Lyophilized formulations comprising fusion protein comprising alpha-galactosidase a

Abstract

The present invention relates to: a lyophilized preparation comprising a fusion protein of alpha-galactosidase A, which not only has storage stability by comprising a composition that provides structural stability to the fusion protein of alpha-galactosidase A, but also has high storage stability by providing structural stability to the fusion protein of alpha-galactosidase A; and a method for preparing the lyophilized preparation of a fusion protein of alpha-galactosidase A. In addition, the fusion protein has excellent stability despite containing a high concentration of the fusion protein.

Description

Technical Field

[0001] The present invention relates to a freeze-dried preparation of a fusion protein containing alpha-galactosidase A, and a preparation method and application thereof. Background Art

[0002] Lysosomes are intracellular organelles involved in the degradation of proteins, various lipids (e.g., glycolipids and cholesterol), and carbohydrates, and the recycling of degradation products as major components of new proteins, membrane components, and other molecules. Diseases associated with their function include lysosomal storage diseases (LSDs).

[0003] Lysosomal storage diseases (LSDs) are diseases caused by genetic defects that encode enzymes that degrade glycolipid or polysaccharide wastes in lysosomes, and the biological activity of lysosomal enzymes is significantly reduced or absent in the cells and tissues of individuals with lysosomal storage diseases. The lack of this degradative enzyme causes a large accumulation of substances, leading to problems with cell function.

[0004] Fabry disease is considered a lysosomal storage disease, a hereditary glycolipid (sphingolipid) metabolic disorder caused by a defect or insufficient activity of the hydrolase α-galactosidase A present in the lysosome. Fabry disease, as a representative disease of α-galactosidase A deficiency, is a disease associated with the X chromosome, and typical Fabry disease patients generally have less than 1% α-galactosidase A activity compared to normal people and have a variety of symptoms, including severe pain in the limbs (acral paresthesia), hypohidrosis, corneal and lens changes, skin lesions (angiokeratoma), renal failure, cardiovascular disease, lung failure, neurological symptoms and stroke.

[0005] Fabry disease causes the gradual accumulation of globotriaosylceramide (Gb3) in most tissues of the body. The accumulation of Gb3 is mainly present in the vascular endothelium. This gradual accumulation of Gb3 in the vascular endothelium can lead to ischemia and infarction in organs such as the kidneys, heart or brain.

[0006] Enzyme replacement therapy (ERT), a representative method for treating lysosomal storage diseases, has been extensively studied (Frances M. Platt et al., J Cell Biol. 2012 November 26; 199 (5): 723-34). In particular, since lysosomal storage diseases are caused by genetic defects in specific enzymes, therapies that replace the defective enzymes are of vital importance. Enzyme replacement therapy is a standard therapy for lysosomal storage diseases, and can have the effect of alleviating existing symptoms or delaying disease progression by replacing the defective enzyme. Therefore, various preparations containing α-galactosidase A for the prevention and treatment of α-galactosidase A deficiency have been studied.

[0007] However, in the case of a preparation, in order to obtain stability, the composition of the preparation varies significantly depending on the structure, physicochemical properties, dosage, etc. of the active ingredient (e.g., protein drug), and therefore it is crucial to develop a specific composition suitable for the active ingredient, and the development of preparations containing α-galactosidase A is insufficient. In particular, in the case of a lyophilized preparation containing a high concentration of α-galactosidase A fusion protein, the storage stability is greatly deteriorated due to problems such as protein precipitation, and therefore conventional lyophilized preparations contain α-galactosidase A fusion protein at a concentration of 5 mg / mL or less. However, it is necessary to develop a highly stable preparation containing a high concentration of α-galactosidase A fusion protein to improve efficacy. Summary of the invention

[0008] Technical issues

[0009] The development of stable lyophilized preparations suitable for long-term storage and having α-galactosidase A activity for a long time is insufficient, and lyophilized preparations containing high concentrations of α-galactosidase A fusion proteins have not yet been developed, so there is still a need to develop lyophilized preparations of novel compositions containing α-galactosidase A fusion proteins.

[0010] Solutions to the Problem

[0011] The object of the present invention is to provide a lyophilized preparation containing α-galactosidase A fusion protein.

[0012] Another object of the present invention is to provide a method for preparing the lyophilized preparation.

[0013] Another object of the present invention is to provide a method for reconstituting a lyophilized formulation.

[0014] Beneficial effects of the invention

[0015] The lyophilized preparation according to the present invention not only has storage stability by comprising a composition that provides structural stability to the α-galactosidase A fusion protein, but also has excellent stability despite comprising a high concentration of the fusion protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of comparison of the cake appearance of the lyophilized preparations according to the addition of amino acids are shown.

[0017] Figure 2 Comparative results of the recovery of native forms of lyophilized formulations according to the addition of amino acids are shown.

[0018] Figure 3 The results of comparison of the cake appearance of the lyophilized formulations according to the changes in amino acid concentration are shown.

[0019] Figure 4 Comparative results of the formation of soluble aggregates in lyophilized formulations according to changes in amino acid concentration before and after lyophilization are shown.

[0020] Figure 5 Comparative results of the recovery of native forms of lyophilized preparations according to changes in amino acid concentrations are shown.

[0021] Figure 6 Chromatogram of the reconstituted lyophilized formulation used to identify soluble aggregates (HMW1) and higher order aggregates (HMW2) formed in the lyophilized formulation based on the change in amino acid concentration. DETAILED DESCRIPTION

[0022] One aspect of the present invention provides a lyophilized formulation comprising an α-galactosidase A fusion protein.

[0023] In a specific embodiment, the lyophilized formulation comprises a mixture prepared by lyophilizing an aqueous solution comprising: a fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region; a buffer; and amino acids.

[0024] In another specific embodiment, the lyophilized preparation is characterized in that it comprises a mixture prepared by lyophilizing an aqueous solution, wherein the aqueous solution comprises a fusion protein at a concentration of 5 mg / mL to 50 mg / mL; a buffer at a pH of 5.5 to 7.0; and an amino acid at a concentration of 0.5% (w / v) to 4.0% (w / v).

[0025] The lyophilized preparation according to any one of the specific embodiments is characterized in that it comprises a mixture prepared by lyophilizing an aqueous solution, wherein the aqueous solution comprises 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; 50 mM to 300 mM sodium chloride; and 0.5% (w / v) to 4.0% (w / v) serine.

[0026] The lyophilized preparation according to any one of the specific embodiments is characterized in that it comprises a mixture prepared by lyophilizing an aqueous solution, wherein the aqueous solution comprises 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; 0.002% (w / v) to 0.1% (w / v) of polysorbate 20; 50 mM to 200 mM of sodium chloride; and 1.0% (w / v) to 4.0% (w / v) of serine.

[0027] The lyophilized preparation according to any one of the specific embodiments is characterized in that it comprises a mixture prepared by lyophilizing an aqueous solution, the aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0, the buffer containing 1 mM to 30 mM histidine; 0.002% (w / v) to 0.1% (w / v) polysorbate 20; 50 mM to 200 mM sodium chloride; and 1.0% (w / v) to 4.0% (w / v) serine.

[0028] The lyophilized preparation according to any one of the specific embodiments is characterized in that it comprises a mixture prepared by lyophilizing an aqueous solution, the aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0, the buffer containing 10 mM to 30 mM histidine; 0.002% (w / v) to 0.1% (w / v) polysorbate 20; 50 mM to 200 mM sodium chloride; 0.5% (w / v) to 4.0% (w / v) sugar or sugar alcohol; and 0.5% (w / v) to 4.0% (w / v) serine.

[0029] The lyophilized preparation according to any one of the embodiments is characterized in that the buffer comprises histidine or a salt thereof, citric acid or a salt thereof, acetic acid or a salt thereof, phosphoric acid or a salt thereof, or any combination thereof.

[0030] The lyophilized formulation according to any of the embodiments is characterized in that the buffer comprises 1 mM to 50 mM histidine.

[0031] The lyophilized formulation according to any one of the embodiments is characterized in that the amino acid is selected from the group consisting of serine, arginine, threonine, glutamine, glycine, alanine, and any combination thereof.

[0032] The lyophilized preparation according to any one of the embodiments is characterized in that the aqueous solution further comprises 0.5% (w / v) to 4.0% (w / v) of sugar.

[0033] The lyophilized preparation according to any one of the embodiments is characterized in that the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, trehalose, or any combination thereof.

[0034] The lyophilized preparation according to any one of the embodiments is characterized in that the aqueous solution further comprises 0.5% (w / v) to 4.0% (w / v) of sugar alcohol.

[0035] The lyophilized preparation according to any one of the embodiments is characterized in that the sugar alcohol is mannitol, sorbitol, or any combination thereof.

[0036] The lyophilized preparation according to any one of the embodiments is characterized in that the aqueous solution further comprises 0.002% (w / v) to 0.1% (w / v) of a nonionic surfactant.

[0037] The lyophilized formulation according to any one of the embodiments is characterized in that the nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and any combination thereof.

[0038] The lyophilized preparation according to any one of the embodiments is characterized in that the aqueous solution further comprises 5 mM to 300 mM of an isotonic agent.

