Liquid preparations containing α-galactosidase A fusion protein

Abstract

The present invention relates to a liquid formulation containing an α-galactosidase A fusion protein and a method for producing the same, and relates to a liquid formulation that not only has storage stability by containing a composition that confers structural stability to the α-galactosidase A fusion protein, but also has excellent stability even when containing a high concentration of the fusion protein.

Description

[Technical field]

[0001] The present invention relates to a liquid preparation containing an α-galactosidase A fusion protein, a method for producing the same, and uses thereof. [Background technology]

[0002] Lysosomes are intracellular organelles that function to degrade proteins, various lipids including glycolipids and cholesterol, and carbohydrates and recycle them as primary components of new proteins, membrane components, and other molecules. Diseases associated with lysosome function include lysosomal storage diseases (LSDs).

[0003] Lysosomal storage diseases (LSDs) are diseases caused by defects in genes that code for enzymes that break down glycolipid or polysaccharide waste products in the lysosomes of cells, and in the cells and tissues of individuals with lysosomal storage diseases, the biological activity of lysosomal enzymes is greatly reduced or almost absent. This lack of degradative enzymes leads to the accumulation of excess substances that are not broken down, which eventually causes problems in the functioning of cells.

[0004] Fabry disease, known as a type of lysosomal storage disease, is a type of inborn error of glycosphingolipid metabolism resulting from the lack or deficiency of the enzyme activity of alpha-galactosidase A, a hydrolase present in lysosomes. Fabry disease, a representative disease of alpha-galactosidase A deficiency, is an X-linked disease, and typical subjects with Fabry disease usually have less than 1% alpha-galactosidase A activity and exhibit a wide range of symptoms, including severe pain in the extremities (acropesthesias), oligohidrosis, corneal and lenticular changes, skin lesions (angiokeratoma), renal failure, cardiovascular disease, pulmonary failure, neurological symptoms, and stroke.

[0005] Fabry disease causes gradual deposition of globotriaosylceramide (Gb3) in most tissues of the body. The deposition of Gb3 is mainly found in the vascular endothelium. This gradual accumulation of Gb3 in the vascular endothelium leads to ischemia and infarction in organs such as the kidney, heart, and brain.

[0006] A representative method for treating lysosomal storage diseases is enzyme replacement therapy (ERT), and many related studies have been conducted (Non-Patent Document 1). In particular, since lysosomal storage diseases are diseases caused by genetic defects in specific enzymes, replacement therapy of the defective enzyme is essential. Enzyme replacement therapy is the standard treatment for lysosomal storage diseases, and by replenishing the deficient enzyme, it has the effect of alleviating existing symptoms and slowing the progression of the disease, so research is being conducted on various dosage forms containing α-galactosidase A for the prevention and treatment of α-galactosidase A deficiency.

[0007] However, in the case of formulations, the composition of the formulation that shows stability varies greatly depending on the structure, physicochemical properties, dosage, etc. of the active ingredient (e.g., protein drug), so there is a strong demand for developing a specific composition suitable for the active ingredient, and at present, the development of formulations containing α-galactosidase A is insufficient. In particular, in liquid formulations containing α-galactosidase A fusion protein at high concentrations, storage stability is significantly reduced due to protein precipitation, etc., so all conventional liquid formulations contain 1 mg / ml or less. However, in order to improve the drug efficacy, it is necessary to develop a formulation that contains α-galactosidase A fusion protein at high concentrations and is highly stable. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2019 / 009684 [Patent Document 2] International Publication No. 97 / 034631 [Patent Document 3] International Publication No. 96 / 032478 [Non-patent literature]

[0009] [Non-Patent Document 1] Frances M. Platt et al., J Cell Biol., 2012 Nov 26;199(5):723-34 [Non-Patent Document 2] Journal of Biological Chemistry,Vol.287,No.34,2012 [Non-Patent Document 3] Lieberman et al.Biochemistry,Vol.48,No.22,2009 [Non-Patent Document 4] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0010] To date, there has been insufficient development of a stable liquid preparation that can maintain the enzymatic activity of α-galactosidase A for a long period of time and can be stored for long periods of time. Furthermore, no liquid preparations containing high concentrations of α-galactosidase A fusion proteins have been developed, so there is still a need to develop liquid preparations containing α-galactosidase A fusion proteins with new compositions. [Means for solving the problem]

[0011] An object of the present invention is to provide a liquid preparation comprising an α-galactosidase A fusion protein. Another object of the present invention is to provide a method for producing the liquid preparation. Effect of the Invention

[0012] Since the liquid preparation according to the present invention contains a composition that confers structural stability to the α-galactosidase A fusion protein, it not only has storage stability but also has excellent stability even when it contains the fusion protein at a high concentration. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of measuring Tm and Tagg values ​​at each pH. [Diagram 2] FIG. 2 is a diagram showing the Tm values ​​confirmed in FIG. 1. [Diagram 3] FIG. 2 is a diagram showing the Tag values ​​confirmed in FIG. 1 in a summarized form. [Figure 4] FIG. 1 shows the results of SE-HPLC analysis of soluble aggregates at each pH of a liquid formulation when stored at room temperature. [Diagram 5] FIG. 1 shows the results of SE-HPLC analysis of fragments at each pH of a liquid formulation when stored at room temperature. [Figure 6] FIG. 1 shows the recovery rate of natural substances at each pH level in a liquid preparation when stored at room temperature, as confirmed by SE-HPLC analysis. [Figure 7] FIG. 1 shows the results of SE-HPLC analysis of soluble aggregates at each pH of a liquid formulation when stored refrigerated. [Figure 8] FIG. 1 shows the results of SE-HPLC analysis of fragments at each pH of a liquid formulation when stored refrigerated. [Figure 9] FIG. 1 shows the recovery rate of natural substances at each pH level in a liquid preparation when stored refrigerated, as confirmed by SE-HPLC analysis. [Figure 10] FIG. 10 shows the results of SE-HPLC ( FIG. 10( a )) and biological activity analysis ( FIG. 10( b )) of α-galactosidase A fusion protein in liquid formulations at pH 6.0 and pH 8.0. [Figure 11] FIG. 1 shows the results of SE-HPLC analysis of soluble aggregates at various tonicity agent concentrations in liquid formulations. [Figure 12] FIG. 1 shows the results of SE-HPLC analysis of fragments at various tonicity agent concentrations in a liquid formulation. [Figure 13] FIG. 13 is a graph showing the recovery rates of natural isotonicity agents at various concentrations in liquid preparations, as confirmed by SE-HPLC analysis. [Figure 14] FIG. 13 is a graph showing the accelerated stability of each type of amino acid in a liquid formulation confirmed by SE-HPLC analysis. [Figure 15] FIG. 1 shows the results of measuring the Tm value at each concentration of amino acids. [Figure 16] FIG. 1 shows the results of SE-HPLC analysis of soluble aggregates at various concentrations of amino acids in liquid formulations. [Figure 17] FIG. 1 shows the recovery rates of natural amino acids at various concentrations in liquid formulations, as confirmed by SE-HPLC analysis. [Figure 18] FIG. 1 shows the results of SE-HPLC analysis of soluble aggregates at various amino acid concentrations in a liquid formulation containing a high concentration (50 mg / ml) fusion protein. [Figure 19] FIG. 1 shows the recovery rates of natural amino acids at various concentrations in a liquid preparation containing a high-concentration (50 mg / ml) fusion protein, as confirmed by SE-HPLC analysis. [Figure 20] FIG. 1 shows a SE-HPLC chromatogram of a liquid formulation containing a high concentration (50 mg / ml) fusion protein. [Figure 21] FIG. 1 shows the results of SE-HPLC analysis of HMW1 and HMW2 aggregates in a liquid formulation containing a high concentration (50 mg / ml) of fusion protein. [Figure 22] FIG. 1 shows the properties of a liquid formulation containing a high concentration (90 mg / ml) fusion protein. [Diagram 23] FIG. 1 shows the turbidity of a liquid preparation containing a high concentration (90 mg / ml) of a fusion protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] One embodiment of the present invention is a liquid formulation comprising an α-galactosidase A fusion protein. In one specific example, the liquid preparation comprises a fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region, a buffer, an isotonicity agent, and an amino acid.

[0015] In another specific example, the liquid formulation is characterized by comprising 10 to 90 mg / ml of the fusion protein, a buffer for maintaining the pH of the liquid formulation in the range of 5.0 to 6.5, and 0.1 to 4.0% (w / v) of an amino acid.

