Stable pharmaceutical preparation

Abstract

To provide a pharmaceutical formulation which enables stable preservation of a drug capable of inhibiting vascular endothelial growth factor (VEGF) and allows for intraocular administration.SOLUTION: A stable pharmaceutical formulation contains: (A) a recombinant fusion protein; (B) a surfactant; (C) a sugar or a derivative thereof; and (D) a buffer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical preparation capable of stably storing a drug capable of inhibiting Vascular Endothelial Growth Factor (VEGF) and capable of being administered intraocularly. [Background technology]

[0002] Release of vascular endothelial growth factor (VEGF) contributes to increased vascular permeability and inappropriate new blood vessel growth in the eye, which can lead to vision loss due to neovascular (wet) age-related macular degeneration, vision loss due to retinal vein occlusion (central retinal vein occlusion or branch retinal vein occlusion) macular edema, vision loss due to diabetic macular edema, and vision loss due to choroidal neovascularization due to pathologic myopia. Thus, inhibiting VEGF is an effective method for treating ophthalmic diseases involving angiogenesis.

[0003] The active ingredient in the VEGF inhibitor is Aflibercept, a type of recombinant fusion protein that can inhibit Type A and Type B VEGF and PIGF (Placental Growth Factor). Pharmaceutical preparations containing this drug must be administered by intraocular injection directly by a licensed physician with experience in intraocular administration. Summary of the Invention [Problem to be solved by the invention]

[0004] To store VEGF inhibitors for long periods of time, a formulation containing histidine, trehalose, and polysorbate 20 was developed as a stabilizer, and sodium chloride was added as an isotonicity agent to achieve an osmotic pressure similar to that inside the eyeball in order to reduce pain during administration. However, it was confirmed that the stability of the drug decreases when sodium chloride is present as an isotonicity agent, and to improve this, the sodium chloride concentration was applied at a level that maintains the appropriate osmotic pressure. The formulation of the present invention has improved drug stabilization compared to the aflibercept liquid formulation of Korean Patent Publication No. 10-2009-0018807, and the improved level was directly compared and evaluated in an experimental example (Experimental Example 1). Therefore, the superiority of the stable pharmaceutical formulation of the present invention, which contains the VEGF inhibitor aflibercept, was finally confirmed.

[0005] Therefore, an object of the present invention is to provide a pharmaceutical preparation which contains the VEGF inhibitor aflibercept and is stable during long-term storage. Another problem to be solved by the present invention is to provide a pharmaceutical preparation having excellent long-term storage stability based on excellent stability under accelerated and harsh conditions. A further object of the present invention is to provide a stable pharmaceutical preparation which can be administered intraocularly. [Means for solving the problem]

[0006] A stable pharmaceutical formulation according to one embodiment of the present invention comprises (A) a recombinant fusion protein, (B) a surfactant, (C) a sugar or derivative thereof, and (D) a buffering agent.

[0007] In one embodiment of the invention, the pharmaceutical formulation may be in liquid form.

[0008] In one embodiment of the present invention, (A) the recombinant fusion protein may comprise a VEGF (Vascular Endothelial Growth Factor) antagonist. Cut. In one embodiment of the invention, (A) the recombinant fusion protein can include the extracellular domain of a human VEGF receptor. In one embodiment of the present invention, (A) the recombinant fusion protein can comprise the extracellular domain 1, 2, or a mixture thereof of a human VEGF receptor. In one embodiment of the invention, (A) the recombinant fusion protein may comprise the Fc portion of immunoglobulin G (IgG), preferably the Fc portion of human immunoglobulin G (IgG). In one embodiment of the present invention, (A) the recombinant fusion protein can comprise (1) an extracellular domain 1 element of a VEGF receptor comprising amino acids 27-129 of SEQ ID NO: 2, (2) an extracellular domain 2 element of a VEGF receptor comprising amino acids 130-231 of SEQ ID NO: 2, and (3) an Fc site element comprising amino acids 232-457 of SEQ ID NO: 2. The amino acids of SEQ ID NO: 2 are encoded by the polynucleotide of SEQ ID NO: 1. In one embodiment of the invention, (A) the recombinant fusion protein can include Aflibercept. In one embodiment of the present invention, the concentration of the (A) recombinant fusion protein may be 5 to 100 mg / ml.

[0009] In one embodiment of the invention, the (B) surfactant may comprise a polysorbate, a poloxamer, or a mixture thereof. In one embodiment of the invention, the (B) surfactant can include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture of two or more thereof. In one embodiment of the invention, the (B) surfactant may include polysorbate 20. In one embodiment of the present invention, the concentration of the surfactant (B) may be 0.01 to 0.1% (w / v).

[0010] In one embodiment of the invention, the (C) sugar can include a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or a mixture of two or more thereof, and the (C) sugar derivative can include a sugar alcohol, a sugar acid, or a mixture thereof. In one embodiment of the present invention, (C) sugar or derivative thereof may include sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof. In one embodiment of the invention, the (C) sugar or derivative thereof may include trehalose. In one embodiment of the present invention, the concentration of (C) the sugar or its derivative may be 1 to 20% (w / v).

[0011] In one embodiment of the present invention, (D) the buffering agent may include an amino acid. In one embodiment of the present invention, (D) the buffering agent may comprise a free amino acid, an amino acid salt, or a mixture thereof. In one embodiment of the invention, (D) the buffering agent may comprise histidine, a histidine salt, or a mixture thereof. In one embodiment of the present invention, the content of the buffer (D) may be 1 to 20 mM.

[0012] In one embodiment of the present invention, the stable pharmaceutical formulation of the present invention may be free of acetic acid, citric acid, phosphoric acid, or mixtures thereof.

[0013] In one embodiment of the present invention, the stable pharmaceutical formulation of the present invention further comprises (E) an isotonicity agent. It can additionally be included. In one embodiment of the present invention, (E) the tonicity agent can include sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl), or a mixture of two or more thereof. In one embodiment of the present invention, the content of the tonicity agent (E) may be 30 mM or less.

[0014] In one embodiment of the present invention, the formulation of the present invention may have a pH of 5.0 to 7.0.

[0015] In one embodiment of the invention, the formulations of the invention may be free of NaF, KBr, NaBr, Na2SO4, NaSCN, K2SO4, or mixtures thereof.

[0016] In one embodiment of the invention, the formulations of the present invention may be chelating agent free.

[0017] A stable pharmaceutical formulation according to one embodiment of the present invention can include (A) 5-100 mg / ml of a recombinant fusion protein comprising (1) one element of the extracellular domain of a VEGF receptor comprising amino acids 27-129 of SEQ ID NO:2, (2) two elements of the extracellular domain of a VEGF receptor comprising amino acids 130-231 of SEQ ID NO:2, and (3) an Fc site element comprising amino acids 232-457 of SEQ ID NO:2, (B) 0.01-0.1% (w / v) of a surfactant, (C) 1-20% (w / v) of a sugar or a derivative thereof, (D) 1-20 mM of a buffer, and (E) 30 mM or less of an isotonicity agent. The amino acids of SEQ ID NO:2 are encoded by a polynucleotide of SEQ ID NO:1.

[0018] A stable pharmaceutical formulation according to one embodiment of the present invention can include: (A) a Vascular Endothelial Growth Factor (VEGF) antagonist; (B) a polysorbate; (C) a sugar or a derivative thereof; and (D) histidine.

[0019] The stable pharmaceutical formulation according to one embodiment of the present invention may additionally comprise (E) sodium chloride.

