Lyophilized preparation containing α-galactosidase A fusion protein

Abstract

The present invention relates to a freeze-dried preparation containing an α-galactosidase A fusion protein and a method for producing the same, and relates to a freeze-dried preparation 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 lyophilized 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 freeze-dried formulations containing α-galactosidase A fusion protein at high concentrations, storage stability is significantly reduced due to protein precipitation, etc., so all conventional freeze-dried formulations contain α-galactosidase A fusion protein at a content of 5 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 a high concentration 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 freeze-dried 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 freeze-dried preparation containing a high concentration of α-galactosidase A fusion protein has been developed, so there is still a need to develop a freeze-dried preparation containing an α-galactosidase A fusion protein with a new composition. [Means for solving the problem]

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

[0012] A further object of the present invention is to provide a method for reconstituting the freeze-dried formulation. Effect of the Invention

[0013] The lyophilized preparation according to the present invention contains a composition that confers structural stability to the α-galactosidase A fusion protein, and therefore not only has storage stability but also excellent stability even when it contains a high concentration of the fusion protein. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a graph comparing cake properties of freeze-dried preparations with and without amino acids. [Diagram 2] FIG. 1 is a graph showing a comparison of the recovery rates of natural freeze-dried preparations with added amino acids. [Diagram 3] FIG. 1 is a graph comparing cake properties of freeze-dried preparations at various amino acid concentrations. [Figure 4] FIG. 13 is a graph comparing the rate of formation of soluble aggregates in freeze-dried preparations at various amino acid concentrations before and after freeze-drying. [Diagram 5] FIG. 1 is a graph comparing the recovery rates of natural amino acids from freeze-dried preparations at various concentrations. [Figure 6] FIG. 13 Chromatograms of reconstituted lyophilized formulations confirm the formation of soluble aggregates (HMW1) and higher order aggregates (HMW2) in the lyophilized formulations at each amino acid concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] One embodiment of the present invention is a lyophilized formulation comprising an α-galactosidase A fusion protein. In one specific example, the lyophilized preparation is characterized by comprising a mixture obtained by lyophilizing an aqueous solution containing a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked, a buffer solution, and an amino acid.

[0016] In another specific example, the lyophilized preparation is characterized by comprising a lyophilized mixture of an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0, and 0.5 to 4.0% (w / v) of an amino acid.

[0017] In the freeze-dried preparation according to any of the above-mentioned specific examples, the freeze-dried preparation is characterized in that it contains a freeze-dried mixture of an aqueous solution containing 5-50 mg / ml of the fusion protein, a buffer solution of pH 5.5-7.0, 50-300 mM sodium chloride, and 0.5-4.0% (w / v) serine.

[0018] In the freeze-dried preparation according to any of the above-mentioned specific examples, the freeze-dried preparation is characterized in that it contains a freeze-dried mixture of an aqueous solution containing 5-50 mg / ml of the fusion protein, a buffer solution of pH 5.5-7.0, 0.002-0.1% (w / v) of polysorbate 20, 50-200 mM sodium chloride, and 1.0-4.0% (w / v) of serine.

[0019] In the freeze-dried preparation according to any of the above-mentioned specific examples, the freeze-dried preparation is characterized in that it contains a freeze-dried mixture of 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0 containing 1 to 30 mM histidine, 0.002 to 0.1% (w / v) polysorbate 20, 50 to 200 mM sodium chloride, and an aqueous solution containing 1.0 to 4.0% (w / v) serine.

[0020] In the freeze-dried preparation according to any of the above-mentioned specific examples, the freeze-dried preparation is characterized in that it contains a freeze-dried mixture of 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0 containing 10 to 30 mM histidine, 0.002 to 0.1% (w / v) polysorbate 20, 50 to 200 mM sodium chloride, 0.5 to 4.0% (w / v) of a sugar or sugar alcohol, and 0.5 to 4.0% (w / v) of serine.

[0021] In the lyophilized formulation according to any of the above-mentioned embodiments, the buffer solution is characterized in that it comprises histidine and its salts, citric acid and its salts, acetic acid and its salts, phosphoric acid and its salts, or a combination thereof.

[0022] In the lyophilized preparation according to any of the above-mentioned specific examples, the buffer solution is characterized in containing 1 to 50 mM histidine. The lyophilized formulation according to any of the previous embodiments, wherein the amino acid is selected from the group consisting of serine, arginine, threonine, glutamine, glycine, alanine, and combinations thereof.

[0023] In the freeze-dried preparation according to any of the above-mentioned specific examples, the aqueous solution is characterized in that it further contains 0.5 to 4.0% (w / v) of a sugar based on the total volume of the aqueous solution. The lyophilized formulation according to any of the above-mentioned embodiments, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, trehalose or a combination thereof.

[0024] In the freeze-dried preparation according to any of the above-mentioned specific examples, the aqueous solution is characterized in that it further contains 0.5 to 4.0% (w / v) of a sugar alcohol based on the total volume of the aqueous solution.