[0016] In a liquid formulation according to any of the above-mentioned specific examples, the liquid formulation is characterized in that it contains 10 to 90 mg / ml of the fusion protein, a buffer for maintaining the pH of the liquid formulation in the range of 5.5 to 6.5, and 0.5 to 4.0% (w / v) of an amino acid.

[0017] In the liquid formulation according to any of the above-mentioned specific examples, the liquid formulation is characterized in that it contains 10-90 mg / ml of the fusion protein, 10-50 mM histidine, and 1.0-4.0% (w / v) serine.

[0018] In any of the liquid formulations according to the above-mentioned specific examples, the liquid formulation is characterized in that it contains 10-90 mg / ml of the fusion protein, 10-50 mM histidine, 50-200 mM sodium chloride, and 1.0-4.0% (w / v) serine.

[0019] In the liquid formulation according to any of the above-mentioned embodiments, the buffer comprises histidine and its salts, citric acid and its salts, acetic acid and its salts, phosphoric acid and its salts, or combinations thereof.

[0020] A liquid formulation according to any of the above-mentioned embodiments, wherein said liquid formulation further comprises an isotonicity agent. In the liquid formulation according to any of the above-mentioned embodiments, the tonicity agent is sodium chloride.

[0021] In any of the above-mentioned specific examples of the liquid preparation, the isotonicity agent is contained in an amount of 50 to 200 mM. The liquid formulation according to any of the previous embodiments, wherein the amino acid is selected from the group consisting of arginine, serine, threonine, glutamine, glycine, alanine, and combinations thereof.

[0022] In the liquid formulation according to any of the above-mentioned specific examples, said liquid formulation is characterized in that it further comprises a non-ionic surfactant at a concentration of 0.005 to 0.1% (w / v). The liquid formulation according to any of the above-mentioned embodiments, wherein the non-ionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

[0023] In the liquid formulation according to any of the above-mentioned embodiments, the α-galactosidase A is characterized in that it comprises the amino acid sequence of SEQ ID NO:1. The liquid formulation according to any of the above embodiments, wherein the immunoglobulin Fc region comprises the amino acid sequence of SEQ ID NO:3.

[0024] In a liquid formulation according to any of the above-mentioned embodiments, the α-galactosidase A fusion protein is characterized in that it comprises the amino acid sequence of SEQ ID NO:4. The liquid formulation according to any of the above embodiments, wherein the immunoglobulin Fc region is derived from IgG4.

[0025] In a liquid formulation according to any of the above embodiments, the immunoglobulin Fc region is characterized in that it is non-glycosylated. In a liquid formulation according to any of the above-mentioned specific examples, the fusion protein is characterized in that two molecules of α-galactosidase A are linked to each monomer of a dimeric immunoglobulin Fc region.

[0026] The liquid formulation according to any of the above-mentioned embodiments, characterized in that the liquid formulation is for the prevention or treatment of Fabry disease. A liquid formulation according to any of the above-mentioned embodiments, characterized in that said liquid formulation is administered by subcutaneous route of administration.

[0027] Another embodiment of the present invention is a method for producing the liquid formulation. In one embodiment, the method for producing the liquid formulation comprises the step of mixing an α-galactosidase A fusion protein, a buffer solution, and an amino acid.

[0028] In another embodiment, the method for producing the liquid formulation further comprises the step of mixing an isotonicity agent, a non-ionic surfactant, a preservative, or a combination thereof. Hereinafter, an embodiment of the present invention will be described.

[0029] In addition, each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0030] Additionally, 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 described herein which equivalents are intended to be encompassed by the present invention.

[0031] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. In addition, amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system.

[0032] Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid Asp,D Cysteine ​​Cys,C Glutamic acid Glu,E Glutamine Gln,Q Glycine Gly,G Histidine Isoleucine Ile,I Leucine Leu,L Lysine Lys,K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin,V One embodiment of the present invention provides a liquid formulation comprising a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked to each other.

[0033] Specifically, the present invention relates to a liquid formulation comprising a pharmacologically effective amount of a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked to each other, a buffer solution, and an amino acid, and further comprising, but not limited to, an isotonicity agent, a non-ionic surfactant, a preservative, or a combination thereof.

[0034] In the present invention, the term "liquid preparation" refers to a drug that has been formulated in a liquid form from a pharmaceutical agent, and includes both liquid preparations for internal use and liquid preparations for external use. The liquid preparation of the present invention contains a substance that can stably maintain and / or store the pharmacologically effective α-galactosidase A fusion protein for a predetermined period of time when the α-galactosidase A fusion protein is formulated into a liquid form. The components contained in the liquid preparation other than the pharmacologically effective α-galactosidase A fusion protein are mixed with a stabilizer. In the present invention, the "stabilizer" refers to a substance that can stably maintain the components, such as the active ingredient, in the preparation for a predetermined period of time.

[0035] The liquid preparation of the present invention is characterized by comprising a stabilizer composition that stabilizes the structure of the α-galactosidase A fusion protein so that the pharmacological effect of the fusion protein is maintained for a long period of time even when stored for a long period of time.

[0036] The α-galactosidase A fusion protein of the present invention is a fusion of α-galactosidase A and an immunoglobulin Fc region, and an unfolding phenomenon occurs in which the protein structure is deformed by various external factors (pH, temperature, osmotic pressure, the presence or absence of a stabilizer, etc.), which causes a loss of enzyme activity and significantly reduces the pharmacological effect of a preparation containing α-galactosidase A. In particular, α-galactosidase A is sensitive to the pH environment, and one of the two domains that form α-galactosidase A is first unfolded (soluble aggregate formation), and then the other domain of α-galactosidase A and the immunoglobulin Fc region are sequentially unfolded, passing through higher-order aggregates, and finally unfolding to CH2 and CH3 of the immunoglobulin Fc region (insoluble aggregate formation), causing precipitation that can be seen with the naked eye. If the structure of the α-galactosidase A fusion protein cannot be maintained due to stress generated during storage of the liquid formulation, and soluble aggregates or higher-order aggregates are formed, the risk of immunogenicity increases, posing a safety issue for the formulation.

[0037] The present inventors have confirmed that for the stability of a liquid formulation, it is important to include a composition that prevents the unfolding of α-galactosidase A fusion protein, and that even if α-galactosidase A is unfolded to form soluble aggregates, the structure of the active site is maintained until insoluble aggregates are formed, and the enzymatic activity of α-galactosidase A is maintained. As a result, it has been elucidated that it is important to adjust the pH and add a stabilizer (such as an amino acid) in order to maximize the structural stability of the α-galactosidase A fusion protein and to prevent the unfolding of the protein from further progressing in the formed soluble aggregates to form insoluble aggregates, and thus the present invention has been completed. The liquid formulation of the present invention can maintain the structure and activity of the fusion protein even when stored for a long period of time, has low immunogenicity, and is excellent in drug safety.

[0038] A specific example of a liquid preparation according to the present invention includes one containing a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked, a buffer solution, and an amino acid. A more specific example is a liquid preparation containing 10 to 90 mg / ml of the fusion protein, a buffer solution, and 0.1 to 4.0% (w / v) of an amino acid, and having a pH of 5.0 to 6.5.

[0039] Another example is a liquid preparation containing 10 to 90 mg / ml of a fusion protein, a buffer solution, and 0.5 to 4.0% (w / v) of an amino acid, and having a pH of 5.5 to 6.5.

[0040] Further examples of the liquid formulation include a liquid formulation containing 10 to 90 mg / ml of the fusion protein, 10 to 50 mM histidine, and 1.0 to 4.0% (w / v) serine, but the liquid formulation of the present invention is not limited thereto.

[0041] The liquid preparation of the present invention contains the α-galactosidase A fusion protein at a concentration of about 1 to 150 mg / ml, about 1 to 130 mg / ml, about 1 to 100 mg / ml, about 5 to 100 mg / ml, about 5 to 90 mg / ml, about 10 to 90 mg / ml, about 10 to 50 mg / ml, or about 50 to 90 mg / ml, but is not limited to these.

[0042] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.01, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about."