[0020] A stable pharmaceutical formulation according to one embodiment of the present invention can include: (A) 5 to 100 mg / ml of a recombinant fusion protein comprising (1) one element of the extracellular domain of a VEGF receptor comprising amino acids 27 to 129 of SEQ ID NO:2, (2) two elements of the extracellular domain of a VEGF receptor comprising amino acids 130 to 231 of SEQ ID NO:2, and (3) an Fc site element comprising amino acids 232 to 457 of SEQ ID NO:2; (B) 0.01 to 0.1% (w / v) polysorbate; (C) 1 to 20% (w / v) of a sugar or a derivative thereof; and (D) 1 to 20 mM histidine.

[0021] The stable pharmaceutical formulation according to one embodiment of the present invention may additionally comprise (E) sodium chloride, up to 30 mM.

[0022] In one embodiment of the present invention, the stable pharmaceutical formulation may have a number of insoluble foreign particles of 10.00 μm or more and less than 400.00 μm or less, measured by HIAC, after storage at 5±3° C. for 9 months.

[0023] In one embodiment of the present invention, the stable pharmaceutical formulation can exhibit a content of the main ingredient of 98% or more after storage at 5±3° C. for 10 days.

[0024] In one embodiment of the present invention, the stable pharmaceutical formulation has a shelf life of 10 days after storage at 5±3° C. It can exhibit a content of high molecular weight components of 1% or less.

[0025] In one embodiment of the present invention, the stable pharmaceutical formulation can exhibit a content of low molecular weight components of less than 0.05% after storage at 5±3° C. for 10 days.

[0026] In one embodiment of the present invention, the stable pharmaceutical formulation can exhibit a charge variant content of 87% or more when stored at 5±3° C. for 9 months.

[0027] In one embodiment of the present invention, the stable pharmaceutical formulation exhibits a VEGF binding affinity of 90% or more when stored at 5±3° C. for 9 months.

[0028] In one embodiment of the present invention, the stable pharmaceutical formulation may be for intraocular administration, preferably for intravitreal administration.

[0029] In one embodiment of the invention, the stable pharmaceutical formulation may not undergo a reconstitution step, a dilution step, or both, prior to use.

[0030] A glass vial according to one embodiment of the present invention is filled with the stable pharmaceutical formulation.

[0031] A pre-filled syringe according to one embodiment of the present invention is filled with the stable pharmaceutical formulation. Effect of the Invention

[0032] The stable pharmaceutical formulations according to the present invention contain recombinant fusion proteins and have low viscosity, maintain excellent stability under long-term storage conditions, accelerated conditions and harsh conditions, and are administrable intraocularly. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] [Stable pharmaceutical formulation] A stable pharmaceutical formulation according to the present invention comprises: (A) a recombinant fusion protein; (B) a surfactant; (C) a sugar or derivative thereof; and (D) a buffering agent.

[0034] In the specification of the present application, the term "free" means that the component is not contained at all. The term also means that the component is substantially free, that is, the component is contained to an extent that does not affect the activity of the fusion protein, the stability and viscosity of the pharmaceutical preparation, for example, 0-1% (w / v), 0-1 ppm (w / v), or 0-1 ppb (w / v) based on the total weight of the pharmaceutical preparation. The pharmaceutical preparation may be in a liquid form, but is not limited thereto.

[0035] (A) Recombinant fusion protein The recombinant fusion protein of the present invention may be a VEGF (Vascular Endothelial Growth Factor) antagonist. The recombinant fusion protein of the present invention may comprise (1) one element of the extracellular domain of a VEGF receptor comprising amino acids 27-129 of SEQ ID NO:2, (2) two elements of the extracellular domain of a VEGF receptor comprising amino acids 130-231 of SEQ ID NO:2, and (3) an Fc site element comprising amino acids 232-457 of SEQ ID NO:2. The amino acids of SEQ ID NO:2 are encoded by the polynucleotide of SEQ ID NO:1.

[0036] The recombinant fusion protein of the present invention can comprise the extracellular domain of a human VEGF receptor, and more specifically, can comprise, but is not limited to, the extracellular domain 1, 2, or a mixture thereof of a human VEGF receptor. The recombinant fusion protein may also include, but is not limited to, the Fc portion of human immunoglobulin G (IgG). The recombinant fusion protein can include, but is not limited to, Aflibercept.

[0037] The concentration of the recombinant fusion protein of the present invention can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the stable pharmaceutical preparation according to the present invention. In one embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 100 mg / ml. In another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 95 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 90 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 85 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 80 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 75 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 70 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 65 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 60 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 55 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 50 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 45 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 5 to 40 mg / ml.

[0038] In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 100 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 90 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 80 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 70 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 60 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 50 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 10 to 40 mg / ml.

[0039] Furthermore, in yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 100 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 90 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 80 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 70 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 60 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 50 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 20 to 40 mg / ml.

[0040] In addition, in yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 100 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 90 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 80 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 70 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 60 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 50 mg / ml. In yet another embodiment of the present invention, the concentration of the recombinant fusion protein may be 30 to 40 mg / ml.

[0041] (B) Surfactant Examples of surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (e.g., polysorbates), polyoxyethylene alkyl ethers (e.g., Brij), alkylphenyl polyoxyethylene ethers (e.g., Triton-X), polyoxyethylene-polyoxypropylene copolymers (e.g., Poloxamer, Pluronic), sodium dodecyl sulfate (SDS), poloxamers, and mixtures thereof.

[0042] In one embodiment of the present invention, the surfactant may include polysorbate, poloxamer, or a mixture thereof, specifically, polyoxyethylene sorbitan fatty acid ester (polysorbate), poloxamer, or a mixture thereof. More specifically, the surfactant may include, but is not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture of two or more thereof. In another embodiment of the present invention, the polysorbate may include, but is not limited to, polysorbate 20.

[0043] In one embodiment of the present invention, the concentration of the surfactant can be freely adjusted within a range that does not adversely affect the stability and viscosity of the stable pharmaceutical preparation according to the present invention. For example, the concentration of the surfactant is preferably 0.001-5% (w / v), 0.005-2% (w / v), more preferably 0.01-1% (w / v), 0.01-0.5% (w / v), 0.01-0.1% (w / v), or 0.01-0.05% (w / v), but is not limited thereto.

[0044] (C) Sugar or sugar derivative The sugars may include monosaccharides, disaccharides, oligosaccharides, polysaccharides, or mixtures of two or more thereof. Examples of monosaccharides include, but are not limited to, glucose, fructose, galactose, etc. Examples of disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, etc. Examples of oligosaccharides include, but are not limited to, fructooligosaccharides, galactooligosaccharides, mannanoligosaccharides, etc. Examples of polysaccharides include, but are not limited to, starch, glycogen, cellulose, chitin, pectin, etc.

[0045] Sugar derivatives can include sugar alcohols, sugar acids, or mixtures thereof. Examples of sugar alcohols include, but are not limited to, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, and the like. Examples of sugar acids include aldonic acids (e.g., glyceric acid), urosonic acids (e.g., neuraminic acid), uronic acids (e.g., glucuronic acid), and aldaric acids (e.g., tartaric acid). , but not limited to these.

[0046] In one embodiment of the present invention, the sugar or its derivatives may include sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof. In another embodiment of the present invention, the sugar or its derivatives may include, but are not limited to, trehalose.

[0047] In one embodiment of the present invention, the concentration of the sugar or its derivative can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical preparation according to the present invention. For example, the concentration of the sugar or its derivative may be 0.1-30% (w / v), 0.5-25% (w / v), or more preferably 1-20% (w / v). According to one embodiment of the present invention, it may be 0.5-20% (w / v) or 1-25% (w / v). According to one embodiment of the present invention, it may be 1-20% (w / v), 2-15% (w / v), 4-13% (w / v), or 8-12% (w / v).