[0025] The lyophilized formulation according to any of the above-mentioned embodiments, wherein the sugar alcohol is mannitol, sorbitol or a combination thereof. In the lyophilized preparation according to any of the above-mentioned specific examples, the aqueous solution is characterized in that it further contains 0.002 to 0.1% (w / v) of a non-ionic surfactant.

[0026] The lyophilized 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.

[0027] In the lyophilized preparation according to any of the above-mentioned specific examples, the aqueous solution is characterized in that it further contains 5 to 300 mM of an isotonicity agent. In the lyophilized formulation according to any of the above-mentioned embodiments, the tonicity agent is characterized in that it is sodium chloride.

[0028] In any of the above-mentioned specific examples of the freeze-dried preparation, the sodium chloride is contained in the aqueous solution at 50 to 200 mM. In the lyophilized 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.

[0029] In the lyophilized formulation according to any of the aforementioned embodiments, the immunoglobulin Fc region is characterized in that it comprises the amino acid sequence of SEQ ID NO:3. In the lyophilized 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.

[0030] In the lyophilized formulation according to any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it is derived from IgG4. In the lyophilized formulation according to any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it is non-glycosylated.

[0031] In any of the lyophilized preparations according to the above-mentioned embodiments, the fusion protein is characterized in that two molecules of α-galactosidase A are linked to each monomer of a dimeric immunoglobulin Fc region.

[0032] The lyophilized formulation according to any of the above-mentioned embodiments is characterized in that the lyophilized formulation is for the prevention or treatment of Fabry disease. Another embodiment of the present invention is a method for producing the lyophilized formulation.

[0033] As one specific example, the method for producing the freeze-dried formulation is characterized by including a step of freeze-drying an aqueous solution containing an α-galactosidase A fusion protein in which α-galactosidase A and an immunoglobulin Fc region are bound, a buffer solution, and an amino acid.

[0034] In another specific example, the method for producing the freeze-dried formulation further comprises a step of mixing an isotonicity agent, a sugar, a sugar alcohol, a non-ionic surfactant, a preservative, or a combination thereof, with the aqueous solution.

[0035] Yet another embodiment of the present invention is a method of reconstituting a lyophilized formulation comprising the step of adding a reconstitution solution to said lyophilized formulation. In one embodiment, the reconstitution solution is distilled water.

[0036] In another specific example, the preparation reconstituted by the above method is characterized in that it contains an α-galactosidase A fusion protein at a concentration of 10 to 100 mg / mL. Hereinafter, an embodiment of the present invention will be described.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 lyophilized preparation comprising a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked to each other.

[0041] Specifically, the present invention relates to a lyophilized 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, the lyophilized mixture being prepared by lyophilizing an aqueous solution containing a buffer and an amino acid, the aqueous solution further comprising, but not limited to, an isotonicity agent, a sugar, a sugar alcohol, a non-ionic surfactant, a preservative, or a combination thereof.

[0042] In the present invention, the term "lyophilized preparation" refers to a drug that has been formulated in a lyophilized form. Specifically, the α-galactosidase A fusion protein is lyophilized together with a substance such as an excipient for stabilizing the α-galactosidase A fusion protein and exists in a solid state. In addition to the α-galactosidase A fusion protein that exerts the pharmacological effect of the lyophilized preparation, the other components contained in the lyophilized preparation are mixed with a stabilizer. In the present invention, the term "stabilizer" refers to a substance that stably maintains the components such as the active ingredient in the preparation for a predetermined period of time.

[0043] In the present invention, the freeze-dried formulation is a concept including freeze-dried materials. The freeze-dried formulation is prepared by freezing and drying in the form of a pre-lyophilized formulation containing a stabilizer for stabilizing the α-galactosidase A fusion protein and a pre-formulation containing the α-galactosidase A fusion protein. In the present invention, the freeze-dried formulation of the α-galactosidase A fusion protein may contain a therapeutically effective amount of the α-galactosidase A fusion protein, and the therapeutically effective amount of the α-galactosidase A fusion protein is placed in a single-use container or a multi-use container, but is not limited thereto. The freeze-dried formulation of the present invention has a composition that can stabilize the α-galactosidase A fusion protein during the freeze-drying process, so that the stability of the formulation is maintained even if it is stored and then reconstituted. In particular, the freeze-dried formulation of the present invention has an effect of maintaining stability even if it contains the α-galactosidase A fusion protein at a high concentration of 5 mg / mL to 50 mg / mL.