[0043] It is generally known that the higher the concentration of a protein contained in a formulation, the more difficult it is to maintain the stability of the formulation, and problems such as protein precipitation occur. However, the liquid formulation of the present invention has a composition that can maintain the structure of the fusion protein even when it contains a fusion protein at a high concentration and does not cause precipitation, and therefore contains a high concentration, for example, about 10 mg / ml or more, about 15 mg / ml or more, about 35 mg / ml or more, about 50 mg / ml, or about 90 mg / ml or more of α-galactosidase A fusion protein as an active ingredient.

[0044] The buffer, which is one of the components contained in the liquid formulation of the present invention, is a solution for maintaining the pH of the liquid formulation so that the α-galactosidase A fusion protein is stabilized. The liquid formulation of the present invention may contain a buffer as a solvent for the liquid formulation, and any buffer may be used as the buffer of the present invention as long as it can maintain a pH that stabilizes the α-galactosidase A fusion protein, which is the target substance to be stabilized.

[0045] In the experimental examples of the present invention, it was confirmed that the structure of the fusion protein changes depending on the pH value of the liquid preparation, and specifically, it was confirmed that the structural stability of the fusion protein is the best in the range of pH 5.5 to 6.5. In addition, it was confirmed that, among the components constituting the fusion protein, α-galactosidase A is more significantly affected by pH than the immunoglobulin Fc region, and unfolding occurs due to changes in pH value. Therefore, it is important to maintain the pH of the liquid preparation at an appropriate level to ensure the structural stability of the α-galactosidase A fusion protein.

[0046] The buffer solution includes, but is not limited to, phosphoric acid and its conjugate salt, i.e., an alkali salt (e.g., phosphate salt: sodium phosphate, potassium phosphate, or its hydrogen or dihydrogen salt), citric acid and its salt (e.g., sodium citrate), acetic acid and its salt (e.g., sodium acetate), histidine and its salt, and mixtures thereof are also used as buffer solutions. For example, the buffer solution is selected from the group consisting of citrate buffer (e.g., sodium citrate buffer), acetate buffer (e.g., sodium acetate buffer), phosphate buffer (e.g., sodium phosphate buffer), histidine buffer, and combinations thereof, and the buffer substance (citric acid and its salt, acetic acid and its salt, histidine and its salt, phosphoric acid and its salt, or combinations thereof) in the buffer solution or liquid formulation is contained in a concentration sufficient to maintain the desired pH of the liquid formulation.

[0047] The pH of the liquid preparation is about 5.0 to 8.0, for example, about pH 5.0 to 7.5, about pH 5.0 to 7.0, about pH 5.0 to 6.5, about pH 5.0 to 6.0, about pH 5.5 to 8.0, about pH 5.5 to 7.5, about pH 5.5 to 7.0, about pH 5.5 to 6.5, about pH 5.5 to 6.3, about pH 5.5 to 6.2, about pH 5.5 to 6.1, about pH 5.5 to 6 0, about pH 5.5 to 5.9, about pH 5.5 to 5.8, about pH 5.5 to 5.7, about pH 5.5 to 5.6, about pH 6.0 to 6.5, or about pH 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 or 6.5, but are not limited to these.

[0048] To achieve the desired pH, the buffer solution may contain phosphate, citric acid, acetic acid, histidine, a salt thereof, or a mixture thereof at 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, 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 particularly limited to these.

[0049] As a specific example, the buffer solution contains 20 mM histidine and has a pH of about 5.5 to 6.5, but is not limited to this. The other types of amino acids contained in the liquid formulation of the present invention as stabilizers maintain the soluble aggregate state of the α-galactosidase A fusion protein in the liquid formulation. If the fusion protein in the soluble aggregate state undergoes further structural modification (unfolding) to form insoluble aggregates, problems such as precipitation and loss of enzyme activity will occur.

[0050] In the experimental examples of the present invention, it was confirmed that the addition of amino acids can prevent the unfolding of α-galactosidase A fusion protein from progressing and changing into higher-order aggregates, and can prevent the formation of insoluble aggregates. It was also confirmed that in a liquid preparation containing a high concentration of α-galactosidase A fusion protein, the prevention of higher-order aggregate formation prevents protein precipitation and has a significant effect on improving stability during storage. Since the formation of soluble aggregates and higher-order aggregates can induce immunogenicity when the preparation is administered to the body, it is necessary to inhibit the formation of soluble aggregates and higher-order aggregates.

[0051] The amino acid contained in the liquid formulation of the present invention is arginine, lysine, serine, threonine, asparagine, glutamine, glycine, proline, alanine, valine, isoleucine, leucine, phenylalanine or a combination thereof, specifically, but not limited to, selected from the group consisting of arginine, serine, threonine, glutamine, glycine, alanine and a combination thereof. Specific example, but not limited to, the amino acid is serine.

[0052] In the liquid preparation of the present invention, the amino acid is present at about 0.1 to 5.0% (w / v), about 0.5 to 5.0% (w / v), about 0.5 to 4.0% (w / v), about 1.0 to 5.0% (w / v), about 1.0 to 4.0% (w / v), about 2.0 to 5.0% (w / v), about 2.5 to 5% (w / v), about 3.0 to 5% (w / v), about 3.5 to 5% (w / v), about 2.0 to 4.5% (w / v), about 2.0 to 4.0% (w / v), or 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 particularly limited thereto.

[0053] The liquid preparation according to the present invention may further contain an isotonicity agent, which is a substance added to adjust the osmotic pressure of the liquid preparation, and any isotonicity agent that contributes to stabilizing the α-galactosidase A fusion protein while maintaining an appropriate osmotic pressure may be contained in the liquid preparation of the present invention.

[0054] The isotonicity agent includes water-soluble inorganic salts, and specifically, the liquid preparation of the present invention includes sodium chloride as an isotonicity agent, but is not limited thereto. In the experimental examples of the present invention, it was confirmed that the isotonicity agent inhibits the formation of soluble aggregates in the liquid formulation of the present invention and helps maintain the native form of the fusion protein.

[0055] The concentration of sodium chloride used in the present invention is about 0 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, about 50 mM to 100 mM, but is not limited thereto, and the components contained in the formulation are adjusted to appropriate contents depending on the types and amounts so that a solution dosage form containing all of the mixtures becomes an isotonic solution.

[0056] The liquid formulation according to the present invention may further comprise a non-ionic surfactant, which lowers the surface tension of the protein solution and prevents the protein from adsorbing to hydrophobic surfaces and from aggregating.

[0057] Specific examples of the nonionic surfactant used in the present invention include polysorbates (e.g., polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), where the number (20) after polyoxyethylene means the total number of oxyethylene groups (-(CH2CHO)-)), poloxamer (PEO-PPO-PEO copolymer, PEO: poly(ethylene oxide), 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.), sodium dodecyl sulphate (SDS), etc., or polysorbate or poloxamer, which may be used alone or in combination of two or more.

[0058] Specifically, the nonionic surfactant is poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80, and may be used in combination, but is not limited thereto.

[0059] The nonionic surfactant in the present invention is contained in the liquid preparation of the present invention at a concentration of about 0.2% (w / v) or less, for example, about 0.001 to about 0.2% (w / v), about 0.001 to about 0.1% (w / v), about 0.005 to about 0.1% (w / v), about 0.001 to about 0.08% (w / v), about 0.002 to about 0.08% (w / v), about 0.005 to about 0.05% (w / v), about 0.01 to about 0.05% (w / v), or about 0.05% (w / v), but is not particularly limited thereto.

[0060] The liquid formulation according to the present invention may further include a sugar. The sugar means a monosaccharide, a disaccharide, a polysaccharide, an oligosaccharide, etc., and can improve the stability of the α-galactosidase A fusion protein in the liquid formulation. Specific examples include mannose, glucose, fructose, galactose, fucose, lactose, maltose, sucrose, trehalose, raffinose, dextran, or a combination thereof. For example, the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, trehalose, or a combination thereof, but is not limited thereto. For example, the sugar is sucrose, but is not particularly limited thereto.

[0061] The liquid preparation according to the present invention may further comprise a sugar alcohol. The sugar alcohol means a substance having multiple hydroxyl groups, and includes those in which the aldehyde group and / or ketone group of a sugar is substituted with an alcohol group, and also includes sugars having polyvalent hydroxyl groups. The sugar or sugar alcohol can improve the stability of the α-galactosidase A fusion protein. For example, the sugar alcohol is at least one selected from the group consisting of mannitol and sorbitol, but is not limited thereto.