[0048] (D) Buffer A buffer is a neutralizing substance that minimizes changes in pH caused by acids or alkalies. Examples of buffers include phosphate, acetate, succinate, gluconate, citrate, as well as the amino acids glutamate and histidine.

[0049] In one embodiment of the present invention, the buffering agent may include an amino acid, more specifically, but is not limited to, a free amino acid, an amino acid salt, or a mixture thereof. In one embodiment of the present invention, the buffering agent may include, but is not limited to, histidine, a histidine salt, or a mixture thereof.

[0050] When histidine salts are used as buffers, they include, for example, histidine chloride, histidine acetate, histidine phosphate, histidine sulfate, etc. It is preferable to include histidine as a buffer in terms of pH adjustment and stability, but the buffer is not limited to this. In addition, other types of acids may be included for pH adjustment, but according to one embodiment of the present invention, the buffer may not include acetic acid, citric acid, phosphoric acid, or a mixture thereof, and the buffer is not limited to this.

[0051] In one embodiment of the present invention, the content of the buffering agent can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical preparation according to the present invention. In one embodiment of the present invention, the content of the buffering agent may be 1 to 20 mM or 1 to 10 mM. In one embodiment of the present invention, the content of the buffering agent may be 2 to 20 mM or 2 to 10 mM. In one embodiment of the present invention, the content of the buffering agent may be 3 to 20 mM or 3 to 10 mM. In one embodiment of the present invention, the content of the buffering agent may be 4 to 20 mM or 4 to 10 mM. In one embodiment of the present invention, the content of the buffering agent may be 5 to 20 mM or 5 to 10 mM. In one embodiment of the present invention, the content of the buffering agent may be 6 to 20 mM or 6 to 10 mM. In one embodiment of the present invention, the content of the buffering agent may be 1 to 20 mM. In one embodiment of the present invention, the content of the buffering agent may be 6.4 to 9.6 mM, but is not limited thereto.

[0052] (E) Tonicity agent In one embodiment of the present invention, (E) a tonicity agent may additionally be included. The isotonicity agent may be sodium chloride, potassium chloride, calcium chloride, or two or more of these. The mixture may include:

[0053] In one embodiment of the present invention, the content of the isotonicity agent can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical preparation according to the present invention. In one embodiment of the present invention, the content of the isotonicity agent may be 30 mM or less. In one embodiment of the present invention, the content of the isotonicity agent may be 0.00001 to 30 mM, 0.0001 to 30 mM, 0.001 to 30 mM, 0.01 to 30 mM, 0.1 to 30 mM, or 1 to 30 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 0.00001 to 30 mM, 0.00001 to 25 mM, or 0.00001 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 0.0001 to 30 mM, 0.0001 to 25 mM, or 0.0001 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 0.001 to 30 mM, 0.001 to 25 mM, or 0.001 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 0.01 to 30 mM, 0.01 to 25 mM, or 0.01 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 0.1 to 30 mM, 0.1 to 25 mM, or 0.1 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 1 to 30 mM, 1 to 25 mM, or 1 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 2 to 30 mM, 2 to 25 mM, or 2 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be 3 to 30 mM, 3 to 25 mM, or 3 to 20 mM. In one embodiment of the present invention, the content of the isotonicity agent may be, but is not limited to, 5 to 18 mM, 6 to 17 mM, or 7.2 to 15.6 mM.

[0054] (F) pH In one embodiment of the present invention, the pH of the stable pharmaceutical composition may be 5.0 to 7.0, 5.0 to 6.5, 5.5 to 7.0, or 5.5 to 6.5. When the pH is within this range, it can have excellent long-term stability and low viscosity. The pH can be adjusted using a buffer. In other words, when the buffer is contained at a predetermined content, it can show a pH in the above range without another pH adjuster. In one embodiment of the present invention, the composition may not contain acetic acid, citric acid, phosphoric acid, or a mixture thereof. When acetic acid (acetate), citric acid (citrate), phosphoric acid, or a mixture thereof is used as a buffer, it may be difficult to show a pH in the above range. When an acid (e.g., hydrochloric acid) or a base (e.g., sodium hydroxide) is additionally contained as another pH adjuster, the stability of the target recombinant fusion protein may be reduced.

[0055] (G) Other Ingredients In one embodiment of the present invention, the stable pharmaceutical preparation may not contain preservatives.Examples of preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, etc. If preservatives are included, they may not be useful for improving stability.

[0056] Additionally, in one embodiment of the present invention, the stable pharmaceutical formulation may be free of NaF, KBr, NaBr, Na2SO4, NaSCN, K2SO4, or mixtures thereof, and may be free of chelating agents, but is not limited thereto.

[0057] (H) "Stable" Pharmaceutical Formulations In the "stable" pharmaceutical formulation of the present invention, the term "stable" refers to the physical stability and / or the ability of the recombinant fusion protein according to the present invention to be maintained during the manufacturing process and / or during storage / preservation. or substantially retains chemical stability and / or biological activity. A variety of analytical techniques for measuring antibody stability are readily available in the art. Stability is measured at a selected temperature for a selected period of time.

[0058] Physical stability can be evaluated by methods known in the art, including measuring the apparent attenuation of light (absorbance or optical density) of a sample, which is related to the turbidity of the formulation. Physical stability can also be measured by measuring the content of high molecular weight components, the content of low molecular weight components, the amount of intact protein, the number of insoluble foreign particles, etc.

[0059] Chemical stability can be assessed, for example, by detecting and quantifying chemically altered forms of the recombinant fusion protein. Chemical stability includes charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography. Charge variants (acidic or basic peaks), etc., can be measured for chemical stability.

[0060] Biological activity can be assessed by methods known in the art, for example, measuring antigen binding affinity by ELISA.

[0061] In one embodiment of the present invention, the term "stable" pharmaceutical formulation refers to a pharmaceutical formulation that satisfies one or more of the following:

[0062] (H)-1 turbidity -After storing for 4 weeks at a temperature of 5±3℃, the absorbance A measured by a spectrophotometer 600 is 0 to 0.0700 or 0 to 0.0400; -After storing the containers at 10℃, 15℃, 20℃, 25℃, 30℃, or 35℃ for 4 weeks under sealed conditions, the absorbance A measured by a spectrophotometer 600 is 0 to 0.0700 or 0 to 0.0400; -After storing for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5%, and sealed conditions, the absorbance A measured with a spectrophotometer 600 is 0 to 0.0700 or 0 to 0.0400;

[0063] (H)-2 Content of main component (main peak) - A pharmaceutical preparation having a content of 98-99% of the main ingredient as determined by SE-HPLC after 9 months of storage at a temperature of 5±3°C; - a pharmaceutical preparation having a content of 89-92% of the main ingredient as measured by SE-HPLC after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5% and under sealed conditions;

[0064] (H)-3 High molecular weight component (peak whose retention time is ahead of the main peak (complete recombinant fusion protein)) - A pharmaceutical preparation having a high molecular weight content of 98-100% as measured by SE-HPLC after storage for 9 months at a temperature of 5±3°C; - a pharmaceutical preparation having 7-10% of high molecular weight components as measured by SE-HPLC after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5% and sealed conditions;

[0065] (H)-4 Low molecular weight component (peak with a retention time later than the main peak (complete recombinant fusion protein)) - A pharmaceutical preparation having a low molecular weight component content of 0.0-0.1% as measured by SE-HPLC after storage for 9 months at a temperature of 5±3°C; -After storing for 4 weeks under sealed conditions at a temperature of 40±2°C and a relative humidity of 75±5%, SE- A pharmaceutical preparation having 0.0-1.2% low molecular weight components as determined by HPLC;