[0044] In addition, a lyophilized preparation containing an α-galactosidase A fusion protein according to the present invention may be stored in a container and reconstituted when administration to an individual is required. In the present invention, "reconstitution" means that the α-galactosidase A fusion protein is made administrable by liquefying the lyophilized substance in a solid state. Here, the concentration of the α-galactosidase A fusion protein contained in the lyophilized preparation of the present invention upon reconstitution is, but is not limited to, 1 mg / mL to 150 mg / mL, 10 mg / mL to 120 mg / mL, or 10 mg / mL to 100 mg / mL. In the lyophilization process, the concentration of the preliminary dosage form and the concentration after reconstitution may be different.

[0045] The lyophilized 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. Such a lyophilized preparation of the present invention is characterized by comprising a mixture obtained by lyophilizing an aqueous solution containing the α-galactosidase A fusion protein, a buffer solution, and an amino acid.

[0046] The α-galactosidase A fusion protein of the present invention is in the form in which α-galactosidase A and an immunoglobulin Fc region are fused, and during the freeze-drying, reconstitution or storage process, various external factors (pH, temperature, osmotic pressure, presence or absence of stabilizers, etc.) cause an unfolding phenomenon in which the protein structure is deformed, resulting in a loss of enzyme activity and significantly inhibiting the pharmacological effect of a preparation containing α-galactosidase A. In particular, α-galactosidase A is first unfolded at one of the two domains that form α-galactosidase A (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 the 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 lyophilized formulation, and soluble aggregates or higher-order aggregates are formed, the risk of immunogenicity increases, posing a safety issue for the formulation.

[0047] The present inventors have confirmed that, for the stability of a lyophilized preparation, it is important to include a composition that prevents the unfolding of α-galactosidase A fusion protein, and that even if a portion of α-galactosidase A is unfolded to form soluble aggregates, the structure of the active site is maintained until insoluble aggregates are formed, and the enzyme activity of α-galactosidase A is maintained. As a result, it has been elucidated that the addition of a stabilizer (such as an amino acid) is important to maximize the structural stability of the α-galactosidase A fusion protein and to prevent further unfolding of the protein in the formed soluble aggregates to form insoluble aggregates, thereby completing the present invention. The lyophilized preparation of the present invention can maintain the structure and activity of the fusion protein even after long-term storage, has low immunogenicity, and is excellent in drug safety.

[0048] A specific example of the freeze-dried preparation according to the present invention is an aqueous solution 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 freeze-dried preparation containing a mixture obtained by freeze-drying an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0, and 0.25 to 4.0% (w / v) of an amino acid.

[0049] Another example is a freeze-dried preparation containing 5 to 50 mg / ml of a fusion protein, a buffer solution of pH 5.5 to 7.0, and 0.5 to 4.0% (w / v) of an amino acid, and having a pH of 5.5 to 7.0.

[0050] Yet another example is a freeze-dried preparation comprising a mixture obtained by freeze-drying an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0, 50 to 200 mM sodium chloride, and 0.5 to 4.0% (w / v) serine.

[0051] Yet another example is a freeze-dried preparation comprising a freeze-dried mixture of an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0, 0.002 to 0.1% (w / v) polysorbate, 50 to 200 mM sodium chloride, and 0.5 to 4.0% (w / v) serine.

[0052] Yet another example is a freeze-dried preparation containing a dried mixture of 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0 containing 1 to 30 mM histidine, 0.002 to 0.1% (w / v) polysorbate 20, 50 to 200 mM sodium chloride, and 1.0 to 4.0% (w / v) serine.

[0053] Further examples include freeze-dried preparations containing a mixture obtained by drying an aqueous solution containing 5-50 mg / ml of fusion protein, a buffer solution of pH 5.5-7.0 containing 1-30 mM histidine, 0.002-0.1% (w / v) polysorbate 20, 50-200 mM sodium chloride, 0.5-4.0% (w / v) sugar or sugar alcohol, and 0.5-4.0% (w / v) serine. However, the freeze-dried preparations of the present invention are not limited to these.

[0054] In the present invention, the term "aqueous solution" refers to a substance that contains an α-galactosidase A fusion protein and allows the α-galactosidase A fusion protein to be stably stored and maintain stability during the lyophilization process and during reconstitution. In particular, the aqueous solution contains an excipient that stabilizes the α-galactosidase A fusion protein, thereby providing stability to the α-galactosidase A fusion protein during the lyophilization process and allowing the preparation of a lyophilized formulation with storage stability. The stabilizing agent may include a buffer and an amino acid. It may further include, but is not limited to, an isotonicity agent, a sugar, a sugar alcohol, a non-ionic surfactant, a preservative, and the like. In a protein such as an α-galactosidase A fusion protein, storage stability is important not only to ensure accurate dosing, but also to suppress potential generation of antigenic substances against the α-galactosidase A fusion protein. The aqueous solution is used in the present invention in combination with a "preliminary dosage form."

[0055] The concentration of the α-galactosidase A fusion protein in the aqueous solution is not particularly limited, since the concentration of the α-galactosidase A fusion protein can be adjusted by adjusting the volume of the reconstitution solution added to the lyophilized preparation. The aqueous solution has the advantage that it can stably freeze-dry even α-galactosidase A fusion protein with a high concentration of about 10 mg / mL or more, specifically about 10 mg / mL to 90 mg / mL, and the stability of the α-galactosidase A fusion protein in the reconstituted solution is maintained.