[0039] The lyophilized formulation according to any one of the embodiments is characterized in that the isotonic agent is sodium chloride.

[0040] The lyophilized preparation according to any one of the embodiments is characterized in that the concentration of sodium chloride contained in the aqueous solution is 50 mM to 200 mM.

[0041] The lyophilized preparation according to any one of the embodiments is characterized in that the α-galactosidase A comprises the amino acid sequence SEQ ID NO:1.

[0042] The lyophilized formulation according to any one of the embodiments is characterized in that the immunoglobulin Fc region comprises the amino acid sequence SEQ ID NO:3.

[0043] The lyophilized formulation according to any one of the embodiments is characterized in that the α-galactosidase A fusion protein comprises the amino acid sequence SEQ ID NO:4.

[0044] The lyophilized formulation according to any one of the embodiments is characterized in that the immunoglobulin Fc region is derived from IgG4.

[0045] The lyophilized formulation according to any one of the embodiments is characterized in that the immunoglobulin Fc region is aglycosylated.

[0046] The lyophilized preparation according to any one of the embodiments is characterized in that the fusion protein has a structure in which two α-galactosidase A molecules are linked to each monomer of the immunoglobulin Fc region in the form of a dimer.

[0047] The lyophilized preparation according to any one of the embodiments is characterized in that the lyophilized preparation is used for preventing or treating Fabry disease.

[0048] Another aspect of the present invention provides a method for preparing the lyophilized preparation.

[0049] In a specific embodiment, the method for preparing the lyophilized preparation is characterized by comprising lyophilizing an aqueous solution comprising an α-galactosidase A fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region; a buffer; and amino acids.

[0050] In another specific embodiment, the method for preparing the lyophilized preparation is characterized by further comprising mixing the aqueous solution with an isotonicity agent, a sugar, a sugar alcohol, a nonionic surfactant, a preservative, or any combination thereof.

[0051] Another aspect of the present invention provides a method for reconstitution of the lyophilized formulation, the method comprising adding a reconstitution solution to the lyophilized formulation.

[0052] In a specific embodiment, the reconstitution solution is distilled water.

[0053] In another specific embodiment, the reconstituted formulation prepared by the method comprises the α-galactosidase A fusion protein at a concentration of 10 mg / mL to 100 mg / mL.

[0054] Mode for Carrying Out the Invention

[0055] Hereinafter, the present invention will be described in more detail.

[0056] At the same time, each of the descriptions and embodiments disclosed herein can be applied to describe different descriptions and embodiments herein. That is to say, all combinations of various factors disclosed herein belong to the scope of the present invention. In addition, the scope of the present invention should not be limited by the detailed description provided below.

[0057] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention. Such equivalents are intended to be encompassed within the scope of the following claims.

[0058] Throughout the specification, not only the conventional single-letter and three-letter codes of naturally occurring amino acids are used, but also the three-letter codes generally allowed for other amino acids, such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine) and α-methyl-glutamic acid. In addition, the amino acids mentioned herein are abbreviated as follows according to the nomenclature of IUPAC-IUB.

[0059] Alanine Ala, A Arginine Arg, R

[0060] Asparagine Asn, N Aspartic acid Asp, D

[0061] Cysteine ​​Cys, C Glutamate Glu, E

[0062] Glutamine Gln, Q Glycine Gly, G

[0063] Histidine His, H Isoleucine Ile, I

[0064] Leucine Leu, L Lysine Lys, K

[0065] Methionine Met, M Phenylalanine Phe, F

[0066] Proline (Pro, P) Serine (Ser, S)

[0067] Threonine Thr, T Tryptophan Trp, W

[0068] Tyrosine Tyr, Y Valine Val, V

[0069] One aspect of the present invention provides a lyophilized preparation comprising a fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region.

[0070] In particular, the present invention relates to a lyophilized preparation comprising a mixture prepared by lyophilizing an aqueous solution containing a pharmacologically effective amount of α-galactosidase A-immunoglobulin Fc region fusion protein, a buffer and amino acids. In addition, the aqueous solution may further contain an isotonic agent, a sugar, a sugar alcohol, a nonionic surfactant, a preservative, or any combination thereof, but is not limited thereto.

[0071] As used herein, the term "lyophilized preparation" refers to a drug prepared by lyophilization. In particular, the α-galactosidase A fusion protein can be lyophilized together with a substance (e.g., an excipient) for stabilizing the fusion protein and exist in a solid state. The components contained in the lyophilized preparation together with the α-galactosidase A fusion protein having the pharmacological efficacy of the lyophilized preparation can be mixed with a stabilizer. As used herein, the term "stabilizer" refers to a substance that stably maintains the components (e.g., active ingredients) of the preparation over a certain period of time.

[0072] In the present invention, a lyophilized preparation is a concept comprising a lyophilized substance. The lyophilized preparation is prepared by freezing a preformulation and drying the resultant, and the preformulation comprises both a pre-lyophilized preparation containing a stabilizer for stabilizing α-galactosidase A fusion protein and an α-galactosidase A fusion protein. In the present invention, the lyophilized preparation of α-galactosidase A fusion protein may contain a therapeutically effective amount of α-galactosidase A fusion protein, and the therapeutically effective amount of α-galactosidase A fusion protein may be contained in a disposable container or a multiple-use container, but is not limited thereto. The lyophilized preparation of the present invention has a composition capable of stabilizing α-galactosidase A fusion protein during the lyophilization process, and even when the stored lyophilized preparation is reconstituted, the stability of the preparation can be maintained. Specifically, the lyophilized preparation of the present invention has the effect of maintaining stability even when a high concentration of α-galactosidase A fusion protein of 5 mg / mL to 50 mg / mL is contained therein.

[0073] Furthermore, the lyophilized preparation containing the α-galactosidase A fusion protein according to the present invention in a state stored in a container can be reconstituted to be administered to an individual.

[0074] As used herein, the term "reconstitution" refers to the process of liquefying a solid lyophilized material so that the α-galactosidase A fusion protein can be administered. In this case, when the lyophilized preparation is reconstituted, the concentration of the α-galactosidase A fusion protein contained in the lyophilized preparation of the present invention may be 1 mg / mL to 150 mg / mL, 10 mg / mL to 120 mg / mL, or 10 mg / mL to 100 mg / mL, but is not limited thereto. The concentration of the preformulation during the lyophilization process may be different from the concentration after reconstitution.

[0075] The lyophilized preparation of the present invention is characterized in that it contains a stabilizer capable of stabilizing the structure of the fusion protein, so that the pharmacological efficacy of the α-galactosidase A fusion protein can be maintained for a long time during long-term storage. The lyophilized preparation of the present invention is characterized in that it contains a mixture prepared by lyophilizing an aqueous solution containing the α-galactosidase A fusion protein, a buffer and amino acids.

[0076] The α-galactosidase A fusion protein of the present invention has a structure in which α-galactosidase A is fused to the immunoglobulin Fc region. The unfolding phenomenon in which the protein structure is deformed due to various external factors (e.g., pH, temperature, osmotic and the presence of stabilizers) occurs during freeze-drying, reconstitution or storage processes, and causes the loss of enzymatic activity, thereby causing the pharmacological efficacy of the preparation containing α-galactosidase to be significantly reduced. Specifically, one of the two domains that make up α-galactosidase A is first unfolded (to form a soluble aggregate), and the other domain of α-galactosidase A and the immunoglobulin Fc region are unfolded in sequence, followed by the formation of a higher-level aggregate, and finally the CH2 and CH3 domains of the immunoglobulin Fc region are unfolded (to form an insoluble aggregate). In this case, a precipitate that can be identified by visual observation is formed. When the structure of the α-galactosidase A fusion protein cannot be maintained due to stress generated when a lyophilized preparation is stored, soluble aggregates or higher-order aggregates are formed and the risk of immunogenicity increases, thereby causing safety issues.

[0077] The present inventors not only found that a composition that inhibits the unfolding of an α-galactosidase A fusion protein is important for the stability of a lyophilized preparation, but also found that even after α-galactosidase A partially unfolds and thereby forms soluble aggregates, the enzymatic activity of α-galactosidase A is maintained because the structure of the active site is retained until insoluble aggregates are formed, and determined that it is important to add a stabilizer (e.g., amino acids) to maintain the structural stability of the α-galactosidase A fusion protein as much as possible and further prevent the formation of insoluble aggregates as the unfolding phenomenon of soluble aggregates proceeds, thereby completing the present invention. The lyophilized preparation of the present invention can have the structure and activity of the fusion protein maintained even after long-term storage, and has high drug safety due to low immunogenicity.

[0078] In a specific example, the lyophilized preparation according to the present invention may include a mixture prepared by lyophilizing an aqueous solution, wherein the aqueous solution includes: a fusion protein in which α-galactosidase A is fused to an immunoglobulin Fc region; a buffer; and amino acids, more specifically, comprising: 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; and amino acids at a concentration of 0.25% (w / v) to 4.0% (w / v).