[0066] (H)-5 Content of one complete recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of the Fc of recombinant human IgG (1VEGF1+1VEGF2+Fc) - a pharmaceutical preparation having a content of 96-98% of the complete extracellular domain 1 of recombinant human VEGF receptor, the extracellular domain 2 of recombinant human VEGF receptor, and a portion of the Fc of recombinant human IgG (1VEGF1+1VEGF2+Fc) as measured by reduced CE-SDS after storage for 9 months at a temperature of 5±3°C; - a pharmaceutical preparation having a content of 90-96% of the complete extracellular domain 1 of recombinant human VEGF receptor, the extracellular domain 2 of recombinant human VEGF receptor, and a portion of the Fc of recombinant human IgG (1VEGF1+1VEGF2+Fc) as measured by reduced CE-SDS after storage for 4 weeks under conditions of temperature 40±2°C, relative humidity 75±5%, and sealed condition;

[0067] (H)-6 Number of insoluble foreign particles - pharmaceutical preparations with a number of insoluble foreign particles (2.00 μm ≦, < 400.00 μm) between 0 and 100, measured by HIAC, after 9 months of storage at a temperature of 5 ± 3 °C; - pharmaceutical preparations with a number of insoluble foreign particles (10.00 μm ≦, < 400.00 μm) between 0 and 50, measured by HIAC, after 9 months of storage at a temperature of 5 ± 3 °C; - pharmaceutical preparations with a number of insoluble foreign particles (25.00 μm ≦, < 400.00 μm) between 0 and 5, measured by HIAC, after 9 months of storage at a temperature of 5 ± 3 °C; - pharmaceutical preparations having a number of insoluble foreign particles (2.00μm≦, <400.00μm) between 0 and 100 particles as measured by HIAC after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5% and sealed conditions; - pharmaceutical preparations having a number of insoluble foreign particles (10.00μm≦, <400.00μm) between 0 and 50 particles as measured by HIAC after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5% and sealed conditions; - pharmaceutical preparations having a number of insoluble foreign particles (25.00μm≦, <400.00μm) between 0 and 5, as measured by HIAC, after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5% and sealed conditions; - a pharmaceutical preparation having a number of insoluble foreign particles (1.00 μm ≦, < 400.00 μm) between 0 and 5,000 particles as measured by MFI after 9 months of storage at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (10.00 μm ≦, < 400.00 μm) between 0 and 100, measured by MFI, after storage for 9 months at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (25.00 μm ≦, < 400.00 μm) between 0 and 15, measured by MFI, after storage for 9 months at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (10.00 μm ≦, <100.00 μm) between 0 and 1,000 particles as measured by MFI after storage for 4 weeks at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (10.00 μm ≦, <100.00 μm) between 0 and 100 particles as measured by MFI after storage for 4 weeks at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (25.00 μm ≦, <100.00 μm) between 0 and 100 particles as measured by MFI after storage for 4 weeks at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (25.00 μm ≦, <100.00 μm) between 0 and 10, measured by MFI, after storage for 4 weeks at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (1.00 μm ≦, <100.00 μm) between 0 and 5,000 particles as measured by MFI after storage for 4 weeks at a temperature of 40 ± 2 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (1.00 μm ≦, <100.00 μm) between 0 and 2,500 particles as measured by MFI after storage for 4 weeks at a temperature of 40 ± 2 °C; -After storing at 40±2℃ for 4 weeks, the insoluble foreign particles (10.00μm) measured by MFI ≦, <100.00 μm) in a pharmaceutical preparation with a number of 0 to 1,000 particles; - a pharmaceutical preparation having a number of insoluble foreign particles (10.00 μm ≦, <100.00 μm) between 0 and 100 particles as measured by MFI after storage for 4 weeks at a temperature of 40 ± 2 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (25.00 μm ≦, <100.00 μm) between 0 and 100 particles as measured by MFI after storage for 4 weeks at a temperature of 40 ± 2 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (25.00 μm ≦, <100.00 μm) between 0 and 10, measured by MFI, after storage for 4 weeks at a temperature of 40 ± 2 °C; - a pharmaceutical preparation having a number of insoluble foreign particles (1.00 μm ≦, < 400.00 μm) between 0 and 20,000 particles as measured by MFI after storage for 4 weeks at a temperature of 40 ± 2°C, a relative humidity of 75 ± 5%, and sealed conditions; - a pharmaceutical preparation having a number of insoluble foreign particles (10.00 μm ≦, < 400.00 μm) between 0 and 70 particles as measured by MFI after storage for 4 weeks at a temperature of 40 ± 2°C, a relative humidity of 75 ± 5% and sealed conditions; - a pharmaceutical preparation having a number of insoluble foreign particles (25.00 μm ≦, < 400.00 μm) between 0 and 15, as measured by MFI, after storage for 4 weeks at a temperature of 40 ± 2°C, a relative humidity of 75 ± 5%, and sealed conditions;

[0068] (H)-7 oxidation rate - pharmaceutical preparations with an oxidation rate of heavy chain Met192 between 0 and 7% as measured by LC-MS after storage for 4 weeks at a temperature of 5 ± 3°C; - pharmaceutical preparations with an oxidation rate of heavy chain Met192 between 0 and 6% as measured by LC-MS after storage for 4 weeks at a temperature of 40 ± 2°C, a relative humidity of 75 ± 5% and under closed conditions;

[0069] (H)-8 Charged variant - A pharmaceutical preparation having a relative content of peak 11 in theoretical peak 4 of 89-91% as measured by cIEF after storage for 4 weeks at a temperature of 5±3°C. - a pharmaceutical preparation having a sum of 11 peaks in the theoretical peak 4 of 86-88% as measured by cIEF after 9 months of storage at a temperature of 5 ± 3 °C; - a pharmaceutical preparation having a total of 86-88% of the theoretical peak 4 in the sum of peak 11 as measured by cIEF after storage for 4 weeks at a temperature of 40 ± 2°C, a relative humidity of 75 ± 5% and under closed conditions; - A pharmaceutical preparation having a relative content of peak 11 in theoretical peak 4 of 86-88% as measured by cIEF after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5%, and sealed conditions.

[0070] (H)-9VEGF binding affinity - a pharmaceutical preparation having a VEGF binding affinity of 75-95% as measured by ELISA after storage for 9 months at a temperature of 5 ± 3°C; - a pharmaceutical preparation that has a VEGF binding affinity of 60-75% as measured by ELISA after storage for 4 weeks at a temperature of 40±2°C, a relative humidity of 75±5% and under closed conditions;

[0071] [Method of manufacturing stable pharmaceutical formulations] The stable pharmaceutical preparation of the present invention can be produced by using known methods and is not limited to a specific method. For example, a pharmaceutical preparation can be produced by adding a buffer to a solution containing a surfactant, a sugar or its derivative, and an isotonic agent, adjusting the pH, and then adding a recombinant fusion protein to this mixed solution. In addition, a pharmaceutical preparation can be produced by producing a solution containing some excipients in the final step of the purification process, and then adding the remaining components. For example, a pharmaceutical preparation can be produced by producing a solution containing a recombinant fusion protein, a buffer, a sugar or its derivative, and an isotonic agent in the final step of the purification process, and then adding a surfactant to this solution.

[0072] Additionally, the preparation may not require a freeze-drying step during production. When the freeze-drying step is not included, for example, the pharmaceutical preparation of the present invention can be produced and, after treatment such as sterilization, directly placed in a sealed container such as a glass vial or a prefilled syringe, which is a primary packaging material.

