg / ml) 3.23 3.21 3.21 3.26 1.85 T0 Purity (Area%) 97.82 97.63 97.82 97.71 96.53 A280 (mg / ml) 9.55 9.72 9.82 9.69 T1W (37C) Purity (%) 96.9 97.65 97.57 97.67 96.78 A280 (mg / ml) 9.54 9.75 9.70 9.66 T2W (25C) Purity (%) 98.09 97.94 98.03 97.71 97.06 A280 (mg / ml) 9.74 9.65 9.91 9.90

[00156] Based on the experimental results above, it was confirmed that no changes were observed in each experimental parameter (SECHPLC, UV, and PAGE) over 24 hours. Furthermore, it was confirmed that the formulation containing mannitol showed stability. <Exemplo Experimental 3-3> Comparative Analysis of Excipients (3)

[00157] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The excipient conditions are shown in Table 26 below. Petition 870250089978, dated 02 / 10 / 2025, page 41 / 104 / 44 Table 26] Identification Buffer Mannitol Trehalose Glycine PS80 (w / v%) (w / v%) (mg / ml) (w / v%) C1 10 mM HisAce pH 6.0 5 - 5 - C2 4 2 5 0.01 C3 4 2 5 - C4 5 5 - -

[00158] The results of the SEC-HPLC analysis, protein concentration, and potency assay are shown in Tables 27 and 28 below. [Table 27] Identification C1 C2 C3 C4 STD Before FD Purity (%) 97.48 95.95 98.20 98.21 97.36 A280 (mg / ml) 3.23 3.21 3.21 3.26 Potency (µg / ml) 1.91±0.18 2.05±0.04 2.16±0.143 1.860±0.29 1.59±0.45 T0 Purity (%) 97.10 96.66 96.49 96.82 A280 (mg / ml) 10.09 9.85 9.88 9.27 Potency (µg / ml) 2.274±0.300 2.812±0.421 2.446±0.216 3.366±0.448 3.059±0.46 Table 28] C1 C2 C3 C4 STD T4W Room temperature (25°C) Purity (%) 96.20 95.97 95.63 96.05 A280 (mg / ml) 1.542 1.445 1.367 1.389 Potency (g / ml) 3.286±0.324 2.450±0.181 2.447±0.714 3.241±0.234 3.059±0.46 T4W (37°C) Purity (%) 94.14 96.24 95.63 96.63 A280 (mg / ml) 1.533 1.427 1.380 1.308 Potency (g / ml) 3.792±0.29 (63% / standard) 3.063±0.07 (77% / standard) 3.451±0.33 (69% / standard) 2.42±0.26 (98% / standard) 2.37±0.29

[00159] Based on the experimental results above, it was confirmed that the formulation showed stability when trehalose was included. <Exemplo Experimental 3-4> Comparative Analysis of Excipients (4)

[00160] The stability of the sGal-9 protein was evaluated in the same way Petition 870250089978, dated 02 / 10 / 2025, page 42 / 104 / 44 in the manner described in<Exemplo Experimental 2-1> The excipient conditions are shown in Table 29 below. [Table 29] Identification Buffer Mannitol Trehalose Glycine PS80 Dithiothreitol (DTT) (w / v%) (w / v%) (mg / ml) (w / v%) (mM) C1 10 mM HisAce pH 6.0 4 2 5 - C2 4 2 5 0.01 C3 4 2 5 0.01 1.0 C4 4 2 5 - 1.0 C5 2 4 5 - -

[00161] The results of the SEC-HPLC analysis, protein concentration and potency assay are shown in Tables 30 to 32 below. [Table 30] ___________________________________________________ C1 C2 C3 C4 C5 STD Before FD Purity (%) 97.59 96.92 96.44 96.94 97.40 95.91 A280 (mg / ml) 1.518 1.718 1.725 1.53 1.587 Potency (mg / ml) 1.689±0.87 2.476±1.133 2.773±0.146 1.515±5.469 2.134±0.15 3.963 After FD (T0) Purity (%) 97.08 97.14 96.28 96.37 96.72 96.46 A280 (mg / ml) 1.296 1.442 1.418 1,353 1,347 Potency (g / ml) 2,862±0.09 2,967±0.19 2,624±0.11 1,921±0.04 3,244±0.19 2,172±0.06 3,077±0.22 Table 31] C1 C2 C3 C4 C5 STD Room temperature (25°C) Purity (%) 96.46 96.78 96.31 96.42 96.49 95.63 A280 (mg / ml) 1.37 1.48 1.49 1.38 1.38 Potency (g / ml) 2.06±0.27 2.60±0.47 2.75±1.39 2.23±0.58 1.74±0.36 2.47 37°C Purity (%) 96.43 96.64 96.14 95.74 96.28 A280 (mg / ml) 1.37 1.47 1.47 1.34 1.30 Potency (ug / ml) 5.84±0.71 6.85±0.90 6.02±1.18 5.01±1.39 2.17±0.58 5.60 Petition 870250089978, dated 02 / 10 / 2025, page 43 / 104 / 44 Table 32] C1 C2 C3 C4 C5 STD Low temperature (5^C) Purity (%) 96.75 96.8 96.02 96.37 96.75 A280 (mg / ml) 9.279 10.111 10.122 9.415 9.372 1.8 Potency (ug / ml) 5.261±2.2 3.358±3.19 1.762±0.37 2.227±0.86 2.789±1.2 1.621±1.16 Room temperature (25C) Purity (%) 96.44 96.75 96.16 96.44 96.68 A280 (mg / ml) 9.284 9.362 10,227 10.241 9.423 1.8 Potency (ug / ml) 2.911±0.04 2.732±0.17 2.897±0.27 3.746±1.3 2.789±1.2 2.106±0.35 37 C Purity (%) 95.07 96.27 95.7 95.56 95.91 A280 (mg / ml) 9.29 9.97 10.11 9.34 9.32 1.8 Potency (ug / ml) 4.337±1.33 5.573±1.12 4.258±1.83 2.978±1.77 4.218±1.04 1.484±0.25