[0056] For example, the aqueous solution of the present invention contains the α-galactosidase A fusion protein at a concentration of about 1 to 90 mg / ml, about 1 to 70 mg / ml, about 1 to 50 mg / ml, about 5 to 50 mg / ml, about 5 to 90 mg / ml, about 10 to 50 mg / ml, or about 10 to 30 mg / ml, but is not limited to these.

[0057] 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."

[0058] It is generally known that the higher the concentration of 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, in the freeze-dried aqueous solution containing the stabilizer composition according to the present invention, the structure of the fusion protein can be maintained during the freeze-drying and reconstitution process even though the fusion protein is contained at a high concentration, and no precipitation occurs, so that the aqueous solution of the present invention contains a high concentration of α-galactosidase A fusion protein as an active ingredient, 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.

[0059] The buffer, which is one of the components contained in the aqueous solution of the present invention, refers to a solution that plays a role in maintaining the pH of the aqueous solution so that the pH of the dosage form does not change suddenly during the lyophilization process or after reconstitution. The aqueous solution is used as a solvent for the α-galactosidase A fusion protein and the stabilizer, and any solution 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.

[0060] The pH value affects the structure of the fusion protein, and the structure of the fusion protein is stably maintained within the range of pH 5.5 to 7.0. If the pH condition deviates from the optimum condition, α-galactosidase A unfolds before the immunoglobulin Fc region, inhibiting the structural stability, so it is important to maintain the pH at an appropriate level to ensure the structural stability of the α-galactosidase A fusion protein.

[0061] 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 hydrogen or dihydrogen salt thereof), 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 aqueous solution is contained in a concentration sufficient to maintain a pH for structural stability of the protein.

[0062] The pH of the aqueous solution 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 The pH range is, but is not limited to, 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, about pH 6.5 to 7.0, 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, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0.

[0063] 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.

[0064] As a specific example, the buffer solution contains about 20 mM histidine and has a pH of about 5.5 to 7.0, or about 5.5 to 6.5, but is not limited thereto. The amino acids contained in the aqueous solution of the present invention prevent the unfolding of the α-galactosidase A fusion protein in the aqueous solution and inhibit the formation of insoluble aggregates. If the structural modification (unfolding) of the fusion protein in the soluble aggregate state proceeds further to form insoluble aggregates, problems such as protein precipitation and loss of enzyme activity will occur.

[0065] In the experimental examples of the present invention, it was confirmed that, compared with a preliminary dosage form not containing amino acids, those containing amino acids (serine) exhibited favorable properties when produced into a lyophilized preparation, and had a superior recovery rate of natural substances. In addition, it was confirmed that the higher the concentration of amino acids, specifically, the more favorable the preparation, which is prepared by lyophilizing an aqueous solution containing 0.5% (w / v) or more of amino acids relative to the total volume of the aqueous solution, not only exhibited favorable properties, but also prevented the unfolding of α-galactosidase A fusion protein, and inhibited the formation of soluble aggregates and higher-order aggregates. Furthermore, the higher the concentration of amino acids, the higher the recovery rate of natural substances. 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.

[0066] The amino acid contained in the lyophilized formulation of the present invention is serine, arginine, lysine, 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 serine, arginine, threonine, glutamine, glycine, alanine, and a combination thereof.Specifically, but not limited to, the amino acid is serine.

[0067] The amino acid of the present invention is present in the aqueous solution at a concentration of about 0.1 to 5.0% (w / v), about 0.25 to 5.0% (w / v), about 0.25 to 4.0% (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 4.0% (w / v), about 2.0 to 5 ... The amount of glycerin present in the blood is, but is not limited to, about 2.5 to 5% (w / v), about 3.0 to 5% (w / v), about 2.0 to 4.5% (w / v), about 2.0 to 4.0% (w / v), or about 0.25% (w / v), about 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).

[0068] The aqueous solution according to the invention may further comprise a non-ionic surfactant, which is defined as a substance that reduces the surface tension of the protein solution and prevents the protein from adsorbing to hydrophobic surfaces and from aggregating after reconstitution.

[0069] 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 sulfate (sodium dodecyl sulphate (SDS), polysorbate or poloxamer, which may be used alone or in combination of two or more thereof.

[0070] 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.

[0071] The nonionic surfactant in the present invention is contained at a concentration of about 0.2% (w / v) or less relative to the total volume of the aqueous solution of the present invention, for example, about 0.001 to about 0.2% (w / v), about 0.001 to about 0.1% (w / v), about 0.002 to about 0.1% (w / v), about 0.01 to about 0.1% (w / v), about 0.01 to about 0.05% (w / v), about 0.02 to about 0.05% (w / v), about 0.025 to about 0.05% (w / v), or about 0.025% (w / v), but is not particularly limited thereto.