[0079] Furthermore, in another example, the lyophilized formulation may include: 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; and an amino acid at a concentration of 0.5% (w / v) to 4.0% (w / v), wherein the pH is 5.5 to 7.0.

[0080] Additionally, in another example, the lyophilized preparation may include a mixture prepared by lyophilizing an aqueous solution, the aqueous solution comprising: 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; 50 mM to 200 mM sodium chloride; and 0.5% (w / v) to 4.0% (w / v) serine.

[0081] In another example, a lyophilized formulation may include a mixture prepared by lyophilizing an aqueous solution, the aqueous solution comprising: 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; 0.002% (w / v) to 0.1% (w / v) of polysorbate; 50 mM to 200 mM of sodium chloride; and 0.5% (w / v) to 4.0% (w / v) of serine.

[0082] In another example, a lyophilized formulation may include a mixture prepared by lyophilizing an aqueous solution comprising: 5 mg / mL to 50 mg / mL of a fusion protein; a buffer having a pH of 5.5 to 7.0 and containing 1 mM to 30 mM histidine; 0.002% (w / v) to 0.1% (w / v) polysorbate 20; 50 mM to 200 mM sodium chloride; and 1.0% (w / v) to 4.0% (w / v) serine.

[0083] In another example, a lyophilized preparation may include a mixture prepared by lyophilizing an aqueous solution, the aqueous solution comprising: 5 mg / mL to 50 mg / mL of a fusion protein; a buffer having a pH of 5.5 to 7.0 and containing 1 mM to 30 mM histidine; 0.002% (w / v) to 0.1% (w / v) polysorbate 20; 50 mM to 200 mM sodium chloride; 0.5% (w / v) to 4.0% (w / v) sugar or sugar alcohol; and 0.5% (w / v) to 4.0% (w / v) serine, but the lyophilized preparations of the present invention are not limited to these examples.

[0084] As used herein, the term "aqueous solution" refers to a substance that contains α-galactosidase A fusion protein and contributes to the stable storage of α-galactosidase A fusion protein and maintains stability during the freeze-drying process and in the case of reconstruction. Specifically, the aqueous solution contains an excipient that stabilizes the α-galactosidase A fusion protein, provides stability for the α-galactosidase A fusion protein during the freeze-drying process, and allows the preparation of a freeze-dried preparation with storage stability. The stabilizer may include a buffer and an amino acid. Although not limited to this, the stabilizer may further include an isotonic agent, a sugar, a sugar alcohol, a nonionic surfactant, a preservative, and the like. In proteins such as α-galactosidase A fusion proteins, storage stability is not only important for accurate dosage, but also for inhibiting the potential production of antigenic substances of α-galactosidase A fusion proteins. In the present invention, the aqueous solution can be used interchangeably with a "preformulation".

[0085] Since the concentration of the α-galactosidase A fusion protein can be controlled by adjusting the volume of the reconstitution solution added to the lyophilized preparation, there is no particular limitation on the concentration of the α-galactosidase A fusion protein in the aqueous solution, but even when the α-galactosidase A fusion protein is contained at a high concentration of about 10 mg / mL or more, particularly about 10 mg / mL to 90 mg / mL, the aqueous solution can be stably lyophilized and the stability of the α-galactosidase A fusion protein can be maintained in the reconstituted solution.

[0086] For example, the aqueous solution of the present invention may contain the α-galactosidase A fusion protein at a concentration of about 1 mg / mL to 90 mg / mL, about 1 mg / mL to 70 mg / mL, about 1 mg / mL to 50 mg / mL, about 5 mg / mL to 50 mg / mL, about 5 mg / mL to 90 mg / mL, about 10 mg / mL to 50 mg / mL, or about 10 mg / mL to 30 mg / mL, but is not limited thereto.

[0087] As used herein, the term "about" refers to all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.01, etc., and includes all values ​​equivalent to the value immediately following the term "about" or a value in a similar range, but is not limited thereto.

[0088] It is generally known that as the concentration of the protein included in the preparation increases, problems such as decreased preparation stability and increased precipitates may occur. However, in the case of freeze-drying the aqueous solution containing the stabilizer composition according to the present invention, although the fusion protein is contained at a high concentration, the structure of the fusion protein does not change during the freeze-drying process and the reconstitution process, and does not cause precipitation, so the aqueous solution can contain a high concentration, for example, a concentration of about 10 mg / mL or more, about 15 mg / mL or more, about 35 mg / mL or more, about 50 mg / mL or more, or about 90 mg / mL or more of α-galactosidase A fusion protein as an active ingredient.

[0089] The buffer, as a component contained in the aqueous solution of the present invention, is a solution that plays a role in maintaining the pH of the aqueous solution to avoid a sharp change in the pH of the preparation during the lyophilization process or after reconstitution. The aqueous solution of the present invention can be used as a solvent for the α-galactosidase A fusion protein and the stabilizer, and any aqueous solution can be used as the buffer of the present invention without limitation, as long as it is at a pH level that stabilizes the α-galactosidase A fusion protein.

[0090] The pH value affects the structure of the fusion protein, and the structure of the fusion protein can be stably maintained at a pH of 5.5 to 7.0. When the pH is outside the appropriate pH range, α-galactosidase A unfolds earlier than the immunoglobulin Fc region, resulting in impaired structural stability. Therefore, it is important to obtain structural stability of the α-galactosidase A fusion protein by maintaining the pH at an appropriate level.

[0091] The buffer may include phosphoric acid and its alkali metal salts, as conjugate salts (e.g., phosphates: sodium phosphate, potassium phosphate, or hydrogen or dihydrogen salts thereof), citric acid and its salts (e.g., sodium citrate), acetic acid and its salts (e.g., sodium acetate), or histidine and its salts, and any mixtures thereof may be used as buffers, but are not limited thereto. For example, the buffer may be selected from citric acid buffers (e.g., sodium citrate buffers), acetic acid buffers (e.g., sodium acetate buffers), phosphate buffers (e.g., sodium phosphate buffers), histidine buffers, and any combination thereof, and the buffer or the substance used as the buffer in the aqueous solution (citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, or any combination thereof) may be included in a concentration sufficient to maintain a pH for structural stability of the protein.

[0092] The pH of the aqueous solution may be about 5.0 to 8.0, for example, about 5.0 to 7.5, about 5.0 to 7.0, about 5.0 to 6.5, about 5.0 to 6.0, about 5.5 to 8.0, about 5.5 to 7.5, about 5.5 to 7.0, about 5.5 to 6.5, about 5.5 to 6.3, about 5.5 to 6.2, about 5.5 to 6.1, about 5.5 to 6.0, about 5.5 to 5.9, About 5.5 to 5.8, about 5.5 to 5.7, about 5.5 to 5.6, about 6.0 to 6.5, or about 6.5 to 7.0, or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0, but is not specifically limited thereto.

[0093] To obtain the target pH, the buffer may include phosphoric acid, citric acid, acetic acid, histidine, or a salt or mixture thereof, and the concentration of the buffer is about 1 mM to about 200 mM, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 40 mM, about 8 mM to about 40 mM, about 5 mM to about 30 mM, or about 5 mM to about 25 mM, about 10 mM to about 50 mM, about 10 mM to about 25 mM, about 15 mM to about 25 mM or about 20 mM, but is not limited thereto.

[0094] In certain embodiments, the buffer may comprise about 20 mM histidine and have a pH of about 5.5 to 7.0 or about pH 5.5 to 6.5, but is not limited thereto.

[0095] The amino acids contained in the aqueous solution of the present invention prevent the unfolding of the α-galactosidase A fusion protein contained in the aqueous solution to inhibit the formation of insoluble aggregates. Once insoluble aggregates are formed from soluble aggregates as structural deformation (unfolding) proceeds further, problems such as protein precipitation and loss of enzymatic activity may occur.

[0096] In the experimental examples of the present invention, it was confirmed that the lyophilized preparation containing amino acids (serine) had a proper appearance and a higher recovery rate of the natural form when compared with a preformulation not containing amino acids. Again, it was confirmed that as the concentration of amino acids increased, in particular, when the lyophilized preparation of the aqueous solution contained 0.5% (w / v) or more amino acids based on the total volume of the aqueous solution, a more proper appearance was obtained and the unfolding of the α-galactosidase A fusion protein was more inhibited, thereby inhibiting the formation of soluble aggregates and higher aggregates. In addition, as the concentration of amino acids increased, the recovery rate of the natural form also increased. Since the formation of soluble aggregates or higher aggregates may cause immunogenicity when the preparation is applied to the body, it is necessary to inhibit the formation of soluble aggregates or higher aggregates.

[0097] The amino acid contained in the lyophilized preparation of the present invention can be serine, arginine, lysine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, phenylalanine or any combination thereof, and can be selected from the group consisting of serine, arginine, threonine, glutamine, glycine, alanine and any combination thereof, but not limited thereto. As a specific example, the amino acid can be serine, but not limited thereto.