[00162] Based on the experimental results above, it was confirmed that there were no significant differences in stability between the candidate excipients. <Exemplo Experimental 3-5>Comparative Analysis of Excipients (5)

[00163] The stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The excipient conditions are shown in Table 33 below. Table 33] Identification Buffer Mannitol Trehalose Glycine PS80 (w / v%) (w / v%) (mg / ml) (w / v%) C1 10mM HisAce pH6.0 4 2 5 - C2 4 2 5 0.01

[00164] The results of the SEC-HPLC analysis, protein concentration and potency assay are shown in Tables 34 to 37 below. Table 34] C1 C2 STD Before FD Purity (%) 96.48 96.60 89.33 A280 (mg / ml) 11.0 11.4 1.9 Potency (ug / ml) 1.94±0.15 2.287±0.506 1.848±0.255 After FD (T0) Purity (%) 95.57 96.16 A280 (mg / ml) 10.8 10.6 Petition 870250089978, dated 02 / 10 / 2025, page 44 / 104 / 44 Power Ug / ml) 1.764±0.173 1.142±0.381 Table 35] C1 C2 STD (1.0 mg / ml) T1M (25°C) Purity (%) 97.4 98.08 97.43 A280 (mg / ml) 10.79 10.86 1.0 Potency (µg / ml) 2.179±0.181 (standard x 127.7%) 3.015±0.041 (standard x 92.27%) 2.782±0.412, 1.723±0.117 T1M (37°C) Purity (%) 97.88 97.51 97.43 A280 (mg / ml) 10.88 11.13 1.0 Potency (µg / ml) 1.324±0.127 (standard x 120.4%) 1.858 ± 0.142 (standard x 96.5%) Table 36] C1 C2 STD (1.8 mg / ml) T2M (5OC) Purity (%) 97.32 97.52 98.72 A280 (mg / ml) 10.82 10.92 1.8 Potency Ug / ml) 2.982±1.34 (74.08%) 2.229±0.62 (99.1%) 2.209±0.21 2.762±0.35 T2M (25°C) Purity (%) 96.6 96.51 98.72 A280 (mg / ml) 10.88 10.94 Potency Ug / ml) 2.766±0.93 (99.86%) 3.048±0.78 (90.62%) T2M (37°C) Purity (%) 97.07 96.67 A280 (mg / ml) 10.86 10.83 Power Ug / ml) 3.641±2.66 (75.8%) 3.609±0.37 (76.5%) Table 37] C1 C2 STD (0.9 mg / ml) T3M Purity (%) 97.89 97.57 (25°C) A280 (mg / ml) 10.75 10.59 Petition 870250089978, dated 02 / 10 / 2025, page 45 / 104 / 44 Potency (pg / ml) 1.512±0.41 (standard difference of 157.7%) 1.776±0.15 (standard difference of 134.3%) 2.385±0.26 T3M (37°C) Purity (%) 97.57 96.83 A280 (mg / ml) 10.64 10.48 Potency (pg / ml) 2.219±0.08 (stdx107.5%) 1.871±0.44 (stdx127.5%)

[00165] Based on the experimental results above, it was confirmed that the presence or absence of PS80 did not result in any significant difference in stability. <Exemplo Experimental 3-6> Comparative Analysis of Excipients (6)

[00166] The lyophilized formulation was prepared in the same manner described in<Exemplos de Preparação 2 e 3> The excipient conditions are shown in Table 38 below. Table 38] Identification Buffer Mannitol Trehalose Glycine PS80 (w / v%) (w / v%) (mg / ml) (w / v%) C1 10mM HisAce pH6.0 4 2 5 0.01 C2 2 2 5 0.01