[0073] [Method of use of stable pharmaceutical preparations] The stable pharmaceutical formulation according to the present invention can be used to treat diseases caused by VEGF leakage, including, but not limited to, neovascular (wet) age-related macular degeneration, vision loss due to retinal vein occlusion (central retinal vein occlusion or branch retinal vein occlusion) macular edema, vision loss due to diabetic macular edema, and vision loss due to choroidal neovascularization caused by pathological myopia.

[0074] The stable pharmaceutical formulations according to the invention may be used once or several times and must be administered by a licensed physician with experience in intraocular administration (injection).

[0075] The concentrations of the recombinant fusion protein and other components in the pharmaceutical preparation are as described above, and the total volume of the pharmaceutical preparation may be 0.05 to 0.3 ml.

[0076] The dosage and administration timing of the pharmaceutical preparations vary depending on the type of disease, the severity and progression of the disease, the patient's health and response to treatment, and the judgment of the treating physician. The recommended dosage is limited to 2 mg (equivalent to about 50 microliters), but the administration interval and administration timing are not limited. For example, after 2 mg of a product containing the pharmaceutical preparation is administered intraocularly based on the concentration of recombinant fusion protein, the same dosage can be administered at least every 2 weeks or at most every 2 months. Administration intervals longer than 16 weeks have not been studied. In one embodiment of the invention, the stable pharmaceutical formulation does not have to go through a reconstitution step, a dilution step, or both, prior to use.

[0077] [Treatment and stabilization methods] The present invention also provides a method for treating a patient suffering from a disease caused by leakage of VEGF using a stable pharmaceutical formulation comprising: (A) a recombinant fusion protein; (B) a surfactant; (C) a sugar or its derivative; and (D) a buffering agent. The present invention further provides a method for stabilizing a stable pharmaceutical formulation comprising: (A) a recombinant fusion protein; (B) a surfactant; (C) a sugar or derivative thereof; and (D) a buffering agent by providing conditions for the pharmaceutical formulation that stabilize the recombinant fusion protein.

[0078] In one embodiment of the method of treatment or stabilization, the pharmaceutical formulation may be in liquid form.

[0079] In one embodiment of the method of treatment or stabilization, (A) the recombinant fusion protein can include a VEGF (Vascular Endothelial Growth Factor) antagonist. In one embodiment of the treatment or stabilization method, (A) the recombinant fusion protein can include the extracellular domain of a human VEGF receptor. In one embodiment of the treatment or stabilization method, (A) the recombinant fusion protein can comprise the extracellular domain 1, 2, or a mixture thereof of the human VEGF receptor. In one embodiment of the method of treatment or stabilization, (A) the recombinant fusion protein may comprise the Fc portion of immunoglobulin G (IgG), preferably the Fc portion of human immunoglobulin G (IgG). In one embodiment of the treatment or stabilization method, (A) the recombinant fusion protein can include (1) one element of the extracellular domain of a VEGF receptor comprising amino acids 27 to 129 of SEQ ID NO:2, (2) two elements of the extracellular domain of a VEGF receptor comprising amino acids 130 to 231 of SEQ ID NO:2, and (3) an Fc site element comprising amino acids 232 to 457 of SEQ ID NO:2. In one embodiment of the method of treatment or stabilization, (A) the recombinant fusion protein can comprise Aflibercept. In one embodiment of the treatment method or stabilization method, the concentration of the recombinant fusion protein (A) may be 5 to 100 mg / ml.

[0080] In one embodiment of the method of treatment or stabilization, the (B) surfactant can include a polysorbate, a poloxamer, or a mixture thereof. In one embodiment of the method of treatment or stabilization, the (B) surfactant can include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture of two or more thereof. In one embodiment of the method of treatment or stabilization, the (B) surfactant can include polysorbate 20. In one embodiment of the treatment method or stabilization method, the concentration of the surfactant (B) may be 0.01 to 0.1% (w / v).

[0081] In one embodiment of the treatment or stabilization method, the (C) sugar can include a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or a mixture of two or more thereof, and the (C) sugar derivative can include a sugar alcohol, a sugar acid, or a mixture thereof. In one embodiment of the method of treatment or stabilization, the (C) sugar or derivative thereof can include sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof. In one embodiment of the method of treatment or stabilization, the (C) sugar or derivative thereof may comprise trehalose. In one embodiment of the treatment method or stabilization method, the concentration of the (C) sugar or a derivative thereof may be 1 to 20% (w / v).

[0082] In one embodiment of the method of treating or stabilizing, (D) the buffering agent can include an amino acid. In one embodiment of the method of treating or stabilizing, (D) the buffering agent can include a free amino acid, an amino acid salt, or a mixture thereof. In one embodiment of the method of treating or stabilizing, (D) the buffering agent can include histidine, a histidine salt, or a mixture thereof. In one embodiment of the treatment method or stabilization method, the content of the buffering agent (D) may be 1 to 20 mM.

[0083] In one embodiment of the method of treatment or stabilization, the stable pharmaceutical formulation of the present invention may be free of acetic acid, citric acid, phosphoric acid, or mixtures thereof.

[0084] In one embodiment of the method of treatment or stabilization, (E) an isotonicity agent may be additionally included. (E) The tonicity agent may include sodium chloride, potassium chloride, calcium chloride, or a mixture of two or more thereof. In one embodiment of the treatment method or stabilization method, the content of the isotonicity agent (E) may be 30 mM or less.

[0085] In one embodiment of the treatment method or stabilization method, the formulation of the present invention may have a pH of 5.0 to 7.0.

[0086] In one embodiment of the method of treatment or stabilization, the formulation of the invention may be free of NaF, KBr, NaBr, Na2SO4, NaSCN, K2SO4, or mixtures thereof.

[0087] In one embodiment of the invention, the formulations of the present invention may be chelating agent free.

[0088] A stable pharmaceutical preparation according to one embodiment of the treatment method or stabilization method can include (A) 5 to 100 mg / ml of a recombinant fusion protein including (1) one element of the extracellular domain of a VEGF receptor including amino acids 27 to 129 of SEQ ID NO: 2, (2) two elements of the extracellular domain of a VEGF receptor including amino acids 130 to 231 of SEQ ID NO: 2, and (3) an Fc site element including amino acids 232 to 457 of SEQ ID NO: 2, (B) 0.01 to 0.1% (w / v) of a surfactant, (C) 1 to 20% (w / v) of a sugar or a derivative thereof, and (D) 1 to 20 mM of a buffer. The pharmaceutical preparation according to one embodiment of the treatment method or stabilization method can additionally include (E) 30 mM or less of an isotonicity agent.

[0089] The pharmaceutical formulation according to one embodiment of the method for treating or stabilizing the present invention may include (A) a vascular endothelial growth factor (VEGF) antagonist; (B) a polysorbate; (C) a sugar or a derivative thereof; and (D) histidine. The pharmaceutical formulation according to one embodiment of the method for treating or stabilizing the present invention may further include (E) sodium chloride.

[0090] A pharmaceutical preparation according to one embodiment of the treatment method or stabilization method can include (A) 5 to 100 mg / ml of a recombinant fusion protein including (1) one element of an extracellular domain of a VEGF receptor including amino acids 27 to 129 of SEQ ID NO: 2, (2) two elements of an extracellular domain of a VEGF receptor including amino acids 130 to 231 of SEQ ID NO: 2, and (3) an Fc site element including amino acids 232 to 457 of SEQ ID NO: 2; (B) 0.01 to 0.1% (w / v) polysorbate; (C) 1 to 20% (w / v) of a sugar or a derivative thereof; and (D) 1 to 20 mM histidine. A pharmaceutical preparation according to one embodiment of the treatment method or stabilization method can additionally include (E) 30 mM or less of sodium chloride.