[0062] The sugar alcohol, sugar or a combination thereof is present at a concentration of about 0.5 to 20% (w / v), about 0.5 to 15% (w / v), about 0.5 to 10% (w / v), about 0.5 to 8% (w / v), about 0.5 to 5% (w / v), about 0.5 to 4% (w / v), about 1 to 20% (w / v), about 1 to 15% (w / v), about 1 to 10% (w / v), about 1 to 8% (w / v), about 1 to 6% (w / v), about 1 to 5% (w / v), 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) relative to the total solution of the liquid preparation, but is not particularly limited thereto.

[0063] The liquid formulation according to the present invention may further include a preservative. The preservative is a substance that substantially reduces the action of bacteria and fungi in the formulation, and is a compound that is included in the formulation to facilitate the production of a multi-dose formulation. Examples of potential preservatives include octadecyldimethyl-benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is a long-chain compound), benzethonium chloride, and the like, and other types of preservatives include, but are not limited to, aromatic alcohols such as phenol alcohol, butyl alcohol, benzyl alcohol, alkyl parabens such as methyl paraben, propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, and the like. The concentration of the preservative is 0.001 to 1.0% (w / v), but is not limited to this.

[0064] In addition, the liquid preparation of the present invention may selectively contain other components or substances known in the art in addition to the above-mentioned buffer solution, isotonicity agent, amino acid, nonionic surfactant, and preservative, as long as the effects of the present invention are not impaired, but is not limited to these.

[0065] A specific example of a liquid formulation according to the present invention includes, but is not limited to, a liquid formulation containing 10 to 90 mg / ml of a fusion protein, 10 to 50 mM histidine, and 1.0 to 4.0% (w / v) serine.

[0066] Other specific examples of liquid formulations according to the present invention include, but are not limited to, liquid formulations containing 10 to 90 mg / ml of fusion protein, 10 to 30 mM histidine, and 2.0 to 4.0% (w / v) serine, and having a pH of 5.5 to 6.0.

[0067] Yet another specific example of the liquid formulation according to the present invention includes, but is not limited to, a liquid formulation containing 10 to 90 mg / ml of the fusion protein, 10 to 30 mM histidine, 50 to 150 mM sodium chloride, and 2.0 to 4.0% (w / v) serine, and having a pH of 5.5 to 6.0.

[0068] Furthermore, the liquid formulation according to the present invention may further comprise a pharma- ceutically acceptable carrier, excipient, etc., and such a carrier, excipient, etc. may be non-naturally occurring. The α-galactosidase A fusion protein, which is an active ingredient contained in the liquid preparation of the present invention, will be described in more detail below.

[0069] The α-galactosidase A fusion protein contained in the liquid formulation of the present invention means a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked, and the fusion protein may be in the form in which two molecules of α-galactosidase A are linked to a dimeric immunoglobulin Fc region via a linker. Specifically, two molecules of α-galactosidase A may be linked to each monomer of the dimeric immunoglobulin Fc region via a linker by covalent bond. In addition, the two molecules of α-galactosidase A form a dimer by non-covalent bond with each other, but are not limited thereto. The fusion protein of the present invention has a long pharmacological effect in the body because the stability of α-galactosidase A is improved by fusing the immunoglobulin Fc region to α-galactosidase A. The liquid formulation of the present invention further contains a stabilizer in addition to such a fusion protein, and therefore has excellent stability and does not lose its pharmacological effect even when stored for a long period of time.

[0070] Specifically, the fusion protein of the present invention includes, but is not limited to, an amino acid sequence of SEQ ID NO: 4 or is encoded by a polynucleotide sequence of SEQ ID NO: 5. The fusion protein of the present invention includes, but is not limited to, a dimer formed by two monomers each including the amino acid sequence of SEQ ID NO: 4. With regard to the fusion protein of the present invention, the specification of Patent Document 1 is incorporated by reference.

[0071] In the present invention, the terms "a fusion protein in which α-galactosidase A is linked to an immunoglobulin Fc region", "α-galactosidase A fusion protein" and "fusion protein" are used interchangeably.

[0072] The fusion protein of the present invention may be expressed in a transformant in a form in which α-galactosidase A is linked to an immunoglobulin Fc region via a linker, and when the immunoglobulin Fc region forms a dimer, α-galactosidase A also forms a dimer through a non-covalent bond.

[0073] Alpha galactosidase A (alpha-Gal A, GLA) of the present invention is an enzyme present in lysosomes such as the spleen, brain, and liver that hydrolyzes the terminal alpha galactosyl moiety in glycolipids and glycoproteins, and is a homodimeric glycoprotein. In particular, it is known to be related to Fabry disease, a lysosomal storage disease. The alpha galactosidase A has a dimeric structure consisting of two domains (TIM barrel domain and β-sheet containing immunoglobulin-like domain) (Non-Patent Documents 2 and 3), and since the higher the pH value, the more the unfolding phenomenon occurs, it is required that a liquid preparation containing it maintains an appropriate pH level. In particular, in the state of soluble aggregates in which one domain is unfolded, the active site is maintained, but if unfolding proceeds further to form insoluble aggregates, activity is lost, so it is required to contain a stabilizer to maintain the state of soluble aggregates and exert enzyme activity. Furthermore, soluble aggregates increase the risk of immunogenicity and are highly likely to induce immune responses when administered to the body. Therefore, it is important to improve the stability of the formulation and ensure safety when administered so as to prevent the formation of soluble aggregates during storage of the formulation as much as possible.

[0074] In the present invention, the α-galactosidase A may be in a natural or recombinant form, specifically comprising the amino acid sequence of SEQ ID NO:1, but is not limited thereto.

[0075] In addition, the present invention also includes analogs of the α-galactosidase A, including fragments of the natural enzyme, and analogs modified by a modification selected from the group consisting of partial amino acid substitution, addition, deletion, modification, and combinations thereof, as long as they have activity equivalent to that of the natural enzyme.

[0076] Furthermore, the α-galactosidase A may comprise an amino acid sequence having 60%, 70%, 80% or more, specifically 90% or more, more specifically 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology to the amino acid sequence of SEQ ID NO:1, and may be obtained from a microorganism by recombinant technology or may be commercially available, but is not limited to these.

[0077] In the present invention, "homology" refers to the degree of similarity to a wild-type amino acid sequence or a wild-type nucleic acid sequence, and the homology comparison is performed by the naked eye or by using a comparison program that is easily available for purchase. Commercially available computer programs can calculate the homology between two or more sequences as a percentage (%). The homology (%) may also be calculated for adjacent sequences.

[0078] Information on the sequence of the α-galactosidase A or a derivative thereof and the nucleotide sequence encoding it can be obtained from known databases such as NCBI. The α-galactosidase A fusion protein of the present invention is prepared by expressing in a transformant a form in which α-galactosidase A is linked to an immunoglobulin Fc region via a linker.

[0079] The linker may be a peptide linker, and the fusion protein of the present invention may be one in which α-galactosidase A and an immunoglobulin Fc region are fused via a peptide linker, with one end of the linker being linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0080] The peptide linker contains one or more amino acids, for example, 1 to 1000 amino acids, and may be any peptide linker known in the art, such as a [GS]x linker, a [GGGS]x linker, or a [GGGGS]x linker, where x is a natural number of 1 or more (for example, 1, 2, 3, 4, 5 or more), but is not limited thereto. Specifically, the peptide linker of the present invention may be composed of an amino acid sequence of 10 to 50 amino acids, more specifically, 20 to 40 amino acids, or may be composed of the amino acid sequence of SEQ ID NO:2.

[0081] For the purposes of the present invention, the position at which the peptide linker is fused to α-galactosidase A and the immunoglobulin Fc region is not limited, so long as the immunoglobulin Fc region is linked while maintaining the activity of α-galactosidase A. Specifically, the position is either one of the termini of α-galactosidase A and the immunoglobulin Fc region, more specifically, the C-terminus of α-galactosidase A or the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

[0082] In the present invention, the "N-terminus" and "C-terminus" refer to the amino-terminus and carboxy-terminus of a protein, respectively, and include, for example, but are not limited to, not only the most terminal amino acid residue of the N-terminus or C-terminus, but also all amino acid residues in the vicinity of the N-terminus or C-terminus, and specifically include the 1st to 10th amino acid residues from the most terminal.

[0083] In the present invention, the peptide linkers are each linked to the immunoglobulin Fc regions of a dimer formed by monomeric immunoglobulin Fc regions, and each linker linked to each immunoglobulin Fc region is independently linked to one molecule of α-galactosidase A, but are not limited thereto.