[00167] In addition, the stability of the sGal-9 protein was evaluated in the same manner described in<Exemplo Experimental 2-1> The aim of the analysis, which included SEC-HPLC, IEX-HPLC, SDS-PAGE, protein concentration and potency assays, was to compare the stability of the sGal-9 protein between 2% and 4% mannitol formulations. The test results are shown in Tables 39 and 40 below. Table 39] Analysis Items SEC IEX A280 (mg / ml) Potency (ug / ml) Area % Area % Before FD C1 97.73 96.88 10.78 1.277±0.102(87.16%), 1.075±0.038(96.59%) C2 97.83 97.04 10.73 1.26±0.051(113.2%), 0.982±0.076(88.23%) After FD (10 / 7) C1 97.47 96.90 10.56 (97.96%) 0.984±0.051(89.05%), 0.992±0.068(89.77%) C2 96.87 96.88 9.95 (92.73%) 1.195±0.064 (108.1%), 1.165±0.018 (104.5%) Petition 870250089978, dated 02 / 10 / 2025, page 46 / 104 / 44 Wstd B8 (1mg / ml) 97.73 96.07 1.113±0.034 1.105±0.096 Table 40] Analysis Items SEC IEX A280 (mg / ml) Potency (ug / ml) Area % Area % 37 C T1M (11 / 7) C1 97.10 99.80 10.26 1.547±0.192 (standard x 117.1%) C2 97.23 99.83 9.89 1.365±0.027 (standard x 132.7%) 37 C T2M (12 / 7) C1 97.21 98.65 10.2 (96.59%) 1.845±0.013 (standard x 72.9%) C2 97.44 98.87 9.7 (97.49%) 1.623±0.050 (standard x 82.87%) 5 C T3M (1 / 10) C1 97.40 99.49 10.33 (97.82%) 1.474±0.03 (standard x 100%) C2 97.28 99.50 9.72 (97.69%) 1.23±0.10 (standard x 116.3%) 25 C T3M (1 / 10) C1 98.10 99.55 10.41 (92.05%) 1.267±0.27 (standard x 112.1%) C2 98.17 99.50 9.91 (99.60%) 1.42±0.12 (standard x 105%) Wstd B8 (1.8mg / ml) 95.19 97.73 98.14 96.07 1.8165 1.811±0.069 1.345±0.048

[00168] Thus, it was confirmed that the sGal-9 protein according to the present invention exhibited 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. INDUSTRIAL APPLICABILITY

[00169] The present invention relates to a pharmaceutical formulation. which comprises a recombinant Galectin-9 protein and can provide a formulation with enhanced stability through the use of a buffer and other excipients. FREE TEXT OF SEQUENCE LISTING

[00170] Sequence: Sequence Identification Number: 1

[00171] Length: 284

[00172] Sequence Type: AA

[00173] Resource Location / Qualifier: Petition 870250089978, dated 02 / 10 / 2025, page 47 / 104 / 44 - REGION, 1..284 > note, sGal-9 - source, 1..284 > mol_type, protein > organism, synthetic construction

[00174] Waste: MAFSGSQAPY LSPAVPFSGT IQGGLQDGLQ ITVNGTVLSS SGTRFAVNFQ TGFSGNDIAF 60 HFNPRFEDGG YVVCNTRQNG SWGPEERKTH MPFQKGMPFD LCFLVQSSDF KVMVNGILFV 120 QYFHRVPFHR VDTISVNGSV QLSYISFQHP PYPMPFITTI LGGLYPSKSI LLSGTVLPSA 180 QRFHINLCSG NHIAFHLNPR FDENAVVRNT QIDNSWGSEE RSLPRKMPFV RGQSFSVWIL 240 CEAHCLKVAV DGQHLFEYYH RLRNLPTINR LEVGGDIQLT HVQT 284

[00175] Sequence Identification Number: 2

[00176] Length: 146

[00177] Sequence type: AA

[00178] Resource Location / Qualifier: - REGION, 1..146 > note, CCRD - source, 1..146 > mol_type, protein > organism, synthetic construction

[00179] Waste: TPAIPPMMYP HPAYPMPFIT TILGGLYPSK SILLSGTVLP SAQRFHINLC SGNHIAFHLN 60 PRFDENAVVR NTQIDNSWGS EERSLPRKMP FVRGQSFSVW Petition 870250089978, dated 02 / 10 / 2025, page 48 / 104 / 44 ILCEAHCLKV AVDGQHLFEY 120 YHRLRNLPTI NRLEVGGDIQ LTHVQT 146

[00180] Sequence Identification Number: 3

[00181] Length: 10

[00182] Sequence Type: AA

[00183] Resource Location / Qualifier: - REGION, 1..10 > note, CCRD 1-10 - source, 1..10 > mol_type, protein > organism, synthetic construct

[00184] Residues: TPAIPPMMYP 10 Petition 870250089978, dated 02 / 10 / 2025, page 49 / 104

Claims

1 / 4 CLAIMS 1. Pharmaceutical formulation, characterized in that it comprises: (1) a stabilized recombinant galectin-9 protein; (2) a buffer; and (3) a stabilizer.