[0098] Based on the total volume of the aqueous solution of the present invention, the amino acid may be about 0.1% (w / v) to 5.0% (w / v), about 0.25% (w / v) to 5.0% (w / v), about 0.25% (w / v) to 4.0% (w / v), about 0.5% (w / v) to 5.0% (w / v), about 0.5% (w / v) to 4.0% (w / v), about 1.0% (w / v) to 5.0% (w / v), about 1.0% (w / v) to 4.0% (w / v), about 2.0% (w / v) to 4.0% (w / v). , about 2.0% (w / v) to 5.0% (w / v), about 2.5% (w / v) to 5% (w / v), about 3.0% (w / v) to 5% (w / v), about 2.0% (w / v) to 4.5% (w / v), about 2.0% (w / v) to 4.0% (w / v), or about 0.25% (w / v), 0.5% (w / v), about 1.0% (w / v), about 2.0% (w / v), about 3.0% (w / v), about 4.0% (w / v) or about 5.0% (w / v), but is not limited to this.

[0099] The aqueous solution according to the present invention may further comprise a nonionic surfactant. A nonionic surfactant refers to a substance that can reduce the surface tension of a protein solution to prevent the protein from being adsorbed or aggregated on a hydrophobic surface after reconstitution.

[0100] Examples of nonionic surfactants useful in the present invention may be polysorbates (e.g., polysorbate 20 (polyoxyethylene (20) sorbitan laurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate); wherein the number 20 after polyoxyethylene indicates the total number of oxyethylene groups (-(CH2CH2O)-)) , poloxamer (PEO-PPO-PEO copolymer; wherein PEO: poly(ethylene oxide) and PPO: poly(propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compounds (e.g., polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C1-C30), polyoxyethylene monoallyl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1-C30) etc.) and sodium dodecyl sulfate (SDS), or may be polysorbate or poloxamer, which are used alone or in combination of at least two of them.

[0101] Particularly, the nonionic surfactant may be poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80, which may be used in combination, but are not limited thereto.

[0102] In the present invention, the nonionic surfactant may be included in the aqueous solution in a concentration of about 0.2% (w / v) or less, such as about 0.001% (w / v) to about 0.2% (w / v), about 0.001% (w / v) to about 0.1% (w / v), about 0.002% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.05% (w / v), about 0.02% (w / v) to about 0.05% (w / v), about 0.025% (w / v) to about 0.05% (w / v), or about 0.025% (w / v), based on the total volume of the aqueous solution of the present invention, but is not limited thereto.

[0103] The aqueous solution according to the present invention may further contain an isotonic agent. An isotonic agent refers to a substance used to appropriately adjust the osmotic pressure when the α-galactosidase A fusion protein is administered to the body after reconstruction. In addition, the isotonic agent may have the additional effect of further stabilizing the α-galactosidase A fusion protein in the aqueous solution. The aqueous solution of the present invention may contain any isotonic agent without limitation, as long as the isotonic agent contributes to the stabilization of the α-galactosidase A fusion protein.

[0104] The isotonic agent may include a water-soluble inorganic salt. Specifically, the aqueous solution of the present invention may include sodium chloride as an isotonic agent, but is not limited thereto.

[0105] The concentration of sodium chloride used in the present invention may be about 0 mM to 300 mM, specifically, about 0.1 mM to 300 mM, about 5 mM to 300 mM, about 5 mM to 200 mM, about 5 mM to 150 mM, about 5 mM to 100 mM, about 10 mM to 200 mM, about 10 mM to 150 mM, about 10 mM to 100 mM, about 30 mM to 200 mM, about 30 mM to 150 mM, about 30 mM to 100 mM, about 50 mM to 200 mM, about 50 mM to 150 mM, or about 50 mM to 100 mM, but is not limited thereto, and may be appropriately adjusted so that the preformulation containing all the mixtures should be isotonic according to the types and amounts of the components contained in the aqueous solution.

[0106] The aqueous solution according to the present invention may further contain sugar. The sugar refers to monosaccharides, disaccharides, polysaccharides and oligosaccharides, and can increase the stability of the α-galactosidase A fusion protein in aqueous solution. Examples of the sugar may include mannose, glucose, fructose, galactose, fucose, lactose, maltose, sucrose, trehalose, raffinose, dextran or any combination thereof, and in a specific embodiment, the sugar may be glucose, fructose, galactose, lactose, maltose, sucrose, trehalose or any combination thereof, but is not limited thereto. For example, the sugar may be sucrose, but is not specifically limited thereto.

[0107] The aqueous solution according to the present invention may further contain sugar alcohol, and the sugar alcohol refers to a substance containing multiple hydroxyl groups, and includes a substance in which the aldehyde group and / or keto group of the sugar is replaced by an alcohol group and a sugar containing multiple hydroxyl groups. Sugar or sugar alcohol can increase the stability of α-galactosidase A fusion protein. For example, the sugar alcohol may include at least one selected from the group consisting of mannitol and sorbitol, but is not limited thereto.

[0108] Sugar alcohol, sugar or any combination thereof can be present in an amount of about 0.5% (w / v) to 20% (w / v), about 0.5% (w / v) to 15% (w / v), about 0.5% (w / v) to 10% (w / v), about 0.5% (w / v) to 8% (w / v), about 0.5% (w / v) to 5% (w / v), about 0.5% (w / v) to 4.0% (w / v), about 1% (w / v) to 5% (w / v), about 1 ... v) to 10% (w / v), about 1% (w / v) to 8% (w / v), about 1% (w / v) to 6% (w / v), about 1% (w / v) to 4% (w / v), or about 0.0% (w / v), about 1.0% (w / v), about 3.0% (w / v), about 4.0% (w / v), about 5.0% (w / v), or about 8.0% (w / v) is present in the aqueous solution, but is not limited thereto.

[0109] The aqueous solution according to the present invention can be combined to contain different types of stabilizers by adjusting the amount of different types of stabilizers. For example, the aqueous solution according to the present invention may contain (i) amino acids without sugar and / or sugar alcohol, or (ii) amino acids and sugar and / or sugar alcohol, but is not limited thereto. Although not limited thereto, in an aqueous solution containing (i) amino acids but not containing sugar and / or sugar alcohol, the content of amino acids may be about 1.0% (w / v) to 4.0% (w / v), and in an aqueous solution containing (ii) amino acids and sugar and / or sugar alcohol, the content of sugar and / or sugar alcohol may be about 0.5% (w / v) to 4.0% (w / v), and the content of amino acids may be about 0.5% (w / v) to 4.0% (w / v).

[0110] The aqueous solution according to the present invention may further include a preservative. A preservative as a substance that significantly reduces the effects of bacteria and fungi in the preparation is added to the preparation to facilitate the production of preparations for multiple administrations. Examples of potential preservatives may include octadecyl dimethyl-benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, wherein the alkyl group is a long-chain compound) and benzethonium chloride. Other types of preservatives may be aromatic alcohols, such as phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol, resorcinol, cyclohexanol, 3-pentanol or meta-cresol, but are not limited thereto. The concentration of the preservative may be 0.001% (w / v) to 1.0% (w / v), but are not limited thereto.

[0111] Meanwhile, in addition to the above-mentioned buffers, isotonic agents, amino acids, nonionic surfactants, sugar alcohols, sugars and preservatives, the aqueous solution (preformulation) used to prepare the lyophilized preparation of the present invention may further optionally contain other components or substances known in the art within the range not impairing the effects of the present invention, but is not limited thereto.

[0112] As a specific example, the aqueous solution (preformulation) used to prepare the lyophilized preparation according to the present invention can be an aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; and 0.5% (w / v) to 4.0% (w / v) of serine, but is not limited thereto.

[0113] As another specific example, the aqueous solution (preformulation) used to prepare the lyophilized preparation according to the present invention can be an aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; 0.002% (w / v) to 0.1% (w / v) of a nonionic surfactant; and 0.5% (w / v) to 4.0% (w / v) of serine, but is not limited thereto.

[0114] As another specific example, the aqueous solution (preformulation) used to prepare the lyophilized preparation according to the present invention can be an aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0 and containing 10 mM to 30 mM of histidine; 0.002% (w / v) to 0.1% (w / v) of polysorbate; and 1.0% (w / v) to 4.0% (w / v) of serine, but is not limited thereto.

[0115] As another specific example, the aqueous solution (preformulation) used to prepare the lyophilized preparation according to the present invention can be an aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; a buffer having a pH of 5.5 to 7.0; 0.002% (w / v) to 0.1% (w / v) of polysorbate 20; 50 mM to 200 mM of sodium chloride; and 1.0% (w / v) to 4.0% (w / v) of serine, but is not limited thereto.

[0116] As another specific example, the aqueous solution (preformulation) used to prepare the lyophilized preparation according to the present invention can be an aqueous solution comprising 5 mg / mL to 50 mg / mL of the fusion protein; 10 mM to 30 mM of histidine; 0.002% (w / v) to 0.1% (w / v) of polysorbate 20; 50 mM to 200 mM of sodium chloride; 0.5% (w / v) to 4.0% (w / v) of a sugar or a sugar alcohol; and 0.5% (w / v) to 4.0% (w / v) of serine, but is not limited thereto.

[0117] In addition, the aqueous solution used to prepare the lyophilized preparation or the lyophilized preparation may further contain a pharmaceutically acceptable carrier, excipient, etc., and such a carrier or excipient may not be naturally occurring.