[0091] According to one embodiment of the method of treatment or stabilization, the stable pharmaceutical preparation may have 50 or less insoluble foreign particles measuring 10.00 μm or more and less than 400.00 μm when stored at 5±3° C. for 9 months, as measured by HIAC.

[0092] According to one embodiment of the method of treatment or stabilization, the stable pharmaceutical preparation may exhibit a content of the main ingredient of 98% or more after storage at 5±3° C. for 10 days.

[0093] A stable pharmaceutical formulation according to one embodiment of the method of treatment or stabilization may exhibit a content of high molecular weight components of 1% or less after storage at 5±3° C. for 10 days.

[0094] The stable pharmaceutical preparation according to one embodiment of the method of treatment or stabilization may exhibit a content of low molecular weight components of 0.05% or less after storage at 5±3° C. for 10 days.

[0095] According to one embodiment of the method of treatment or stabilization, the stable pharmaceutical preparation may exhibit a charge variant content of 87.5% or more when stored at 5±3° C. for 9 months.

[0096] The stable pharmaceutical preparation according to one embodiment of the method of treatment or stabilization may exhibit a VEGF binding affinity of 90% or more when stored at 5±3° C. for 9 months.

[0097] In one embodiment of the method of treatment or stabilization, the stable pharmaceutical formulation may be for intraocular administration, preferably for intravitreal administration.

[0098] In one embodiment of the method of treatment or stabilization, the stable pharmaceutical formulation may not undergo a reconstitution step, a dilution step, or both, prior to use.

[0099] According to one embodiment of the treatment or stabilization method, a glass vial is filled with the stable pharmaceutical formulation.

[0100] According to one embodiment of the method of treatment or stabilization, a pre-filled syringe is filled with the stable pharmaceutical formulation.

[0101] [product] The present invention also provides an article of manufacture comprising: the stable pharmaceutical formulation; and a container housing the stable pharmaceutical formulation in a sealed state. The stable pharmaceutical formulation is as described above.

[0102] In one embodiment of the present invention, the container may be made of, but is not limited to, glass, polymer (plastic), metal, and other materials.In one embodiment of the present invention, the container may be, but is not limited to, a bottle, a vial, a cartridge, a syringe (pre-filled syringe), or a tube.In one embodiment of the present invention, the container may be, but is not limited to, a glass or polymer vial, or a glass or polymer pre-filled syringe.

[0103] The specific product forms of the vial, cartridge, prefilled syringe, etc. and the method of filling the stable pharmaceutical preparation into the vial, cartridge, prefilled syringe, etc. can be easily obtained or practiced by those having ordinary skill in the art to which the present invention belongs. For example, U.S. Patent Nos. 4,861,335 and 6,331,174 disclose specific product forms and filling methods of prefilled syringes. The vial, cartridge, prefilled syringe, etc., which are commercially available, can be used as they are, or a product made to order can be used in consideration of the physical properties, administration site, dosage, etc. of the stable pharmaceutical preparation.

[0104] In one embodiment of the invention, the article of manufacture may further comprise instructions providing a method of use, storage, or all of the above, of the stable pharmaceutical formulation, including a method of treating a disease caused by VEGF leakage, including a route of administration, dosage, and timing of administration. In one embodiment of the invention, the product may include other tools necessary from a commercial and user standpoint, such as needles, syringes, etc.

[0105] The present invention will be described in detail with reference to the following examples. The following examples are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited to the following examples.

[0106] Working Example In connection with the recombinant fusion proteins used in the following experimental examples, aflibercept was used that was cultured and purified at Celltrion Laboratories.

[0107] The following methods were used to measure the physical stability, chemical stability and biological activity of the pharmaceutical preparations used in the following experimental examples. -Turbidity The absorbance at 600 nm was measured using a UV-Vis spectrophotometer. -Main ingredient content The content of the main component (main peak;%) was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC). - High molecular weight component content The content of high molecular weight components (pre-peak;%) was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC). -Low molecular weight component content The content of low molecular weight components (post-peak;%) was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC). -Contains one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of the Fc of recombinant human IgG (1VEGF1+1VEGF2+Fc). The contents (%) of one complete recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of the Fc of recombinant human IgG (1VEGF1 + 1VEGF2 + Fc) were measured using reduced capillary electrophoresis-sodium dodecyl sulfate (RCE-SDS). - Number of insoluble foreign particles (sub-visible particles) The number of insoluble foreign particles was measured using Micro Flow Imaging (MFI) and a light-obscuration type particle counter (model name: HIAC9703). -Oxidation The oxidation rate (%) of Met192 in the heavy chain was measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS). - Charge transformation product (relative content of peak 11 in peak 4 out of the total 12 peaks) The acidic and basic peaks (%) were measured by capillary iso-electric focusing (cIEF). -VEGF binding affinity Enzyme-Linked ImmunoSorbent The VEGF binding affinity (%) was measured by ELISA (ELISA Assay).

[0108] Experimental Example 1: Comparison of histidine buffer concentrations; Comparison of pH 5.5-pH 6.5; Comparison of sodium chloride isotonicity agent concentrations; Comparison of trehalose concentrations; and confirmation of the superiority of the preparation of the present invention In relation to the pharmaceutical preparations used in Experimental Example 1, each buffer solution was prepared according to each pH, ​​and then trehalose and sodium chloride were added thereto, and the recombinant fusion protein and surfactant were added thereto to prepare the samples shown in Table 1. The specific content of each component is as shown in Table 1. Examples 1-9 and Comparative Example 1 were supplemented with aflibercept purified by Celltrion Research Institute, and Comparative Example 2 was a sample in which aflibercept produced by another company was added to the same pharmaceutical preparation as Comparative Example 1, which is Regeneron's Eylea product. The target volume of each sample filled into a glass vial was 0.278 ml. The pharmaceutical preparations prepared in Examples 1-9 and Comparative Examples 1-2 were stored at temperatures of 5±3°C and 40±2°C and 75±5% relative humidity. After 0 weeks and 2 weeks at 5±3°C, The stability was measured after 4 weeks, and after 2 and 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%. The results are shown in Table 2-15.

[0109] [Table 1]

[0110] [Table 2]

[0111] From Table 2, it can be seen that all of Examples 1-9, which set the range of the formulation of the present invention, are transparent pharmaceutical formulations with very low turbidity even after 4 weeks at a temperature condition of 5±3°C. In particular, it can be seen that the absorbance is 0.0400 or less even after 4 weeks at a temperature condition of 40±2°C and relative humidity condition of 75±5%. It can be seen that the results are similar to those of the existing formulations (Comparative Examples 1-2).

[0112] [Table 3]

[0113] From Table 3, it can be seen that the contents of the main components of all Examples 1-9, which set the range of the formulation of the present invention, are similar to the results of Comparative Examples 1-2 after 4 weeks at a temperature of 5±3° C. In contrast, the contents of the main components of Examples 1-9 are higher than those of Comparative Examples 1-2 after 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%, indicating that the formulation of the present invention is an improved pharmaceutical formulation in which the target recombinant fusion protein can be stored more stably.

[0114] [Table 4]

[0115] From Table 4, it can be seen that after 4 weeks at a temperature of 5±3° C., the contents of the high molecular weight components in all of Examples 1-9, which define the range of the formulation of the present invention, are similar to the results of Comparative Examples 1-2. In contrast, after 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%, the contents of the high molecular weight components in Examples 1-9 are lower than those in Comparative Examples 1-2, indicating that the formulation of the present invention is an improved pharmaceutical formulation in which the target recombinant fusion protein can be stored more stably.