[0072] The aqueous solution according to the present invention may further contain an isotonicity agent. The isotonicity agent refers to a substance that plays a role in maintaining an appropriate osmotic pressure when the α-galactosidase A fusion protein is reconstituted and administered to the body. The isotonicity agent also has an additional effect of making the α-galactosidase A fusion protein more stable in the aqueous solution phase. Any isotonicity agent that contributes to stabilizing the α-galactosidase A fusion protein may be contained in the aqueous solution of the present invention.

[0073] The tonicity agent includes water-soluble inorganic salts, and specifically, the aqueous solution of the present invention includes sodium chloride as an isotonicity agent, but is not limited thereto. The concentration of sodium chloride used in the present invention is about 0 to 300 mM, specifically about 0.1 mM to 300 mM, such as 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, and about 50 mM to 100 mM, but is not limited thereto, and the components contained in the aqueous solution are adjusted to appropriate contents depending on the types and amounts so that a preliminary dosage form containing all of the mixtures becomes an isotonic solution.

[0074] The aqueous solution 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 aqueous solution. 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.

[0075] The aqueous solution according to the present invention may further contain 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.

[0076] 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 4.0% (w / v), about 1 to 10% (w / v), about 1 to 8% (w / v), about 1 to 6% (w / v), about 1 to 4% (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 volume of the aqueous solution, but is not particularly limited thereto.

[0077] The aqueous solution according to the present invention may contain a combination of different types of stabilizers with their contents adjusted. As specific examples, the aqueous solution according to the present invention may (i) not contain sugar and / or sugar alcohol and contain amino acid, or (ii) contain sugar and / or sugar alcohol and contain amino acid, but is not limited thereto. (i) In the aqueous solution containing no sugar and / or sugar alcohol and containing amino acid, the amino acid may be contained at about 1.0 to 4.0% (w / v), and (ii) In the aqueous solution containing sugar and / or sugar alcohol and containing amino acid, the sugar and / or sugar alcohol may be contained at about 0.5 to 4.0% (w / v) and the amino acid may be contained at about 0.5 to 4.0% (w / v).

[0078] The aqueous solution according to the present invention may further comprise 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 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.

[0079] In addition, the aqueous solution (preliminary dosage form) for producing the lyophilized formulation 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, sugar alcohol, sugar, and preservative, as long as the effects of the present invention are not impaired, but is not limited to these.

[0080] A specific example of an aqueous solution (preliminary dosage form) for producing a lyophilized formulation according to the present invention includes, but is not limited to, an aqueous solution containing 5 to 50 mg / ml of a fusion protein, a buffer solution of pH 5.5 to 7.0, and 0.5 to 4.0% (w / v) serine.

[0081] Another specific example of the aqueous solution (preliminary dosage form) for producing the lyophilized formulation according to the present invention includes, but is not limited to, an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0, 0.002 to 0.1% (w / v) of a nonionic surfactant, and 0.5 to 4.0% (w / v) of serine.

[0082] Yet another specific example of the aqueous solution (preliminary dosage form) for producing the lyophilized formulation according to the present invention includes, but is not limited to, an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0 containing 10 to 30 mM histidine, 0.002 to 0.1% (w / v) polysorbate, and 1.0 to 4.0% (w / v) serine.

[0083] Yet another specific example of the aqueous solution (preliminary dosage form) for producing the lyophilized formulation according to the present invention includes, but is not limited to, an aqueous solution containing 5 to 50 mg / ml of the fusion protein, a buffer solution of pH 5.5 to 7.0, 0.002 to 0.1% (w / v) of polysorbate 20, 50 to 200 mM sodium chloride, and 1.0 to 4.0% (w / v) of serine.

[0084] Yet another specific example of the aqueous solution (preliminary dosage form) for producing the lyophilized preparation according to the present invention includes, but is not limited to, an aqueous solution containing 5 to 50 mg / ml of the fusion protein, 10 to 30 mM histidine, 0.002 to 0.1% (w / v) polysorbate 20, 50 to 200 mM sodium chloride, 0.5 to 4.0% (w / v) of a sugar or sugar alcohol, and 0.5 to 4.0% (w / v) of serine.

[0085] In addition, the aqueous solution for producing the freeze-dried formulation according to the present invention or the freeze-dried formulation may further contain a pharma- ceutically acceptable carrier, excipient, etc., and such a carrier or excipient may be non-naturally occurring.

[0086] The aqueous solution for preparing the lyophilized formulation of the present invention is frozen and dried under suitable freezing and drying conditions known in the art. The drying may be completed in one step or in two or more steps.