2. Pharmaceutical formulation according to claim 1, characterized in that the recombinant stabilized galectin-9 protein comprises an amino acid sequence represented by SEQ ID NO:

1.

3. Pharmaceutical formulation according to claim 1, characterized in that the recombinant stabilized galectin-9 protein comprises a protein with at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

1.

4. Pharmaceutical formulation according to claim 1, characterized in that the recombinant stabilized galectin-9 protein comprises a deletion of the first N-terminal amino acid residue of SEQ ID NO:

1.

5. Pharmaceutical formulation according to claim 1, characterized in that the recombinant stabilized galectin-9 protein is present at a concentration of 1 to 20 mg / mL. 6.Pharmaceutical formulation according to claim 1, characterized in that 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.

7. Pharmaceutical formulation according to claim 1, characterized in that the buffer is one or more selected from the group consisting of phosphate, histidine, acetate and combinations thereof.

8. Pharmaceutical formulation according to claim 1, characterized in that the buffer comprises histidine acetate.

9. Pharmaceutical formulation according to claim 1, characterized in that the buffer has a pH of 4.5 to 7.

10. Pharmaceutical formulation according to claim 1, characterized in that the buffer has a pH of 5.5 to 6.

5. 11.Pharmaceutical formulation according to claim 1, characterized in that the buffer is present in a concentration of 5 to 20 mM.

12. Pharmaceutical formulation according to claim 1, characterized in that the buffer is present in a concentration of 5 to 15 mM.

13. Pharmaceutical formulation according to claim 1, characterized in that the stabilizer comprises: (i) at least one carbohydrate or sugar; and (ii) at least one amino acid or a pharmaceutically acceptable salt thereof.

14. Pharmaceutical formulation according to claim 13, characterized in that 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.Pharmaceutical formulation according to claim 13, characterized in that the carbohydrate or sugar is selected from the group consisting of trehalose and mannitol.

16. Pharmaceutical formulation according to claim 13, characterized in that 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. Pharmaceutical formulation according to claim 13, characterized in that the amino acid is glycine.

18. Pharmaceutical formulation according to claim 13, characterized in that each carbohydrate or sugar is present in an amount of 1 to 10% w / v. 19.Pharmaceutical formulation according to claim 13, characterized in that the carbohydrate or sugar comprises trehalose and mannitol, each in an amount of 1 to 5% w / v.

20. Pharmaceutical formulation according to claim 13, characterized in that the amino acid or pharmaceutically acceptable salt thereof is present in an amount of 1 to 20 mg / mL.

21. Pharmaceutical formulation according to claim 13, characterized in that the stabilizer further comprises polysorbate.

22. Pharmaceutical formulation according to claim 13, characterized in that the stabilizer further comprises 0.001 to 0.05% w / v of polysorbate 20, polysorbate 80 or a combination thereof.

23. Pharmaceutical formulation according to claim 13, characterized in that the pharmaceutical formulation is in a liquid or lyophilized dosage form. 24.Bottle, characterized by being pre-filled with the pharmaceutical formulation as defined in any one of claims 1 to 23.

25. Syringe, characterized by being pre-filled with the pharmaceutical formulation as defined in any one of claims 1 to 23.

26. Pharmaceutical formulation according to any one of claims 1 to 23, characterized by comprising a therapeutically effective amount thereof, for use in the prevention or treatment of a bone disease.

27. Pharmaceutical formulation according to any one of claims 1 to 23, characterized by comprising a therapeutically effective amount thereof, for use in the prevention or treatment of cancer or an autoimmune disease. 28.Use of the pharmaceutical formulation as defined in any one of claims 1 to 23, characterized by being for the manufacture of a medicament for the prevention or treatment of a bone disease.

29. Use of the pharmaceutical formulation as defined in any one of claims 1 to 23, characterized by being for the manufacture of a medicament for the prevention or treatment of cancer or an autoimmune disease.

30. Use of the pharmaceutical formulation as defined in any one of claims 1 to 23, characterized by being for the prevention or treatment of a bone disease.

31. Use of the pharmaceutical formulation as defined in any one of claims 1 to 23, characterized by being for the prevention or treatment of cancer or an autoimmune disease. Petition 870250089978, dated 02 / 10 / 2025, pp. 53 / 104.

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