[0116] [Table 5]

[0117] From Table 5, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the contents of low molecular weight components in all of Examples 1-9, which define the range of the formulation of the present invention, are pharmaceutical formulations similar to the results of Comparative Examples 1-2. In addition, after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, it can be seen that the contents of low molecular weight components in Examples 2-9 are pharmaceutical formulations at a similar level to Comparative Example 1. In contrast, throughout Example 1, the condition of pH 5.5 showed that the contents of low molecular weight components were similar to the results of Comparative Examples 1-2. It was found that the content was slightly less favorable than that of Comparative Example 1-2, and therefore it was found that the target pH and pH range needed to be adjusted slightly higher. The results of the experiment in which the pH range was adjusted are included in Experimental Example 2.

[0118] [Table 6]

[0119] From Table 6, it can be seen that after 4 weeks at a temperature of 5±3° C., the contents of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc) of all Examples 1-9, which define the range of the formulation of the present invention, are pharmaceutical formulations similar to the results of Comparative Example 1-2. In addition, after 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%, the contents of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc) of Examples 2-9 are pharmaceutical formulations at a similar level to Comparative Example 1-2. However, it was found through Example 1 that the pH 5.5 condition was somewhat unfavorable compared to Comparative Example 1-2 in terms of the content of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of the Fc of recombinant human IgG (1VEGF1+1VEGF2+Fc), and therefore the target pH and pH range needed to be adjusted slightly higher. The results are included in Experimental Example 2.

[0120] [Table 7]

[0121] From Table 7, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the number of insoluble foreign particles (2.00μm≦, <400.00μm) measured by HIAC in all Examples 1-9, which set the range of the formulation of the present invention, is a very clean pharmaceutical formulation similar to the result of Comparative Example 1. In addition, after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, the number of insoluble foreign particles (2.00μm≦, <400.00μm) measured by HIAC in Examples 1-9 is a pharmaceutical formulation at a similar level to Comparative Example 1.

[0122] [Table 8]

[0123] From Table 8, it can be seen that after 4 weeks at a temperature of 5±3° C., the number of insoluble foreign particles (10.00 μm≦, <400.00 μm) measured by HIAC for all Examples 1-9, which set the range of the formulation of the present invention, is very clean pharmaceutical formulations, similar to the results of Comparative Example 1. In addition, after 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%, the number of insoluble foreign particles measured by HIAC for all Examples 1-9, which set the range of the formulation of the present invention, is very clean pharmaceutical formulations, similar to the results of Comparative Example 1. It can be seen that the number of insoluble foreign particles (10.00 μm≦, <400.00 μm) measured by HIAC for No. 1-9 is a pharmaceutical preparation having a similar level to that of Comparative Example 1.

[0124] [Table 9]

[0125] From Table 9, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the number of insoluble foreign particles measured by HIAC in all of Examples 1-9, which set the range of the formulation of the present invention (25.00 μm≦, <400.00 μm), is a very clean pharmaceutical formulation similar to the result of Comparative Example 1. In addition, after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, the number of insoluble foreign particles measured by HIAC in Examples 1-9 is at a similar level to Comparative Example 1.

[0126] [Table 10]

[0127] From Table 10, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the number of insoluble foreign particles measured by MFI in all of Examples 1-9, which set the range of the formulation of the present invention (1.00 μm≦, <100.00 μm), is a very clean pharmaceutical formulation similar to the result of Comparative Example 1. In addition, after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, the number of insoluble foreign particles measured by MFI in Examples 1-9 is a pharmaceutical formulation at a similar level to Comparative Example 1.

[0128] [Table 11]

[0129] From Table 11, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the number of insoluble foreign particles measured by MFI in all of Examples 1-9, which set the range of the formulation of the present invention (10.00μm≦, <100.00μm), is a very clean pharmaceutical formulation similar to the result of Comparative Example 1. In addition, after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, the number of insoluble foreign particles measured by MFI in Examples 1-9 is a pharmaceutical formulation at a similar level to Comparative Example 1.

[0130] [Table 12]

[0131] From Table 12, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the number of insoluble foreign particles measured by MFI in all of Examples 1-9, which set the range of the formulation of the present invention (25.00μm≦, <100.00μm), is a very clean pharmaceutical formulation similar to the result of Comparative Example 1. In addition, after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, the number of insoluble foreign particles measured by MFI in Examples 1-9 is a pharmaceutical formulation at a similar level to Comparative Example 1.

[0132] [Table 13]

[0133] From Table 13, it can be seen that after 4 weeks at a temperature of 5±3°C, all of the Examples 1-9, which set the range of the formulation of the present invention, are pharmaceutical formulations with oxidation rates (Met192) similar to the results of Comparative Example 1-2. In addition, after 4 weeks at a temperature of 40±2°C and a relative humidity of 75±5%, the oxidation rates (Met192) of Examples 1-9 are pharmaceutical formulations with a similar level to that of Comparative Example 1-2. It is clear that this is a pharmaceutical preparation.

[0134] [Table 14]

[0135] From Table 14, it can be seen that after 4 weeks at 5±3°C, all the charge variants of Examples 1-9 (relative content of peak 11 in peak 4 of the total 12 peaks) that define the range of the formulation of the present invention are pharmaceutical formulations similar to the results of Comparative Example 1-2. It can also be seen that after 4 weeks at 40±2°C and 75±5% relative humidity, the charge variants of Examples 1-9 (relative content of peak 11 in peak 4 of the total 12 peaks) are pharmaceutical formulations at a similar level to Comparative Example 1-2.

[0136] [Table 15]

[0137] From Table 15, it can be seen that after 4 weeks at a temperature of 5±3° C., all of the Examples 1-9, which define the range of the formulation of the present invention, are pharmaceutical formulations with VEGF binding affinity similar to the results of Comparative Example 1-2. In addition, after 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%, the VEGF binding affinity of Examples 1-9 is also at a similar level to that of Comparative Example 1-2. It is known to be a drug.

[0138] Example 2: Adjustment of target pH and pH range to improve low molecular weight content In relation to the pharmaceutical preparation used in Experimental Example 2, each buffer solution was prepared according to each pH, ​​and then trehalose and sodium chloride were added thereto, and the recombinant fusion protein and surfactant were added to prepare the samples in Table 16 below. The specific content of each component is as shown in Table 16. The following example was added with aflibercept purified by Celltrion Research Institute, and Comparative Example 1 is a sample containing Regeneron's Eylea product and aflibercept manufactured by another company. The difference between Example 2 and Example 10 is the protein concentration, and the difference between Examples 10-14 is the pH. The target volume of glass vials filled with each sample was 0.278 ml. The pharmaceutical preparations prepared in Examples 2, 10 to 14 and Comparative Example 1 were stored at 5±3° C. and 50±2° C., and their stability was measured after 0 days and 10 days at 5±3° C., and after 5 days and 10 days at 50±2° C. The results are shown in Tables 17-24.

[0139] [Table 16]

[0140] [Table 17]

[0141] From Table 17, it can be seen that the contents of the main components of Examples 2 and 10 to 14, which set the range of the formulation of the present invention, after 10 days at a temperature condition of 5±3°C are pharmaceutical formulations similar to the results of Comparative Example 1. In contrast, after 10 days at a temperature condition of 50±2°C, the contents of the main components of Examples 2 and 10 to 14 are higher than that of Comparative Example 1, indicating that the formulations of the present invention are improved pharmaceutical formulations in which the target recombinant fusion protein can be stored more stably.