[0084] The immunoglobulin Fc region, which is one moiety of the enzyme fusion protein of the present invention, may be a dimer formed from monomeric immunoglobulin Fc regions. In the present invention, the term "immunoglobulin Fc region" refers to a region that excludes the variable regions of the heavy and light chains of an immunoglobulin and includes the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3). For the purposes of the present invention, such an Fc region includes, but is not limited to, a mutated hinge region. Specifically, the immunoglobulin Fc region is a natural immunoglobulin Fc region in which at least one amino acid has been altered in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0085] The immunoglobulin Fc region is a substance used as a carrier in the manufacture of drugs, and in order to stabilize proteins and prevent them from being removed by the kidney, research into fusion proteins using the immunoglobulin Fc region has been actively conducted recently. Immunoglobulins are major components of blood, and there are five types, IgG, IgM, IgA, IgD, and IgE, but the type mainly used in research into fusion proteins is IgG, which is classified into four subtypes, IgG1 to IgG4.

[0086] Such an immunoglobulin Fc region includes a hinge region in the heavy chain constant region, and the hinge region allows the monomeric immunoglobulin Fc region to form a dimer, but is not limited thereto. Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region including a part or the whole of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding the variable regions of the heavy and light chains of the immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of a natural one. Furthermore, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0087] Specifically, the immunoglobulin Fc region of the present invention is a combination of at least one of the following domains: 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; and 5) a combination of an immunoglobulin hinge region (or a part of a hinge region) with a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain. However, the combination is not limited thereto. More specifically, the immunoglobulin Fc region is composed of a hinge region, a CH2 domain, and a CH3 domain, but is not limited thereto.

[0088] In the present invention, the "hinge sequence" refers to a site located in a heavy chain that forms an immunoglobulin Fc domain dimer through a disulfide bond, and the immunoglobulin Fc domain of the present invention may be in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of the hinge sequence.

[0089] Specifically, the hinge region may be mutated to include only one cysteine ​​(Cys) residue by deleting a portion of the hinge region, or may be substituted with a proline (Pro) residue for the serine (Ser) residue involved in chain exchange. More specifically, the second serine residue in the hinge sequence may be substituted with a proline residue, but is not limited thereto. The immunoglobulin Fc region of the present invention includes, but is not limited to, the amino acid sequence of SEQ ID NO:3.

[0090] The immunoglobulin Fc region of the present invention includes not only native sequences obtained by papain digestion of immunoglobulins, but also derivatives and substitutions thereof, for example, variants such as sequences that differ from native sequences by deletion, insertion, non-conservative or conservative substitution, or combinations thereof of at least one amino acid residue of the native sequence, provided that the derivatives, substitutions, and variants have the ability to bind to FcRn.

[0091] 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, which are known to be important for binding, are used as sites suitable for modification.

[0092] Various derivatives are also used, such as derivatives in which the site forming disulfide bonds has been removed, derivatives in which several amino acids have been deleted from the N-terminus of native Fc, and derivatives in which a methionine residue has been added to the N-terminus of native Fc. Furthermore, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in Patent Documents 2 and 3, etc.

[0093] Amino acid substitutions in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 4). The most common substitutions are the following amino acid residues: 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, Asp / Gly. In some cases, modifications may be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0094] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have improved structural stability against heat, pH, and the like of the Fc region. In addition, such an Fc region may be obtained from a natural source isolated from the living body of an animal such as a human or a cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be a recombinant product or derivative thereof obtained from a transformed animal cell or a microorganism. Here, the method of obtaining from a natural source may be a method of obtaining by isolating the whole immunoglobulin from the living body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. These can be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0095] Furthermore, the immunoglobulin Fc region may have a natural sugar chain, an increased sugar chain compared to the natural one, a decreased sugar chain compared to the natural one, or a form in which the sugar chain has been removed. For increasing, decreasing, or removing such immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms are used. Here, an immunoglobulin Fc region from which the sugar chains have been removed from Fc has a significantly reduced binding ability to complement (c1q), and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. For these reasons, an immunoglobulin Fc region from which the sugar chains have been removed or which has been deglycosylated is in a form suitable for its original purpose as a drug carrier.

[0096] In the present invention, "deglycosylation" refers to an Fc region from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0097] On the other hand, the immunoglobulin Fc region of the present invention is of human origin or animal origin, such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment is of human origin.

[0098] The immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, or IgM, or may be a combination or hybrid of these. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to extend the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in an even more 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 includes, but is not limited to, the amino acid sequence of SEQ ID NO: 3. More specifically, the immunoglobulin Fc region includes a monomer of the amino acid sequence of SEQ ID NO: 3, and the immunoglobulin Fc region is a homodimer of a monomer of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0099] On the other hand, the term "combination" in the present invention means that, when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin is bound to a single-chain polypeptide of a different origin, i.e., a dimer or multimer can be prepared from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0100] The immunoglobulin Fc region of the α-galactosidase A fusion protein contained in the liquid formulation of the present invention may be in a dimeric form, specifically, a structure in which two polypeptide chains are linked by a disulfide bond. More specifically, the two chains are linked to each other via a nitrogen atom of one of the chains, but this is not limited thereto. The linkage via the nitrogen atom is linked to the ε-amino atom of lysine or the amino group at the N-terminus by reductive amination, but this is not limited thereto. As a specific example, the immunoglobulin Fc region is linked to the nitrogen atom of proline at the N-terminus, but this is not limited thereto. One Fc region in the dimeric form is covalently linked to two molecules of α-galactosidase A via two linkers, but this is not limited thereto.

[0101] Unless otherwise specified in this specification, the detailed description of the specification and the claims regarding α-galactosidase A or the fusion protein according to the present invention apply to the category including not only α-galactosidase A or the fusion protein, but also all of the salts (e.g., pharma- ceutically acceptable salts) or solvates thereof. Thus, even if the specification only describes "α-galactosidase A" or "fusion protein", the description also applies to a specific salt thereof, a specific solvate thereof, and a specific solvate of a specific salt thereof. These salt forms may be, for example, any pharma- cetically acceptable salt. The type of the salt is not particularly limited. However, it is preferable that the salt is in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0102] The term "pharmacologically acceptable" refers to a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reactions, and the like, within the scope of medical judgment. The "pharmaceutically acceptable salt" in the present invention includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids 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, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Suitable salts derived from bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, etc.

[0103] Furthermore, the term "solvate" in the present invention means a complex formed between α-galactosidase A, the fusion protein or a salt thereof according to the present invention and a solvent molecule. The fusion protein can be prepared or produced by a method well known in the art. Specifically, the fusion protein can be prepared or produced by culturing an animal cell into which an animal cell expression vector has been inserted and purifying it from the culture, or can be synthesized based on the sequence and used, but is not limited thereto.

[0104] The liquid formulation of the present invention is intended for the prevention, treatment or amelioration of diseases that can be prevented, treated or ameliorated by administration of an α-galactosidase A fusion protein, such as, but not limited to, α-galactosidase A deficiency.

[0105] The α-galactosidase A fusion protein of the present invention may be used as a drug for enzyme replacement therapy (ERT), which can prevent or treat diseases by restoring the reduced enzyme function by supplementing the deficient or insufficient enzyme that causes the disease.

[0106] Specifically, the liquid preparation of the present invention is used for the prevention, treatment, or amelioration of α-galactosidase A deficiency. The 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 diseases such as Fabry Disease, Angiokeratoma Diffuse, Angiokeratoma Corporis Diffusum, and Hereditary Dystopic Lipidosis.

[0107] Fabry disease is a type of lysosomal storage disease, which develops when the X chromosome is inactivated due to sex chromosome recessive inheritance. It is a congenital metabolic disorder of glycolipids caused by a lack or insufficiency of alpha-galactosidase A activity. It is known that abnormalities in alpha-galactosidase A cause abnormal accumulation of Gb3 (globotriaosylceramide) in blood vessel walls and various parts of the body, such as the skin, kidneys, heart, and nervous system, which affects blood flow and reduced nutritional supply. Symptoms such as hypohidrosis, acroparesthesia, severe pain, angiokeratoma, corneal opacity, cardiac ischemia, myocardial infarction, and kidney abnormalities appear, and ultimately the kidneys are unable to perform their original functions, leading to death.