[0087] The freeze-dried preparation of the present invention is obtained by preparing a preliminary dosage form containing an appropriate concentration of α-galactosidase A fusion protein in consideration of the intended dosage, freeze-drying it, and reconstituting it so that it can be preferably administered to an individual. Furthermore, the preliminary dosage form may be diluted to increase its volume and freeze-dried, and then diluted with a reconstitution solution having a smaller volume than the volume at the time of freeze-drying when reconstituted, but is not limited thereto. The freeze-dried preparation of the present invention means a mixture obtained by freeze-drying an aqueous solution containing α-galactosidase A fusion protein and a stabilizer (such as a buffer solution or amino acid), and also means a dosage form in which the freeze-dried preparation is reconstituted in a reconstitution solution, but is not limited thereto.

[0088] The α-galactosidase A fusion protein, which is an active ingredient contained in the lyophilized preparation of the present invention, will be described in more detail below. The α-galactosidase A fusion protein contained in the freeze-dried preparation 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 immunoglobulin Fc region that forms a dimer by a covalent bond via a linker. In addition, the two molecules of α-galactosidase A form a dimer by non-covalent bonding with each other, but are not limited thereto. The fusion protein of the present invention exerts a long-lasting 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 freeze-dried preparation of the present invention is obtained by freeze-drying a preliminary formulation that further contains a stabilizer to such a fusion protein, thereby imparting excellent stability to the fusion protein and preventing the loss of pharmacological effect even when stored for a long period of time.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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 formulation 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] The immunoglobulin Fc region of the α-galactosidase A fusion protein 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 by reductive amination to the ε-amino atom of lysine or the amino group at the N-terminus, but this is not limited thereto. As a specific example, the immunoglobulin Fc region is linked via a nitrogen atom of proline at its 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.

[0120] 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.

[0121] 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.

[0122] 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 to these methods.

[0123] The lyophilized preparation 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.

[0124] 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.

[0125] Specifically, the lyophilized 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.

[0126] 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.

[0127] 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 lyophilized 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 lyophilized preparation containing the same.

[0128] In a specific embodiment of the present invention, the lyophilized formulation of the present invention is used for the prevention or treatment of Fabry disease, but is not limited thereto. The lyophilized preparation of the present invention is administered via, but is not limited to, a subcutaneous route. Specifically, the lyophilized preparation of the present invention in a lyophilized state is reconstituted to be suitable for subcutaneous administration, but is not limited to, the lyophilized preparation of the present invention.

[0129] In the present invention, "administration" refers to introducing the reconstituted lyophilized formulation into a patient by any suitable method, and the route of administration of the lyophilized formulation may be any common route that can deliver the α-galactosidase A fusion protein of the lyophilized formulation to a target in the body, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration.

[0130] 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 lyophilized preparation of the present invention may be formulated so that the above dosage can be appropriately administered, or may be reconstituted so that the above dosage can be administered. However, the dosage of the fusion protein is determined by taking into consideration various factors such as the age, weight, health condition, sex, severity of disease, diet, and excretion rate of the patient, as well as the route of administration and number of treatments, 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 lyophilized preparation of the present invention.

[0131] Another embodiment of the present invention provides an aqueous solution or a pre-lyophilized formulation comprising an α-galactosidase A fusion protein, a buffer and an amino acid for producing a lyophilized formulation.

[0132] The aqueous solution or pre-lyophilized preparation may further contain at least one selected from an isotonicity agent, a sugar or sugar alcohol, and a nonionic surfactant, but is not limited thereto.

[0133] The aqueous solution, the lyophilized preparation and the components constituting them are as described above. Another embodiment of the present invention provides a method for producing the lyophilized formulation. Specifically, the method may include a step of lyophilizing an aqueous solution containing an α-galactosidase A fusion protein, a buffer solution, and an amino acid.

[0134] The aqueous solution or lyophilized formulation further comprises at least one component selected from the group consisting of, but not limited to, an isotonicity agent, a sugar or sugar alcohol, and a non-ionic surfactant.

[0135] The aqueous solution, the lyophilized preparation and the components constituting them are as described above. Yet another aspect of the present invention provides a method of reconstituting the lyophilized formulation, comprising adding a reconstitution solution to the lyophilized formulation.

[0136] The aqueous solutions, lyophilized formulations, and reconstitution are as described above. In the present invention, the term "reconstitution solution" refers to a solution that is added to a solid freeze-dried preparation to reconstitute it. An example of such a reconstitution solution is distilled water (water), but is not particularly limited thereto.

[0137] The reconstituted formulation contains the α-galactosidase A fusion protein at a concentration suitable for administration to an individual, specifically, but not limited to, the α-galactosidase A fusion protein at a concentration of 10-100 mg / mL, 10-90 mg / mL, 10-50 mg / mL, or 10-30 mg / mL.

[0138] The reconstituted formulation may be, but is not limited to, a lyophilized formulation according to the present invention that has been reconstituted to be suitable for subcutaneous administration to an individual. 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

[0139] Preparation of 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).

[0140] 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.

[0141] 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.