[0118] The aqueous solution used to prepare the lyophilized preparation of the present invention can be frozen and dried under appropriate freezing and drying conditions well known in the art. Drying can be completed by one process or two or more processes.

[0119] In addition, the lyophilized preparation of the present invention can be prepared by freeze-drying a preformulation containing an appropriate concentration of α-galactosidase A fusion protein in consideration of the desired dose, and reconstructing the resultant to be suitable for administration to an individual. In addition, the preformulation can be diluted to increase the volume and freeze-dried, and then reconstituted by diluting with a smaller volume of a reconstitution solution than the reconstitution solution of the lyophilized preparation, but is not limited thereto. In the present invention, the lyophilized preparation can be a mixture prepared by freeze-drying an aqueous solution containing α-galactosidase A fusion protein and a stabilizer (e.g., a buffer and an amino acid), and can also be a reconstituted preparation prepared by reconstructing the lyophilized preparation using a reconstitution solution, but is not limited thereto.

[0120] Meanwhile, hereinafter, the α-galactosidase A fusion protein as an active ingredient contained in the lyophilized preparation of the present invention will be described in more detail.

[0121] The α-galactosidase A fusion protein contained in the lyophilized preparation of the present invention refers to a fusion protein in which α-galactosidase A is connected to the immunoglobulin Fc region, and the fusion protein may have a structure in which two α-galactosidase A molecules are connected to the dimeric immunoglobulin Fc region via a linker. In particular, two α-galactosidase A molecules can be covalently connected to each monomer of the immunoglobulin Fc region in the form of a dimer via a linker. In addition, the two α-galactosidase A molecules can form a dimer via a non-covalent bond, but are not limited thereto. The fusion protein of the present invention has an improved stability of α-galactosidase A due to the fusion of the immunoglobulin Fc region with α-galactosidase A, and thus can maintain pharmacological efficacy for a long time in vivo. The lyophilized preparation of the present invention does not lose its pharmacological efficacy even after long-term storage by the following means: excellent stability is provided to the fusion protein by freeze-drying the preformulation containing the fusion protein and the stabilizer.

[0122] In particular, the fusion protein of the present invention may have an amino acid sequence of SEQ ID NO: 4, or may be encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 5, but is not limited thereto. The fusion protein of the present invention may be in the form of a dimer formed by two monomers comprising the amino acid sequence of SEQ ID NO: 4, but is not limited thereto. For the fusion protein of the present invention, the specification of WO 2019 / 009684 is incorporated as a reference.

[0123] As used herein, the terms "a-galactosidase A fusion protein fused to an immunoglobulin Fc region", "a-galactosidase A fusion protein" and "fusion protein" may be used interchangeably.

[0124] The fusion protein of the present invention is expressed in a transformant in a form in which α-galactosidase A is linked to the immunoglobulin Fc region via a linker so that when the immunoglobulin Fc region forms a dimer, α-galactosidase A forms a dimer via a non-covalent bond.

[0125] The α-galactosidase A (α-Gal A, GLA) of the present invention is an enzyme that is present in the lysosomes of the spleen, brain, liver, etc. and hydrolyzes the α-galactosyl part of the end of glycolipids and glycoproteins, and is a homodimeric glycoprotein. Specifically, it is known that α-galactosidase A is associated with Fabry disease as a lysosomal storage disease. α-galactosidase A has a dimer structure composed of two domains (TIM barrel domain and immunoglobulin-like domain containing β-sheet) (Journal of Biological chemistry, Vol. 287, No. 34, 2012; Lieberman et al. Biochemistry, Vol. 48, No. 22, 2009), and since the unfolding phenomenon is more likely to occur as the pH increases, it is necessary to appropriately adjust the pH of the preparation containing α-galactosidase A. Specifically, although the active site is maintained in the state of soluble aggregates formed when a domain is unfolded, once insoluble aggregates are formed as the unfolding is further performed, the activity will be lost, so the preparation needs to include a stabilizer to keep the enzyme in an active state by maintaining a soluble aggregate state. In addition, since soluble aggregates increase the risk of immunogenicity, when soluble aggregates are applied to the body, there is a high risk of inducing an immune response. Therefore, it is important to improve the stability of the preparation to prevent the formation of soluble aggregates during the storage of the preparation as much as possible, to improve medication safety.

[0126] In the present invention, the α-galactosidase A may be in a natural form or a recombinant form, and may specifically comprise the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0127] In addition, the α-galactosidase A of the present invention includes fragments of the natural form or analogs thereof, in which one or several amino acids are changed by one selected from the group consisting of substitution, addition, deletion, modification and any combination thereof, but not limited thereto, as long as the α-galactosidase A has the same or equivalent activity as the natural form of the enzyme.

[0128] In addition, α-galactosidase A may comprise an amino acid sequence having at least 60%, 70% or 80%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 1, and may be obtained from a microorganism by using a genetic engineering method or may be commercially available, but is not limited thereto.

[0129] As used herein, the term "homology" refers to the degree of identity with a wild-type amino acid sequence or a wild-type nucleotide sequence, and homology comparisons can be performed by visual observation or using a commercially available comparison program. Using a commercially available computer program, the homology between two or more sequences can be expressed as a percentage (%). The homology (%) between adjacent sequences can be calculated.

[0130] The sequence of α-galactosidase A or its derivatives and the genetic information of the nucleotide sequence encoding the same can be obtained from known databases (eg, National Center for Biotechnology Information (NCBI)).

[0131] The α-galactosidase A fusion protein of the present invention can be prepared by expressing the fusion protein in a transformant in a form in which α-galactosidase A is linked to the immunoglobulin Fc region via a linker.

[0132] The linker is a peptide linker, and the fusion protein of the present invention may be in a form in which α-galactosidase A is linked to the immunoglobulin Fc region via a peptide linker. One end of the linker may be linked to one of the chains of the immunoglobulin Fc region in the dimer form, but is not limited thereto.

[0133] The peptide linker may comprise one or more amino acids, for example 1 to 1000 amino acids, and includes any peptide linker known in the art, such as [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, wherein x is a natural number of 1 or greater (e.g., 1, 2, 3, 4, 5 or more), but is not limited thereto. In particular, the peptide linker of the present invention may consist of 10 to 50 amino acids, more particularly 20 to 40 amino acids, and may have an amino acid sequence of SEQ ID NO: 2.

[0134] In view of the purpose of the present invention, there is no particular limitation on the sites of α-galactosidase A and immunoglobulin Fc region to which the peptide linker is linked, as long as the activity of α-galactosidase A is maintained after being linked to the immunoglobulin Fc region. In particular, the sites may be both ends of α-galactosidase A and immunoglobulin Fc region, more particularly, the C-terminus of α-galactosidase A and the N-terminus of immunoglobulin Fc region, but are not limited thereto.

[0135] As used herein, the term "N-terminus" or "C-terminus" refers to the amino terminus and carboxyl terminus of the corresponding protein. Examples thereof include, but are not limited to, not only the last amino acid residue at the N-terminus or the C-terminus, but also amino acid residues near the N-terminus or the C-terminus, in particular, starting from the last amino acid until the 10th amino acid residue.

[0136] In the present invention, the peptide linker can be connected to each monomer in the monomer of the immunoglobulin Fc region in the dimer form, and the linkers respectively connected to the immunoglobulin Fc region monomers in the dimer can be independently connected to α-galactosidase A, but are not limited thereto.

[0137] The immunoglobulin Fc region, as one of the parts constituting the enzyme fusion protein of the present invention, may be a dimer formed of immunoglobulin Fc region monomers.

[0138] As used herein, the term "immunoglobulin Fc region" refers to a region comprising a heavy chain constant domain 2 (CH2) and / or a heavy chain constant domain 3 (CH3) other than the heavy chain and light chain variable domains of an immunoglobulin. In view of the purposes of the present invention, the immunoglobulin Fc region may include a modified hinge region, but is not limited thereto. In particular, the immunoglobulin Fc region may be an immunoglobulin Fc region in which one or more amino acids are changed from the native form of the immunoglobulin Fc region by one selected from the group consisting of: substitution, addition, deletion, modification, and any combination thereof, but is not limited thereto.

[0139] Immunoglobulin Fc region is the material used as carrier in drug production. In order to stabilize protein and prevent the protein from being eliminated by kidney, immunoglobulin Fc region has been used to carry out extensive research on fusion protein in recent years. Immunoglobulin is the main component of blood, and there are five different types, i.e. IgG, IgM, IgA, IgD and IgE. The most frequently used type for fusion protein research is IgG, and it is classified into four subtypes (IgG1 to IgG4).

[0140] The immunoglobulin Fc region may include a hinge region in the heavy chain constant domain, and the immunoglobulin Fc region monomer may form a dimer via the hinge region, but is not limited thereto. In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region comprising part or all of the heavy chain constant domain 1 (CH1) and / or the light chain constant domain 1 (CL1) other than the heavy chain and light chain variable domains of the immunoglobulin, as long as the immunoglobulin Fc region has substantially the same or enhanced effect compared to the natural form. In addition, the immunoglobulin Fc region may be a region in which a considerable portion of the amino acid sequence corresponding to CH2 and / or CH3 is eliminated.