[0108] In the present invention, "prevention" refers to any action of suppressing or delaying the onset of a disease by administering the fusion protein or a liquid preparation containing the same, and "treatment" in the present invention refers to any action of improving, reversing, or favorably changing the symptoms of a target disease by administering the fusion protein or a liquid preparation containing the same.

[0109] In a specific embodiment of the present invention, the liquid formulation of the present invention is used for the prevention or treatment of Fabry disease, but is not limited thereto. Furthermore, the liquid preparation of the present invention has a dosage form and composition suitable for subcutaneous administration, and is administered via a subcutaneous administration route, but is not limited thereto.

[0110] In the present invention, "administration" means introducing a liquid formulation into a patient by any suitable method, and the route of administration of the liquid formulation may be any common route that can deliver the α-galactosidase A fusion protein of the liquid formulation to a target in the body, including, but not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0111] The preferred total dosage of the fusion protein of the present invention is about 0.0001 mg to 500 mg per kg of patient body weight per day, and the liquid preparation is formulated so that the above dosage can be appropriately administered. However, the dosage of the fusion protein is determined by taking into consideration various factors such as the administration route and number of treatments, as well as the patient's age, body weight, health condition, sex, severity of disease, diet, and excretion rate, and therefore, taking these factors into consideration, a person having ordinary knowledge in the art would be able to determine an appropriate effective dosage for a specific use of the liquid preparation of the present invention.

[0112] Another embodiment of the present invention provides a method for producing the liquid formulation. Specifically, the method may include the step of mixing an α-galactosidase A fusion protein, a buffer solution, and an amino acid.

[0113] The liquid formulation further comprises at least one component selected from the group consisting of, but not limited to, an isotonicity agent and a non-ionic surfactant. The liquid preparation and its constituent elements are as described above.

[0114] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples. EXAMPLES

[0115] Size exclusion liquid chromatography (SE-HPLC) For size exclusion chromatography, samples were first diluted to 1.0 mg / ml with mobile phase (1x PBS, Lonza) and then sterile filtered. 200 μL of the filtered sample was injected into a vial insert and inserted into a screw top vial.

[0116] The mobile phase was then connected to a pump, and then the mobile phase was run through a Waters e2695 and Waters 2489 instrument (Waters Japan) at a flow rate of 0.5 mL / min, and an analytical column (TSKgel G3000SWXL, Tosoh) was installed. The mobile phase was run at a rate of 0.5 mL / min for 30 minutes or more, equilibrated until the detector signal stabilized, and when the temperature of the autosampler dropped to 4°C, the sample was set in the sampler. 10 μL of the sample was injected, and then the mobile phase was run for 35 minutes, and the detection peak at 214 nm was confirmed.

[0117] Analyses were performed using Empower Pro software on a PC. EXAMPLES

[0118] Analysis of thermal unfolding and thermal aggregation When the temperature rises and the protein unfolds, the degree of protein unfolding can be measured by detecting the emission wavelength from tryptophan exposed on the surface. An UNcle (Unchained Labs) device was used for differential scanning fluorimetry based on the intrinsic fluorescence intensity. To analyze the thermal stability of the protein using this device, Tm (thermal unfolding) and Tagg (thermal aggregation) were measured as follows.

[0119] 8.8 μL of sample was injected in duplicate into a Uni sample loader (Unchained Labs), and the temperature was then raised from 25°C to 95°C at a rate of 1°C / min. As the temperature was raised, the intensity of the wavelengths emitted by the excitation wavelength of 266 nm (250-720 nm) was measured. Fluorescence data analysis was performed using UNcle Analysis software (Unchained Labs).

[0120] The temperature at the maximum value of the fluorescence emission peak in the above analysis is defined as Tm, and the protein aggregation onset temperature at which protein static light scattering at 473 nm (SLS473) begins is measured and defined as Tagg. EXAMPLES

[0121] Analysis of biological activity (pNP-Gal activity) In the activity assay of α-galactosidase A (α-Gal A), 4-nitrophenyl-α-D-galactopyranoside (pNP-Gal) was used as a chromogenic substrate. Upon hydrolysis, the pNP-Gal is decomposed by the action of α-galactosidase A into galactose and 4-nitrophenol. 4-Nitrophenol gives a bright yellow color and provides visual evidence of hydrolysis.

[0122] The buffers used in the analysis were as follows: 1) Assay buffer: 20 mM citric acid, 30 mM sodium phosphate, 0.1% BSA, 0.67% absolute ethanol (pH 4.6) 2) Substrate solution: 24.1 mg / mL pNP-Gal (Sigma) in assay buffer 3) Stop solution: 130 mM glycine, 83 mM sodium carbonate (pH 10.6) In vitro activity was measured as follows.

[0123] Samples (300ng / mL, 150ng / mL) diluted with assay buffer were prepared, and a standard solution of 4-Nitrophenol (Sigma) diluted with assay buffer was prepared. 50μL of sample and STD (standard solution) were repeatedly loaded into each well of an assay plate (Corning). The assay plate and substrate solution were incubated at 37℃ for 30 minutes, temperature equilibrium was achieved, and 50μL of substrate solution was loaded into the assay plate. The assay plate was incubated at 37℃ for 20 minutes, and then 100μL of stop solution was loaded to stop the reaction and stabilize it. The absorbance at 405nm was then analyzed (Multimode Plate Reader, PerkinElmer). EXAMPLES

[0124] Turbidity Analysis Turbidity was analyzed by Lunatic (Unchained Labs). 2.0 μL of sample was injected into a Lunatic plate (Unchained Labs) and the turbidity was measured at 350 nm. The turbidity of placebo buffer was calculated by subtracting it from the turbidity value of the sample.

[0125] The experimental results of the above embodiment were analyzed as follows. Experimental Example 1: Preparation of a fusion protein of α-galactosidase A and immunoglobulin Fc region A fusion protein of α-galactosidase A and immunoglobulin Fc domain (hereinafter referred to as α-galactosidase A fusion protein, sequence number 4) was prepared in which natural α-galactosidase A (sequence number 1) and immunoglobulin Fc domain (sequence number 3) are linked via a linker (sequence number 2).

[0126] Specifically, a polynucleotide (SEQ ID NO: 5) encoding the α-galactosidase A fusion protein was inserted into the XOGC vector, an expression vector, using a restriction enzyme to prepare a vector that expresses the α-galactosidase A fusion protein.

[0127] The DNA and protein sequences of the α-galactosidase A fusion protein are shown in Table 1. In the protein sequences in Table 1, underlines indicate signal sequences, bold indicates amino acid substitutions, and italics indicate linkers. The α-galactosidase A fusion protein of the present invention is a dimer of two monomers containing the amino acid sequence of SEQ ID NO:4.

[0128] [Table 1-1]

[0129] [Table 1-2]

[0130] A cell line capable of mass-producing α-galactosidase A fusion protein was constructed by transfecting a vector (pX0GC-alpha galactosidase-Fc) expressing the α-galactosidase A fusion protein prepared as described above into a CHO-S cell line.

[0131] Specifically, CHO-S cells were cultured in suspension in a 1 L Erlenmeyer flask (Corning, cat. No. 431147) using a serum-free medium (FreeStyle CHO Expression Medium, Thermo Fisher, cat. No. 12651014), and the cells in the culture vessel reached 5 × 10 8 When the cells were grown to 1000 cells, they were transformed using Freestyle Max (Thermo Fisher, cat. No. 16447-100). That is, 10 mL of OptiPro SFM (Thermo Fisher, cat. No. 12309-019) was placed in each of two tubes, 500 μg of DNA was placed in one tube, and 500 μL of Freestyle Max was placed in the other tube. Both solutions were mixed and left to stand at room temperature for 10 minutes, and then added to cells that had been previously replaced with fresh Freestyle CHO expression medium (Thermo Fisher, cat. No. 12651014). The cells were cultured in an incubator at 37, 5% CO2, and 125 rpm for about 96 hours to produce α-galactosidase A fusion protein.

[0132] The α-galactosidase A fusion protein thus prepared had a form in which each of the two monomeric immunoglobulin Fc regions constituting a dimeric immunoglobulin Fc region was linked to one α-galactosidase A, and the dimeric immunoglobulin Fc region was fused to two α-galactosidase A enzymes.

[0133] Experimental Example 2: Confirmation of the stability of α-galactosidase A fusion protein under various pH conditions of liquid formulation Experimental Example 2-1: Preparation of dosage form In order to understand the structural changes of the α-galactosidase A fusion protein caused by pH change, liquid preparations were prepared by changing the pH as follows.