[0142] [Table 1-1]

[0143] [Table 1-2]

[0144] 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.

[0145] Specifically, CHO-S cells were cultured in suspension in a 1 L Erlenmeyer flask (Corning, cat. No. 431147) using serum-free medium (FreeStyle CHO Expression Medium, Thermo Fisher, cat. No. 12651014), and the cells in the culture vessel reached 5 × 10 8 Once the cells had grown to 100 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 the cells were placed in a fresh Freestyle CHO expression medium (Thermo Fisher, cat. No. 12651014) that had been replaced in advance. The cells were incubated at 37, 5% CO 2 The cells were cultured in an incubator at 125 rpm for approximately 96 hours to produce an α-galactosidase A fusion protein.

[0146] 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. EXAMPLES

[0147] Confirmation of stability of freeze-dried preparations with added amino acids Example 2-1: Preparation and reconstitution of freeze-dried formulations In order to confirm the change in stability of the freeze-dried preparation containing the α-galactosidase A fusion protein prepared in Example 1 due to the addition of amino acids, each freeze-dried preparation was prepared and the properties and size exclusion chromatography analysis of the freeze-dried preparation were performed.

[0148] To prepare the freeze-dried preparations, 1.0 ml of the solution of the preparation having the composition shown in Table 2 was dispensed into glass vials (3 ml), which were semi-stoppered with a rubber stopper, and then loaded onto the shelves of a freeze dryer (Freeze dryer, Lyostar 3, SP Scientific). Freeze drying was then performed under the conditions shown in Table 3, and the freeze-dried preparations thus prepared were capped with aluminum caps after the completion of the aluminum freeze-drying.

[0149] [Table 2]

[0150] [Table 3]

[0151] The stability of the freeze-dried preparation thus prepared was confirmed by reconstitution with 1.0 ml of DW (distilled water) and then by SE-HPLC analysis. Example 2-2: Size exclusion liquid chromatography (SE-HPLC) For size exclusion chromatography, the reconstituted lyophilized formulation was first diluted to 1.0 mg / ml with mobile phase (1x PBS, Lonza) and then subjected to sterile filtration. 200 μL of the filtered sample was then injected into a vial insert and inserted into a screw top vial.

[0152] 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. An analytical column (TSKgel G3000SWXL, Tosoh) was attached, and the mobile phase was run at a rate of 0.5 ml / min for 30 minutes or more to allow equilibration until the detector signal stabilized. When the temperature of the autosampler dropped to 4°C, the sample was placed 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. Analysis was performed using Empower Pro software on a PC.

[0153] Example 2-3: Confirmation of stability of freeze-dried preparations with added amino acids First, the cake properties of the freeze-dried preparations prepared in Example 2-1 were compared. Although some collapse occurred in the preparation containing a high concentration of isotonicity agent (NaCl), the freeze-dried preparations generally did not show any serious collapse and showed favorable properties. When reconstituted, the freeze-dried preparation containing serine showed a transparent property and was confirmed to have excellent properties (Figure 1).

[0154] In addition, the recovery rate of natural substances in the freeze-dried formulations prepared was confirmed by size exclusion chromatography. As a result, the recovery rate of natural substances was below 100% in all formulations without added serine, and the recovery rate decreased as the concentration of the isotonicity agent (NaCl) increased. In contrast, the recovery rate of natural substances in the formulations with added serine was 102.0%, confirming that the composition was stable (Figure 2).

[0155] Here, the term "natural product" refers to the α-galactosidase A fusion protein prepared in Example 1, which is a normal dimeric fusion protein that has not undergone aggregation or degradation. These definitions are similarly applied to the following examples.

[0156] The results of comparing the stability of the freeze-dried preparations containing an amino acid (serine) confirmed in this manner are summarized in Table 4.

[0157] [Table 4]

[0158] Example 3: Confirmation of stability of lyophilized preparations at various amino acid concentrations Example 3-1: Preparation and reconstitution of lyophilized formulations First, in order to confirm the change in stability of the freeze-dried formulation containing the α-galactosidase A fusion protein prepared in Example 1 at each amino acid concentration, each freeze-dried formulation was prepared and the properties and size exclusion chromatography analysis of the freeze-dried formulation were performed.

[0159] The solution of the formulation having the composition shown in Table 5 was dispensed into glass vials (3 ml) in 0.5 ml portions, semi-stoppered with a rubber stopper, and then loaded onto the shelves of a freeze dryer (Freeze dryer, Lyostar 3, SP Scientific). Freeze drying was then carried out under the conditions shown in Table 6, and the freeze-dried formulations thus produced were capped with aluminum caps after completion of the aluminum freeze-drying.

[0160] [Table 5]

[0161] [Table 6]

[0162] The stability of the freeze-dried preparation was confirmed by reconstitution with 0.25 ml of DW (distilled water) and then by SE-HPLC analysis. The composition of the reconstituted freeze-dried preparation is shown in Table 7 below. The SE-HPLC analysis was performed in the same manner as in Example 2-2.