[0141] In particular, the immunoglobulin Fc region of the present invention may include 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; or 5) a combination of one or more domains selected from a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region (or a portion of the hinge region), but is not limited thereto. More particularly, the immunoglobulin Fc region may include a hinge region, a CH2 domain, and a CH3 domain, but is not limited thereto.

[0142] As used herein, the term "hinge sequence" refers to a site located at the heavy chain and forming a dimer of the immunoglobulin Fc region via an inter-disulfide bond. In the present invention, the dimer is formed by two immunoglobulin Fc chain molecules via the hinge sequence of the immunoglobulin Fc region.

[0143] In particular, the hinge region may be a hinge region in which a portion of the hinge region is deleted to have only one cysteine ​​(Cys) residue, or a hinge region in which a serine (Ser) residue participating in chain exchange is substituted by a proline (Pro) residue. More particularly, the hinge region may be a hinge region in which the second serine residue is substituted by a proline residue, but is not limited thereto. The immunoglobulin Fc region of the present invention may have an amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0144] The immunoglobulin Fc region of the present invention includes not only the native sequence obtained by papain digestion of immunoglobulin, but also its derivatives, substitution products and variants, such as sequences different from the native sequence and obtained by modifying one or more amino acid residues by deletion, addition, non-conservative or conservative substitution, or a combination thereof. The derivatives, substitution products and variants are those derivatives, substitution products and variants that have the ability to bind to FcRn.

[0145] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important for ligation can be used as suitable modification sites.

[0146] In addition, other various types of derivatives may be used, including derivatives in which sites capable of forming disulfide bonds are deleted or certain amino acid residues are eliminated from the N-terminus of the native Fc form, and a methionine residue is added to the N-terminus of the native Fc form. In addition, in order to remove effector functions, complement binding sites, such as C1q binding sites, may be deleted, and antibody dependent cell mediated cytotoxicity (ADCC) sites may be deleted. The technology for preparing such sequence derivatives of immunoglobulin Fc regions is disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478.

[0147] Amino acid exchanges in proteins and peptides that generally do not alter the activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common amino acid residue exchanges are exchanges between Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. If desired, the Fc region can be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation and amidation.

[0148] The above-mentioned sequence derivatives of the immunoglobulin Fc region are derivatives having biological activities equivalent to those of the immunoglobulin Fc region of the present invention and improved structural stability against heat, pH and the like.

[0149] In addition, these immunoglobulin Fc regions can be obtained from natural forms separated from humans and other animals (including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs), or can be recombinants or derivatives thereof obtained from transformed animal cells or microorganisms. In this regard, they can be obtained from natural immunoglobulins by separating complete immunoglobulins from living humans or animals and treating the complete immunoglobulins with proteases. Papain digests natural immunoglobulins into Fab regions and Fc regions, and pepsin digests natural immunoglobulins into pF'c fragments and F(ab)2 fragments. These fragments can be subjected to size exclusion chromatography to separate Fc or pF'c. In a more specific embodiment, the human immunoglobulin Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0150] In addition, the immunoglobulin Fc region may have native glycans or glycans that are increased or decreased compared to the native type, or be in a deglycosylated form. The increase, reduction or removal of the glycans of the immunoglobulin Fc can be achieved by any method commonly used in the art, such as a chemical method, an enzymatic method, and a genetic engineering method using a microorganism. In this regard, the immunoglobulin Fc region obtained by removing the glycans shows a significantly reduced binding affinity to complement c1q and a reduced or lost antibody-dependent cellular toxicity or complement-dependent cellular toxicity, and thus does not induce an unnecessary immune response in a living organism. Based on this, the deglycosylated or non-glycosylated immunoglobulin Fc region may be more suitable as a drug carrier for its own purpose.

[0151] As used herein, the term "deglycosylated" refers to an Fc region from which glycans have been removed using an enzyme, and the term "aglycosylated" refers to an Fc region that is not glycosylated and produced in a prokaryote, more specifically E. coli.

[0152] Meanwhile, the immunoglobulin Fc region may be derived from humans or animals, such as cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs. In a more specific embodiment, the immunoglobulin Fc region may be derived from humans.

[0153] In addition, the immunoglobulin Fc region can be derived from IgG, IgA, IgD, IgE or IgM, or any combination or hybrid thereof. In a more specific embodiment, the immunoglobulin Fc region is derived from IgG or IgM, the most abundant proteins in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to enhance the half-life of ligand-binding proteins. In still even more specific embodiments, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto. The immunoglobulin Fc region of the present invention may have an amino acid sequence of SEQ ID NO: 3, but is not limited thereto. More particularly, the immunoglobulin Fc region may include a monomer having an amino acid sequence of SEQ ID NO: 3, and the immunoglobulin Fc region may be a homodimer of a monomer having an amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0154] Meanwhile, as used herein, the term "combination" means that when a dimer or multimer is formed, a connection is formed between a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin and a single-chain polypeptide of a different origin. That is, two or more Fc fragments selected from the group consisting of an IgG Fc fragment, an IgA Fc fragment, an IgM Fc fragment, an IgD Fc fragment, and an IgE Fc fragment can be used to prepare a dimer or multimer.

[0155] The immunoglobulin Fc region of the α-galactosidase A fusion protein can be in the form of a dimer, and in particular can have a structure in which two polypeptide chains are connected to each other via a disulfide bond. More particularly, the two chains can be connected via a nitrogen atom of one of the two chains, but are not limited thereto. The connection via the nitrogen atom can be formed via the reductive amination of the ε-amino group of lysine or the N-terminal amino group, but is not limited thereto. In a specific embodiment, the immunoglobulin Fc region can be connected to a linker via a nitrogen atom of a proline at the N-terminus, but is not limited thereto. An Fc region in the form of a dimer can be covalently connected to two α-galactosidase A molecules via two linkers, but is not limited thereto.

[0156] Unless otherwise stated, the detailed description of α-galactosidase A or fusion protein disclosed in the specification or claims of the present invention is applicable not only to α-galactosidase A or fusion protein, but also to its salt (e.g., pharmaceutically acceptable salt), or its solvate form. Therefore, although only "α-galactosidase A" or "fusion protein" is described in this specification, its description may also be applicable to its specific salt, its specific solvate, and the solvate of the specific salt. Such salts may be in the form of, for example, pharmaceutically acceptable salts. There is no particular limitation on the type of salt. However, the salt may be in a form that is safe and effective for mammals, but is not limited thereto.

[0157] The term "pharmaceutically acceptable" refers to substances that are effective for their intended use within the range of pharmacological discretion and do not induce undue toxicity, irritation, allergic response, or the like.

[0158] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid or base. Examples of suitable acids may include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Examples of salts derived from appropriate bases may include alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., magnesium and ammonium salts).

[0159] Furthermore, as used herein, the term "solvate" refers to a complex of the α-galactosidase A, the fusion protein or a salt thereof according to the present invention and solvent molecules.

[0160] The fusion protein can be prepared or produced by any method known in the art, and in particular, can be obtained from animal cells into which an expression vector is inserted after culturing and purifying the animal cells, or can be synthesized based on the sequence of the fusion protein, but is not limited thereto.

[0161] The lyophilized preparation of the present invention can be used to prevent, treat and alleviate diseases, such as α-galactosidase A deficiency, and the symptoms of the disease can be prevented, treated or alleviated by administering α-galactosidase A fusion protein, but is not limited thereto.

[0162] The α-galactosidase A fusion protein of the present invention can be used as a drug for enzyme replacement therapy (ERT). Enzyme replacement therapy can prevent or treat diseases by restoring enzyme dysfunction by supplementing defective or insufficient enzymes that cause the disease.

[0163] In particular, the lyophilized preparation of the present invention can be used to prevent, treat or alleviate α-galactosidase A deficiency. α-galactosidase A deficiency is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A), and includes Fabry disease, diffuse angiokeratoma, diffuse angiokeratoma corporis, or hereditary ectopic liposis.

[0164] Fabry disease, as one of the lysosomal storage diseases, is a recessive hereditary disease caused by X chromosome inactivation. Fabry disease is a congenital glycolipid (sphingolipid) metabolic disorder caused by defective or insufficient α-galactosidase A activity. It is known that the abnormal accumulation of globotriaosylceramide (Gb3) in the blood vessel walls and various parts of the body (such as skin, kidneys, heart and nervous system) caused by abnormal α-galactosidase A seriously affects blood circulation and reduces the supply of nutrients. It causes symptoms such as hypohidrosis, paresthesia of the extremities, severe pain, vascular keratosis, corneal opacity, cardiac ischemia, myocardial infarction and renal failure, and ultimately the kidneys cannot function normally, leading to death.

[0165] As used herein, the term "prevention" refers to all actions that inhibit or delay a disease by administering a fusion protein or a lyophilized formulation comprising the fusion protein. As used herein, the term "treatment" refers to all actions that improve or beneficially change the symptoms of a target disease by administering a fusion protein or a lyophilized formulation comprising the fusion protein.