[0134] [Table 2]

[0135] Experimental Example 2-2: Measurement of Tm and Tagg upon pH change For each dosage form in Table 2, the Tm and Tagg values ​​were measured using the UNCLE device of Example 2.

[0136] As a result, as shown in FIG. 1, it was confirmed that the lower the pH of the liquid preparation (closer to pH 5.5), the higher the Tm value, and the higher the pH (closer to pH 8.0), the higher the Tagg value.

[0137] Specifically, the Tm values ​​were confirmed to be 69.8, 69.7, 66.6, 62.3, 57.8 and 52.6°C at pH 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, respectively (Figure 2), and the Tagg values ​​were confirmed to be 66.6, 67.5, 66.9, 69.0, 70.0 and 70.1°C (Figure 3).

[0138] Here, a higher Tm value means a higher structural stability of the protein, and a higher Tagg value means a higher colloidal stability of the protein.

[0139] These results confirmed that the structural stability of the α-galactosidase A fusion protein was excellent at low pH levels close to pH 5.5, and specifically, was particularly excellent at pH levels of 5.5 to 6.5.

[0140] Furthermore, from Figures 1 to 3, it was confirmed that the degree of change in Tm value due to pH was greater than the degree of change in Tagg, and that the Tm value has a higher pH-sensitivity than the Tagg value.

[0141] Experimental Example 2-3: SE-HPLC analysis with pH change (accelerated stability at room temperature) The stability of each liquid formulation with different pH prepared in Experimental Example 2-1 was compared by a room temperature accelerated stability test. Specifically, the liquid formulation of Experimental Example 2-1 was analyzed by size exclusion liquid chromatography. All samples were analyzed at the initial time point and after 7 days of storage at 25°C (65% RH).

[0142] As a result, after one week at room temperature, it was confirmed that as the pH increased, the amount of soluble aggregates (HMW) increased relative to the entire fusion protein (Figure 4), and that the amount of fragments also increased (Figure 5).

[0143] In addition, after one week at room temperature, the recovery of the native form was analyzed, and it was confirmed that the recovery of the native form decreased with increasing pH (Figure 6).

[0144] Here, the soluble aggregate (HMW) is a form that is soluble due to the unfolding of a portion of the domain of α-galactosidase A and maintains the activity of α-galactosidase A, the native form refers to the α-galactosidase A fusion protein prepared in Experimental Example 1, which is a normal dimeric fusion protein that has not undergone aggregation or decomposition, and the fragment refers to a substance in which one or two molecules of α-galactosidase A have been separated from the Fc region in the native form, losing the structure of the α-galactosidase A fusion protein of the present invention. These definitions are similarly applied to the following examples.

[0145] Experimental Example 2-4: SE-HPLC analysis of pH changes (long-term stability in refrigerator, 6 months) The long-term refrigerated stability of each liquid formulation with different pH prepared in Experimental Example 2-1 for six months was confirmed. Specifically, the liquid formulations of Experimental Example 2-1 were analyzed by size exclusion liquid chromatography. All samples were analyzed at the initial time point and after six months of storage at 5°C.

[0146] As a result, after six months of refrigerated storage, it was confirmed that the amount of soluble aggregates (HMW) increased as the pH increased (Figure 7), and that the amount of fragments also increased (Figure 8).

[0147] In addition, after six months of refrigerated storage, the recovery rate of the native form was analyzed and it was confirmed that the recovery rate of the native form decreased with increasing pH (Figure 9).

[0148] From Experimental Examples 2-3 and 2-4, it was confirmed that the higher the pH of the liquid preparation, the more soluble aggregates formed and the more unfolding of the α-galactosidase A fusion protein occurred, and that the structure of the α-galactosidase A fusion protein became difficult to recover during storage. These experimental results also suggest that the structural stability of the α-galactosidase A fusion protein is excellent in a liquid preparation at pH 5.5 to 6.5.

[0149] Experimental Example 2-5: Confirmation of the enzyme activity of α-galactosidase A fusion protein The biological activity of the α-galactosidase A fusion protein in the liquid formulation was confirmed in the same manner as in Example 3. Specifically, liquid formulations #2 (pH 6.0) and #6 (pH 8.0) in Experimental Example 2-1 were analyzed by SE-HPLC and pNP-Gal activity assay. Liquid formulation #2 (pH 6.0) was analyzed at the initial time point of storage at 25°C, and liquid formulation #6 (pH 8.0) was analyzed after 7 days of storage at 25°C.

[0150] As a result, as shown in Figure 10(a), the natural content (%) of the pH 6.0 liquid preparation at the initial stage of storage was 96.7%, and the natural content (%) of the pH 8.0 liquid preparation stored at room temperature for 7 days was 64.0% in SE-HPLC. In addition, as shown in Figure 10(b), the pNP-Gal activity showed a slight difference at the level of analytical deviation (<20%). Therefore, it was confirmed that even if there was a slight difference in the purity of the α-galactosidase A fusion protein between the pH 6.0 and 8.0 liquid preparations, the difference in the activity of α-galactosidase A was not large.

[0151] This suggests that even if some domains of α-galactosidase A are unfolded to form soluble aggregates, the enzyme activity is maintained. Experimental Example 3: Confirmation of the stability of α-galactosidase A fusion protein by adding an isotonic agent to the liquid formulation Experimental Example 3-1: Preparation of dosage form In order to understand the structural changes of the α-galactosidase A fusion protein caused by the presence or absence of an isotonicity agent (NaCl) and changes in its concentration, a liquid preparation was prepared as follows.

[0152] [Table 3]

[0153] Experimental Example 3-2: SE-HPLC analysis of liquid formulations at various concentrations of isotonicity agent (accelerated stability at room temperature) The stability of each liquid formulation with different concentrations of the isotonicity agent prepared in Experimental Example 3-1 was confirmed by a room temperature accelerated stability test. Specifically, the liquid formulation of Experimental Example 3-1 was analyzed by size exclusion liquid chromatography. All samples were analyzed at the initial time point, and after 7 and 14 days of storage at 25°C.

[0154] As a result, after 2 weeks at room temperature, it was confirmed that the soluble aggregates (HMW) of the fusion protein decreased to 9.4, 9.2, 8.8, 8.8, and 8.7% as the concentration of the isotonicity agent (NaCl) increased to 0, 50, 100, 200, and 300 mM, respectively. In addition, there was no difference in the percentage of soluble aggregates in liquid formulations containing 100 mM or more NaCl (Figure 11).

[0155] After 2 weeks at room temperature, the percentage of the fragments decreased to 7.3, 6.0, 5.5, 4.6, and 4.6% of the total fusion protein as the concentration of the isotonicity agent (NaCl) increased to 0, 50, 100, 200, and 300 mM, respectively. Furthermore, in liquid preparations containing 200 mM or more NaCl, there was no significant difference in the percentage of the fragments (Figure 12).

[0156] Furthermore, after 2 weeks at room temperature, the recovery of the native form was analyzed, and it was confirmed that the recovery of the native form increased with increasing NaCl concentration, with no significant difference in liquid formulations containing 200 mM or more NaCl (Figure 13).

[0157] Experimental Example 4: Confirmation of the stability of α-galactosidase A fusion protein by adding amino acids to liquid formulations Experimental Example 4-1: SE-HPLC analysis of liquid formulations with various types of stabilizers (amino acids, sugars, sugar alcohols) (accelerated stability at 40°C) In order to understand the structural changes of the α-galactosidase A fusion protein caused by the type of stabilizer in a liquid preparation containing stabilizers (amino acids, sugars, and sugar alcohols), a liquid preparation was prepared as follows.

[0158] [Table 4-1]

[0159] [Table 4-2]

[0160] Accelerated stability studies were conducted to confirm the stability of each liquid formulation containing different types of stabilizers. Specifically, the liquid formulations in Table 4 were analyzed by size exclusion liquid chromatography. All samples were analyzed after 15 hours of storage at 40°C.

[0161] As a result, it was confirmed that the percentage of soluble aggregates in liquid formulations #1 and #3 containing arginine and L-serine was 9.7 and 9.8%, respectively, which was lower than that of liquid formulations containing other types of amino acids (Figure 14).

[0162] Experimental Example 4-2: Tm analysis by amino acid concentration In order to analyze the influence of the concentrations of arginine and serine, the stability effects of which were confirmed in Experimental Example 4-1, on the liquid preparation, a liquid preparation was prepared as follows.