[0163] [Table 7]

[0164] Example 3-2: Confirmation of stability of freeze-dried preparations at various amino acid concentrations A comparison of the cake properties of the freeze-dried preparations at each amino acid concentration revealed that no serious collapse phenomenon was observed in any of the freeze-dried preparations, and the cake properties were favorable (Figure 3).

[0165] Size exclusion chromatography analysis also confirmed that as the serine concentration in the lyophilized formulation increased, the amount of soluble aggregates decreased (Figure 4), the recovery rate of native aggregates increased (Figure 5), and the progression from soluble aggregates (HMW1) to higher-order aggregates (HMW2) was prevented (Figure 6). In particular, the recovery rate of native aggregates exceeded 100% in lyophilized formulations containing serine at a concentration of 2.0% or more, compared to the lyophilized preparations.

[0166] Here, the soluble aggregate is a form that has solubility due to the unfolding of a part of the domain of α-galactosidase A and maintains the activity of α-galactosidase A, and the higher-order aggregate means an aggregate formed by further unfolding of the protein in the soluble aggregate. The natural product means the α-galactosidase A fusion protein prepared in Example 1, which is a normal dimer-form fusion protein that has not undergone aggregation or decomposition. The risk of immunogenicity increases as the size of the aggregate increases due to the formation of higher-order aggregates, so the safety of the lyophilized preparation can be improved by preventing the formation of higher-order aggregates by adding serine.

[0167] All these experimental results suggest that lyophilized preparations containing amino acids are safer, and that a higher amino acid concentration, in particular, can prevent aggregate formation and is advantageous for maintaining the purity of the α-galactosidase A fusion protein.

[0168] The stability of the freeze-dried preparations containing the amino acid (serine) confirmed in this manner was compared at various amino acid concentrations, and the results are summarized in Table 8.

[0169] [Table 8]

[0170] 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 freeze-dried preparation comprising a fusion protein in which α-galactosidase A and an immunoglobulin Fc region are linked, The freeze-dried preparation comprises: 5-50 mg / ml of the fusion protein; A buffer solution having a pH of 5.5 to 7.0; and 0.5 to 4.0% (w / v) of an amino acid, Lyophilized 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 freeze-dried formulation according to claim 1.

3. The buffer contains 1 to 50 mM histidine. The freeze-dried formulation according to claim 1.

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

5. The aqueous solution further comprises 0.5 to 4.0% (w / v) sugar. The freeze-dried formulation according to claim 1.

6. The sugar is glucose, fructose, galactose, lactose, maltose, sucrose, trehalose or a combination thereof; The freeze-dried preparation according to claim 5.

7. The aqueous solution further comprises 0.5 to 4.0% (w / v) sugar alcohol. The freeze-dried formulation according to claim 1.

8. The sugar alcohol is mannitol, sorbitol or a combination thereof. The freeze-dried formulation according to claim 7.

9. The aqueous solution further comprises 0.002 to 0.1% (w / v) of a non-ionic surfactant. The freeze-dried formulation according to claim 1.

10. 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 freeze-dried formulation according to claim 9.

11. The aqueous solution further comprises 5 to 300 mM of an isotonicity agent. The freeze-dried formulation according to claim 1.

12. The tonicity agent is sodium chloride. The freeze-dried formulation according to claim 11.

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

1. The freeze-dried formulation according to claim 1.

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

4. The freeze-dried formulation according to claim 1.

15. 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 freeze-dried formulation according to claim 1.

16. The freeze-dried preparation comprises: 5-50 mg / ml of the fusion protein; 10 to 30 mM histidine; 0.002-0.1% (w / v) polysorbate 20; 50 to 200 mM sodium chloride; and 1.0 to 4.0% (w / v) serine. The freeze-dried formulation according to claim 1.

17. The freeze-dried preparation comprises: 5-50 mg / ml of the fusion protein; 10 to 30 mM histidine; 0.002-0.1% (w / v) polysorbate 20; 50 to 200 mM sodium chloride; 0.5 to 4.0% (w / v) sugar or sugar alcohol; and 0.5 to 4.0% (w / v) serine. The freeze-dried formulation according to claim 1.

18. The lyophilized preparation is for the prevention or treatment of Fabry disease. The freeze-dried formulation according to claim 1.

19. The method includes a step of freeze-drying an aqueous solution containing an α-galactosidase A fusion protein in which α-galactosidase A and an immunoglobulin Fc region are bound, a buffer solution, and an amino acid; A method for producing the lyophilized preparation according to any one of claims 1 to 18.

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

21. The reconstitution solution is distilled water.

21. The method of claim 20.

22. The formulation reconstituted by the method contains α-galactosidase A fusion protein at a concentration of 10 to 100 mg / mL.

21. The method of claim 20.

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