[0166] In a specific embodiment of the present invention, the lyophilized preparation of the present invention can be used to prevent or treat Fabry disease, but is not limited thereto.

[0167] In addition, the lyophilized preparation of the present invention can be administered via a subcutaneous route, but is not limited thereto. In particular, the lyophilized preparation according to the present invention can be reconstituted to be suitable for subcutaneous administration, but is not limited thereto.

[0168] As used herein, the term "administering" means introducing the reconstituted lyophilized formulation into the patient's body by any suitable method, and there is no particular limitation on the administration route of the lyophilized formulation, but the lyophilized formulation can be administered by any general route as long as the α-galactosidase A fusion protein of the lyophilized formulation reaches the target in the living body, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration or rectal administration can be used, but it is not limited thereto.

[0169] The preferred daily dose of the fusion protein of the present invention can be about 0.0001mg to 500mg per 1kg patient body weight, and the lyophilized preparation of the present invention can be formulated so as to appropriately apply or reconstruct the daily dose, so as to apply the daily dose. However, the dosage of the fusion protein is a dosage effective for the patient determined by considering various factors (such as the patient's age, weight, health status and gender; Disease severity; Diet; and excretion rate and its route of administration and number of administrations), and therefore those of ordinary skill in the art can take these factors into account to determine the appropriate effective dose for the specific use of the lyophilized preparation of the present invention.

[0170] Another aspect of the present invention provides an aqueous solution or a pre-lyophilized preparation, wherein the aqueous solution or the pre-lyophilized preparation comprises an α-galactosidase A fusion protein, a buffer and an amino acid, and the aqueous solution or the pre-lyophilized preparation is used for preparing a lyophilized preparation.

[0171] The aqueous solution or pre-lyophilized preparation may further include at least one selected from an isotonicity agent, a sugar or a sugar alcohol, and a nonionic surfactant, but is not limited thereto.

[0172] The aqueous solution, pre-lyophilized preparation, lyophilized preparation, and components constituting the aqueous solution, pre-lyophilized preparation, lyophilized preparation are as described above.

[0173] Another aspect of the present invention provides a method for preparing the lyophilized preparation.

[0174] In particular, the preparation method may include lyophilizing an aqueous solution comprising an α-galactosidase A fusion protein, a buffer and an amino acid.

[0175] The aqueous solution or lyophilized preparation may further include at least one selected from an isotonicity agent, sugar or sugar alcohol, and a nonionic surfactant, but is not limited thereto.

[0176] The aqueous solution, lyophilized preparation and components constituting the aqueous solution and lyophilized preparation are as described above.

[0177] Another aspect of the present invention provides a method for reconstituting a lyophilized formulation, the method comprising adding a reconstitution solution to the lyophilized formulation.

[0178] Aqueous solutions, lyophilized formulations, and reconstitution are as described above.

[0179] As used herein, the term "reconstitution solution" refers to a solution for reconstitution of a lyophilized preparation by adding the reconstitution solution to a solid lyophilized preparation. Examples of the reconstitution solution may include distilled water (water), but are not limited thereto.

[0180] The reconstituted formulation may include an α-galactosidase A fusion protein at a concentration suitable for administration to an individual, and specifically may include an α-galactosidase A fusion protein in an amount of 10 mg / mL to 100 mg / mL, 10 mg / mL to 90 mg / mL, 10 mg / mL to 50 mg / mL, or 10 mg / mL to 30 mg / mL, but is not limited thereto.

[0181] The reconstituted preparation can be obtained by reconstitution of the lyophilized preparation of the present invention so as to be suitable for subcutaneous administration to an individual, but is not limited thereto.

[0182] Hereinafter, the present invention will be described in more detail in conjunction with the following examples and experimental examples. However, the following embodiments and experimental examples are only presented to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0183] Example 1: Preparation of fusion protein of α-galactosidase A and immunoglobulin Fc region

[0184] A fusion protein of α-galactosidase A and immunoglobulin Fc region (hereinafter referred to as α-galactosidase A fusion protein, SEQ ID NO: 4) was prepared, in which native α-galactosidase A (SEQ ID NO: 1) was linked to immunoglobulin Fc region (SEQ ID NO: 3) via a linker (SEQ ID NO: 2).

[0185] Specifically, a polynucleotide encoding α-galactosidase A fusion protein (SEQ ID NO: 5) was inserted into the XOGC vector as an expression vector by using restriction enzymes to prepare a vector expressing the α-galactosidase A fusion protein.

[0186] The DNA and protein sequences of the α-galactosidase A fusion protein are shown in Table 1 below. In the protein sequence of Table 1 below, the underline indicates the signal sequence, the bold letters indicate the amino acid substitution, and the italic letters indicate the linker. The α-galactosidase A fusion protein of the present invention is in the form of a dimer, which is composed of two monomers having the amino acid sequence SEQ ID NO: 4.

[0187]

Table 1

[0188]

[0189]

[0190]

[0191] The CHO-S cell line was transfected with a vector (pX0GC-α-galactosidase-Fc) expressing the α-galactosidase A fusion protein prepared as described above to prepare a cell line capable of producing a large amount of α-galactosidase A fusion protein.

[0192] Specifically, CHO-S cells were cultured in suspension in a 1 L conical flask (Corning, catalog number 431147) using serum-free medium (FreeStyle CHO Expression Medium, Thermo Fisher, catalog number 12651014) until the number of cells in the culture vessel reached 5 × 10 8 The cells were then transformed with FreeStyle Max (Thermo Fisher, catalog number 16447-100). That is, after 10 mL of OptiPro SFM (Thermo Fisher, catalog number 12309-019) was added to each of the two test tubes, 500 μg of DNA was added to one test tube and 500 μL of FreeStyleMax was added to another test tube, the two solutions were mixed afterwards, and the mixture was allowed to stand at room temperature for 10 minutes. Then, the culture medium was replaced with a new FreeStyle CHO expression medium (Thermo Fisher, catalog number 12651014). The cells were incubated for about 96 hours at 37 ° C, 5% CO2, 125 rpm to prepare α-galactosidase A fusion protein.

[0193] In the α-galactosidase A fusion protein prepared as described above, each of the two immunoglobulin Fc region monomers constituting the dimer is linked to α-galactosidase A to form a structure in which the immunoglobulin Fc region in the form of a dimer is fused to two α-galactosidase A molecules.

[0194] Example 2: Identification of the stability of lyophilized formulations based on the addition of amino acids

[0195] Example 2-1: Preparation and reconstitution of lyophilized formulations

[0196] For the lyophilized preparation containing α-galactosidase A fusion protein prepared in Example 1, in order to determine the change in stability of the lyophilized preparation according to the addition of amino acids, various lyophilized preparations were prepared and subjected to appearance analysis and size exclusion chromatography.

[0197] In order to prepare the lyophilized preparation, each of the solutions of the preparations having the compositions shown in Table 2 below was dispensed into glass vials (3 ml) at 1.0 ml, half-turned with a rubber stopper, and loaded onto the shelf of a freeze dryer (Lyostar 3, SPscientific). Subsequently, lyophilization was performed under the conditions shown in Table 3 below, and the prepared lyophilized preparation was sealed with an aluminum cap after aluminum lyophilization was completed.

[0198]

Table 2

[0199]

[0200]

[0201]

Table 3

[0202] Loading capacity freezing One-time drying Secondary drying Temperature(℃) 4.0 -60 -25 20 Heating rate (℃ / min) 0 1.0 1.0 1.0 Holding time (min) 30 300 2000 1200 Pressure (mTorr) not applicable not applicable 40 40

[0203] The stability of the prepared lyophilized formulations was determined by SE-HPLC analysis after reconstitution with 1.0 ml of distilled water (DW).

[0204] Example 2-2: Size Exclusion Liquid Chromatography (SE-HPLC)

[0205] For size exclusion chromatography, the reconstituted lyophilized formulation was first diluted to a concentration of 1.0 mg / mL using mobile phase (1xPBS, Lonza) followed by sterile filtration and 200 μL of the filtered sample was injected into a vialinsert and prepared in a screw cap vial.

[0206] Subsequently, after the mobile phase is connected to the pump, the assay column (TSKgel G3000SWXL, Tosoh) is loaded onto Waters e2695 and Waters 2489 devices (manufactured by Waters, Japan), while the mobile phase is made to flow at a flow rate of 0.5mL / min. The sample is balanced by making the mobile phase flow at a flow rate of 0.5mL / min for 30 minutes or longer until the detector signal stabilizes. When the temperature of the automatic sampler is reduced to 4°C, the sample is inserted into the sampler. After injecting 10 μL of the sample, the detection peak was identified at 214nm when the mobile phase was made to flow for 35 minutes. The Empower Pro software analysis results using a personal computer (personal computer, PC) were used.

[0207] Example 2-3: Identification of the stability of lyophilized formulations based on the addition of amino acids

[0208] First, as a result of comparison between the cake-like appearances of the lyophilized preparations prepared in Example 2-1, although partial collapse was observed in the preparation containing a high concentration of an isotonic agent (NaCl), severe collapse was not observed in the lyophilized preparation, and an appropriate appearance was observed. In the case of reconstitution, the lyophilized preparation to which serine was added exhibited a transparent appearance, indicating an excellent appearance ( Figure 1 ).