[0163] [Table 5-1]

[0164] [Table 5-2]

[0165] Using the UNCLE device of Example 2, Tm was measured for liquid preparations #1 to #12 in Table 5. As a result, the Tm value increased in proportion to the increase in the concentration of serine added, whereas no correlation was confirmed between the concentration of arginine added and the Tm value. In addition, it was confirmed that the liquid formulations to which arginine was added at concentrations of 2.0 and 4.0% had lower Tm values ​​than the liquid formulations to which serine was added at concentrations of 2.0 and 4.0%. Furthermore, it was confirmed that no synergistic effect was observed when serine and arginine were mixed and used as a stabilizer (Figure 15).

[0166] Experimental Example 4-3: SE-HPLC analysis of liquid formulations at various amino acid concentrations (accelerated stability at room temperature) The stability of liquid formulations containing arginine and serine at each amino acid concentration was confirmed by accelerated stability testing at room temperature.

[0167] Specifically, the room temperature accelerated stability was confirmed by measuring the rate of soluble aggregate formation and the recovery rate of native aggregates in liquid formulations containing serine and arginine at various concentrations as shown in Table 5. The liquid formulations in Table 5 were analyzed by size exclusion liquid chromatography. All liquid formulations were analyzed at the initial time point, and after 1 day, 4 days, and 7 days of storage at 25°C.

[0168] As a result, in the case of the addition of serine, the formation rate of soluble aggregates decreased as the concentration of added serine increased over the storage period, and it was confirmed that even when serine was added in a small amount of 0.5% (w / v), the formation of soluble aggregates was more effective than that of the control groups (liquid preparations #1 and #2). It was also confirmed that the formation of aggregates was more effective than that of liquid preparations containing arginine as a stabilizer. As in Figure 15, there was no significant difference in stability between the mixture of arginine and serine and the one containing only serine (Figure 16).

[0169] In addition, in the liquid preparations containing serine, the recovery rate of natural substances increased as the concentration of added serine increased, and it was confirmed that even when serine was added at a small amount of 0.5% (w / v), the recovery rate of natural substances was superior to that of the control groups (liquid preparations #1 and #2). Furthermore, it was confirmed that the liquid preparations containing arginine as a stabilizer were superior in storing natural substances. As in Figure 15, there was no significant difference in stability between the liquid preparations containing a mixture of arginine and serine and the liquid preparations containing only serine (Figure 17).

[0170] These experimental results suggest that the addition of amino acids, especially serine, improves the structural stability of the α-galactosidase A fusion protein, prevents the unfolding of α-galactosidase A, and possibly inhibits the formation of soluble aggregates, thereby contributing to the stability of the liquid formulation.

[0171] Experimental Example 5: Confirmation of the stability of high-concentration α-galactosidase A fusion protein In order to confirm that stability is maintained even when a liquid formulation containing a high concentration of α-galactosidase A fusion protein is prepared based on the composition of the liquid formulation whose stability was confirmed in the above-mentioned experimental example, the following experiment was performed.

[0172] Experimental Example 5-1: Confirmation of the stability of high concentration (50 mg / ml) α-galactosidase A fusion protein (accelerated stability at room temperature) To confirm the stability of a liquid formulation containing 50 mg / ml of α-galactosidase A fusion protein, a liquid formulation was prepared as follows.

[0173] [Table 6]

[0174] The liquid formulations in Table 6, prepared as described above, were analyzed by size-exclusion liquid chromatography. All liquid formulations were analyzed at the initial time point, and after 1 day, 4 days, and 7 days of storage at 25° C.

[0175] As a result, with the passage of the storage period, the rate of soluble aggregate (HMW) formation for the entire fusion protein decreased as the concentration of added serine increased, and it was confirmed that even when serine was added in a small amount of 0.5% (w / v), it was more effective at preventing the formation of soluble aggregates than the control groups (liquid preparations #1 and #2) (Figure 18).

[0176] Furthermore, as the concentration of added serine increased, the recovery rate of the natural product also increased, and it was confirmed that even when serine was added at a small amount of 0.5% (w / v), the recovery rate of the natural product was superior to that of the control groups (liquid preparations #1 and #2) (Figure 19).

[0177] Such an inhibitory effect of serine on the formation of soluble aggregates was greater in liquid formulations containing 50 mg / ml of fusion protein than in liquid formulations containing 10 mg / ml of fusion protein, suggesting the importance of serine in liquid formulations containing high concentrations of fusion protein.

[0178] In addition, the analysis of SE-HPLC chromatogram after 7 days of storage at 25°C confirmed that the progression of soluble aggregates (HWM1) to higher-order aggregates (HMW2) was reliably prevented as the concentration of serine increased (Figure 20). The higher-order aggregates refer to aggregates formed by further unfolding of proteins in soluble aggregates. The risk of immunogenicity increases as the size of the aggregates increases, so the addition of serine can prevent the formation of higher-order aggregates, thereby improving the safety of the lyophilized formulation.

[0179] Similarly, as the concentration of serine increased, the formation of higher order aggregates (HMW2) was significantly reduced compared to the formation of soluble aggregates (HMW1) (FIG. 21). Experimental Example 5-2: Confirmation of the stability of high concentration (90 mg / ml) α-galactosidase A fusion protein (3-week refrigerated stability) In order to confirm the stability of a liquid preparation containing a higher concentration (90 mg / ml) of α-galactosidase A fusion protein than in Experimental Example 5-1, a liquid preparation was prepared as follows.

[0180] [Table 7]

[0181] The formulations in Table 7 were analyzed by visual inspection and UV spectroscopy to determine appearance and turbidity. Analyses were performed at the initial point and after 21 days of storage at 2-8°C.

[0182] As a result of comparing the properties of liquid formulations containing a high concentration (90 mg / ml) of the fusion protein, liquid formulation #1, which did not contain any amino acid as a stabilizer, showed a cloudy property, whereas liquid formulation #2, which contained 2.0% (w / v) serine, showed a clear and transparent property (Figure 22).

[0183] In addition, as a result of comparing the turbidity, the measured turbidity of liquid formulation #1, which did not contain an amino acid as a stabilizer, was 49.9, while the measured turbidity of liquid formulation #2, which contained 2.0% (w / v) serine, was 2.8. Therefore, it was confirmed that the inclusion of an amino acid (serine) as a stabilizer improved the turbidity of the liquid formulation, and thus improved the stability of the liquid formulation (FIG. 23).

[0184] These experimental results suggest that the liquid formulation of the present invention is stable even when it contains a high concentration of the α-galactosidase A fusion protein. From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A liquid preparation comprising a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked, The liquid formulation comprises: 10-90 mg / ml of the fusion protein; a buffer for maintaining the pH of the liquid formulation in the range of 5.5 to 6.5; 0.5 to 4.0% (w / v) amino acid; Liquid formulation.

2. The buffer comprises histidine and its salts, citric acid and its salts, acetic acid and its salts, phosphoric acid and its salts, or a combination thereof. The liquid formulation according to claim 1.

3. The liquid formulation further comprises 5 to 200 mM of an isotonicity agent. The liquid formulation according to claim 1.

4. The tonicity agent is sodium chloride. The liquid formulation according to claim 3.

5. The amino acids are selected from the group consisting of arginine, serine, threonine, glutamine, glycine, alanine, and combinations thereof. The liquid formulation according to claim 1.

6. The liquid formulation further comprises a non-ionic surfactant at a concentration of 0.005 to 0.1% (w / v). The liquid formulation according to claim 1.

7. The non-ionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof; The liquid formulation according to claim 6.

8. The α-galactosidase A comprises the amino acid sequence of SEQ ID NO:

1. The liquid formulation according to claim 1.

9. The fusion protein comprises the amino acid sequence of SEQ ID NO:

4. The liquid formulation according to claim 1.

10. The fusion protein is a protein in which two molecules of α-galactosidase A are linked to each monomer of a dimeric immunoglobulin Fc region. The liquid formulation according to claim 1.

11. The liquid formulation comprises: 10-90 mg / ml of the fusion protein; 10 to 50 mM histidine; and 1.0 to 4.0% (w / v) serine; The liquid formulation according to claim 1.

12. The liquid formulation is for the prevention or treatment of Fabry disease. The liquid formulation according to claim 1.

13. The liquid formulation is administered via a subcutaneous route. The liquid formulation according to claim 1.

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