[0209] In addition, as a result of identifying the recovery rate of the native form from the prepared lyophilized preparation by size exclusion chromatography, it was confirmed that the recovery rate of the native form in the preparation without adding serine was 100% or less, and as the concentration of the isotonic agent (NaCl) increased, the recovery rate of the native form decreased, but the recovery rate of the native form in the preparation containing serine was 102.0%, indicating that the preparation was stable ( Figure 2 ).

[0210] In this regard, the native form of the α-galactosidase A fusion protein prepared in Example 1 is a fusion protein in the form of a complete dimer without aggregation or degradation. The definition can be applied to the following examples in the same manner.

[0211] The results of the stability comparison of the lyophilized formulations with the addition of amino acid (serine) are shown in Table 4 below.

[0212]

Table 4

[0213]

[0214] Example 3: Identification of the stability of lyophilized formulations based on amino acid concentration

[0215] Example 3-1: Preparation and reconstitution of lyophilized formulations

[0216] First, in order to determine changes in stability of the lyophilized preparations according to the concentration of amino acids in the lyophilized preparations containing the α-galactosidase A fusion protein prepared in Example 1, various lyophilized preparations were prepared and subjected to appearance analysis and size exclusion chromatography.

[0217] Each of the preparations having the composition shown in Table 5 below was dispensed into a glass vial (3 ml) at 0.5 ml, half-turned with a rubber stopper, and loaded on the shelf of a freeze dryer (Lyostar 3, SP scientific). Subsequently, lyophilization was performed under the conditions shown in Table 6 below, and the prepared lyophilized preparation was capped with an aluminum cap after aluminum lyophilization was completed.

[0218]

Table 5

[0219]

[0220]

Table 6

[0221] Loading capacity freezing One-time drying Secondary drying Temperature(℃) 4.0 -60 -25 20 Heating rate (℃ / min) 0 1.0 1.0 1.0 Holding time (min) 30 300 2000 1200 Pressure (mTorr) not applicable not applicable 40 40

[0222] After reconstitution with 0.25 ml of distilled water (DW), the stability of the prepared lyophilized formulation was determined by SE-HPLC analysis. The composition of the reconstituted lyophilized formulation is shown in Table 7 below, and SE-HPLC analysis was performed in the same manner as in Example 2-2.

[0223]

Table 7

[0224]

[0225] Example 3-2: Identification of stability of lyophilized formulations based on amino acid concentration

[0226] As a result of comparing the cake-like appearance of the lyophilized preparations according to the concentration of amino acids, no severe collapse was observed in all the lyophilized preparations, and a suitable appearance ( Figure 3 ).

[0227] Furthermore, based on size exclusion chromatography, it was confirmed that the formation of soluble aggregates decreased with increasing serine concentration in the lyophilized formulations ( Figure 4 ), the recovery rate of the natural form increased ( Figure 5 ), and the progression from soluble aggregates (HMW1) to higher-order aggregates (HMW2) was more inhibited ( Figure 6 ). Specifically, in a lyophilized preparation containing serine at a concentration of 2.0% or more, it was confirmed that the recovery rate of the native form was more than 100% when compared with that before lyophilization.

[0228] In this regard, soluble aggregates formed as some domains of α-galactosidase A unfold while having solubility and α-galactosidase A activity, and higher-order aggregates are aggregates formed as soluble aggregates unfold. The native form refers to the α-galactosidase A fusion protein prepared in Example 1, and is a fusion protein in the form of a complete dimer that has not yet undergone aggregation or degradation. With the formation of higher-order aggregates and the increase in aggregate size, the risk of immunogenicity increases, and therefore the safety of the lyophilized preparation can be improved by adding serine to inhibit the formation of higher-order aggregates.

[0229] These experimental results indicate that the lyophilized preparation containing amino acids has high safety, and in particular, as the amino acid concentration increases, the formation of aggregates is more inhibited, making it easier to maintain the purity of the α-galactosidase A fusion protein.

[0230] The results of the stability comparison of the lyophilized formulations containing an amino acid (serine) confirmed as described above are listed in Table 8 below.

[0231]

Table 8

[0232] serial number Pie-shaped appearance Appearance after dissolution Recovery rate of natural form (%) 1 Partially collapsed Clear and transparent, no bubbles 90.4 2 Partially collapsed Clear and transparent, no bubbles 93.5 3 Partially collapsed Clear and transparent, no bubbles 96.9 4 Partially collapsed Clear and transparent, no bubbles 101.9 5 Partially collapsed Clear and transparent, no bubbles 103.8

[0233] The above description of the present invention is provided only for illustrative purposes, and it should be understood by those skilled in the art that various changes and modifications can be made without changing the technical concept and basic features of the present invention. Therefore, it is obvious that the above-mentioned embodiments are illustrative in all aspects and do not limit the present invention. In addition, the scope of the present invention should be limited by the appended claims rather than detailed description, and it should be understood that all modifications or changes derived from the meaning and scope of the present invention and their equivalents are included in the scope of the present invention.

Claims

1. A lyophilized preparation comprising a fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region, the lyophilized preparation comprising a mixture prepared by lyophilizing an aqueous solution, the aqueous solution comprising: The fusion protein at a concentration of 5 mg / mL to 50 mg / mL; A buffer having a pH of 5.5 to 7.0; and Amino acids at concentrations ranging from 0.5% (w / v) to 4.0% (w / v).

2. The lyophilized preparation according to claim 1, wherein the buffer comprises histidine or a salt thereof, citric acid or a salt thereof, acetic acid or a salt thereof, phosphoric acid or a salt thereof, or any combination thereof. The lyophilized formulation according to claim 1 , wherein the buffer comprises 1 mM to 50 mM histidine.

4. The lyophilized formulation according to claim 1, wherein the amino acid is selected from the group consisting of serine, arginine, threonine, glutamine, glycine, alanine, and any combination thereof. The lyophilized preparation according to claim 1 , wherein the aqueous solution further comprises sugar at a concentration of 0.5% (w / v) to 4.0% (w / v).

6. The lyophilized preparation according to claim 5, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, trehalose, or any combination thereof. The lyophilized preparation according to claim 1 , wherein the aqueous solution further comprises a sugar alcohol at a concentration of 0.5% (w / v) to 4.0% (w / v). The lyophilized preparation according to claim 7 , wherein the sugar alcohol is mannitol, sorbitol, or any combination thereof. 9 . The lyophilized preparation according to claim 1 , wherein the aqueous solution further comprises a nonionic surfactant at a concentration of 0.002% (w / v) to 0.1% (w / v).

10. The lyophilized formulation according to claim 9, wherein the nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and any combination thereof. The lyophilized formulation according to claim 1 , wherein the aqueous solution further comprises an isotonic agent at a concentration of 5 mM to 300 mM. The lyophilized preparation according to claim 11 , wherein the isotonic agent is sodium chloride. The lyophilized formulation according to claim 1 , wherein the α-galactosidase A comprises the amino acid sequence of SEQ ID NO:

1. The lyophilized formulation according to claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

4. 15 . The lyophilized preparation according to claim 1 , wherein the fusion protein has a structure in which two α-galactosidase A molecules are linked to each monomer of the immunoglobulin Fc region in a dimer form.

16. The lyophilized formulation according to claim 1, wherein the lyophilized formulation comprises a mixture prepared by lyophilizing an aqueous solution comprising: The fusion protein at a concentration of 5 mg / mL to 50 mg / mL; 10 mM to 30 mM histidine; 0.002% (w / v) to 0.1% (w / v) polysorbate 20; 50 mM to 200 mM sodium chloride; and 1.0% (w / v) to 4.0% (w / v) serine.

17. The lyophilized formulation according to claim 1, wherein the lyophilized formulation comprises a mixture prepared by lyophilizing an aqueous solution comprising: The fusion protein at a concentration of 5 mg / mL to 50 mg / mL; 10 mM to 30 mM histidine; 0.002% (w / v) to 0.1% (w / v) polysorbate 20; 50 mM to 200 mM sodium chloride; A sugar or sugar alcohol at a concentration of 0.5% (w / v) to 4.0% (w / v); and 0.5% (w / v) to 4.0% (w / v) serine. The lyophilized preparation according to claim 1 , wherein the lyophilized preparation is used for preventing or treating Fabry disease.

19. A method for preparing a lyophilized formulation according to any one of claims 1 to 18, the method comprising lyophilizing an aqueous solution comprising an α-galactosidase A fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region; a buffer; and amino acids.

20. A method for reconstituting a lyophilized formulation according to any one of claims 1 to 18, the method comprising adding a reconstitution solution to the lyophilized formulation according to any one of claims 1 to 18.

21. The method of claim 20, wherein the reconstitution solution is distilled water.

22. The method of claim 20, wherein the reconstituted formulation prepared by the method comprises the α-galactosidase A fusion protein at a concentration of 10 mg / mL to 100 mg / mL.

Images