[0142] [Table 18]

[0143] From Table 18, it can be seen that after 10 days at a temperature condition of 5±3°C, the contents of high molecular weight components in Examples 2 and 10 to 14, which set the range of the formulation of the present invention, are lower than Comparative Example 1. In addition, even after 10 days at a temperature condition of 50±2°C, the contents of high molecular weight components in Examples 2 and 10 to 14 are lower than Comparative Example 1, which shows that the formulation of the present invention is an improved pharmaceutical formulation in which the target recombinant fusion protein can be stored more stably.

[0144] [Table 19]

[0145] From Table 19, it can be seen that the contents of low molecular weight components in Examples 2 and 10 to 14, which set the range of the formulation of the present invention, are similar to the results of Comparative Example 1 after 10 days at a temperature condition of 5±3°C. In contrast, it can be seen that the contents of low molecular weight components in Examples 2 and 10 to 14 are slightly higher than Comparative Example 1 after 10 days at a temperature condition of 50±2°C. From the contents of low molecular weight components in Examples 11 to 14, it can be seen that the lower the pH, the higher the low molecular weight content. From Example 11, it can be seen that the formulation at pH 5.5 has a characteristic that the content of low molecular weight components increases rapidly by about 4% or more beyond the fluctuation level of the analytical method compared to Example 13, which is a pharmaceutical formulation at the target pH. Therefore, it can be confirmed that it is better to set the pH range of the formulation of the present invention to 5.9 to 6.5.

[0146] [Table 20]

[0147] From Table 20, it can be seen that after 10 days at a temperature condition of 5±3°C, the contents of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a part of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc) in Examples 2 and 10 to 14, which set the range of the formulation of the present invention, are pharmaceutical formulations similar to the results of Comparative Example 1. In contrast, after 10 days at a temperature condition of 50±2°C, it can be seen that the contents of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a part of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc) in Examples 2 and 10 to 14 are slightly lower than those of Comparative Example 1. The contents of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and recombinant human IgG Fc in Examples 11 to 14 are slightly lower than those of Comparative Example 1. Looking at the content of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a part of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc), it can be seen that the lower the pH, the lower the content of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a part of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc). Looking at Example 11, it can be confirmed that the content of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a part of recombinant human IgG Fc (1VEGF1+1VEGF2+Fc) in the formulation at pH 5.5 is rapidly reduced by about 10% or more beyond the analytical method fluctuation level compared to Example 13, which is a pharmaceutical formulation at the target pH. Therefore, it was confirmed that the pH range of the formulation of the present invention is better when set to 5.9 to 6.5.

[0148] [Table 21]

[0149] From Table 21, it can be seen that after 10 days at a temperature condition of 5±3°C, the number of insoluble foreign particles (1.00μm≦, <100.00μm) measured by MFI of Examples 2 and 10-14, which sets the range of the formulation of the present invention, is absolutely low, and it is a very clean pharmaceutical formulation. Also, after 10 days at a temperature condition of 50±2°C, the number of insoluble foreign particles (1.00μm≦, <100.00μm) measured by MFI of Examples 2 and 10-14 is absolutely low, and it is a very clean pharmaceutical formulation.

[0150] [Table 22]

[0151] As shown in Table 22, after 10 days at a temperature of 5±3° C., the number of insoluble foreign particles measured by MFI for Examples 2, 10 to 14, which set the range of the formulation of the present invention (10.00 μm≦, <10 It can be seen that the number of insoluble foreign particles (10.00 μm≦, <100.00 μm) measured by MFI in Examples 2 and 10 to 14 is absolutely low even after 10 days at a temperature and condition of 50±2°C, and it can be seen that the pharmaceutical preparations are very clean.

[0152] [Table 23]

[0153] From Table 23, it can be seen that after 10 days at a temperature condition of 5±3° C., the number of insoluble foreign particles (25.00 μm≦, <100.00 μm) measured by the MFI of Examples 2 and 10 to 14, which set the range of the formulation of the present invention, is absolutely low, making it a very clean pharmaceutical formulation. Also, after 10 days at a temperature and condition of 50±2° C., the number of insoluble foreign particles (25.00 μm≦, <100.00 μm) measured by the MFI of Examples 2 and 10 to 14, which set the range of the formulation of the present invention, is absolutely low, making it a very clean pharmaceutical formulation.

[0154] [Table 24]

[0155] From Table 24, it can be seen that the VEGF binding affinity of Examples 2 and 10-14, which set the range of the formulation of the present invention, after 10 days at a temperature condition of 5±3°C, is a pharmaceutical formulation similar to the result of Comparative Example 1. In contrast, it can be seen that the VEGF binding affinity of some of Examples 2 and 10-14, which set the range of the formulation of the present invention, is slightly higher than that of Comparative Example 1 after 10 days at a temperature condition of 50±2°C. From the VEGF binding affinity of Examples 11-14, it can be seen that the lower the pH, the lower the VEGF binding affinity. From Example 11, it can be seen that the formulation of pH 5.5 has a characteristic that the VEGF binding affinity is rapidly reduced by about 38% or more, exceeding the fluctuation level of the analytical method, compared to Example 13, which is a pharmaceutical formulation of the target pH. Therefore, it can be confirmed that the pH range of the formulation of the present invention is better when set to 5.9-6.5.

[0156] Experimental Example 3: Further comparison for reinforcing the concentration range of sodium chloride isotonicity agent and confirmation of the superiority of the formulation of the present invention In relation to the pharmaceutical preparations used in Experimental Example 2, each buffer solution was prepared according to the concentration of sodium chloride, and then the recombinant fusion protein and surfactant were added to prepare the samples in Table 25. The specific contents of each component are as shown in Table 25. Examples 15-16 and Comparative Example 1 are samples to which aflibercept purified by Celltrion Research Institute was added. The target volume of each sample filled into a glass vial was 0.278 ml. The pharmaceutical preparations prepared in Examples 15-16 and Comparative Example 1 were stored at 5±3° C. and 40±2° C. and 75±5% relative humidity, and their stability after 4 weeks at 5±3° C., and after 2 and 4 weeks at 40±2° C. and 75±5% relative humidity was measured. The results are shown in Tables 26-31.

[0157] [Table 25]

[0158] [Table 26]

[0159] From Table 26, it can be seen that the turbidity of Examples 15-16 under low concentration sodium chloride conditions is very low even after 4 weeks at a temperature of 5±3°C, and the pharmaceutical formulations are transparent. In particular, it can be seen that the absorbance is below 0.0400 even after 4 weeks at a temperature of 40±2°C and a relative humidity of 75±5%. It can be seen that the results are similar to those of the existing formulation (Comparative Example 1).

[0160] [Table 27]

[0161] From Table 27, it can be seen that after 4 weeks at 5±3°C, the charge transformation bodies of Examples 15-16 under low sodium chloride conditions (relative content of peak 11 in peak 4 among all 12 peaks) are pharmaceutical preparations similar to the results of Comparative Example 1. It can also be seen that after 4 weeks at 40±2°C and 75±5% relative humidity, the charge transformation bodies of Examples 15-16 (relative content of peak 11 in peak 4 among all 12 peaks) are pharmaceutical preparations at a similar level to Comparative Example 1.

[0162] [Table 28]

[0163] From Table 28, it can be seen that after 4 weeks at a temperature condition of 5±3°C, the number of insoluble foreign particles measured by MFI in Examples 15-16 under low concentration sodium chloride conditions (1.00 μm≦, <100.00 μm) is a very clean pharmaceutical preparation similar to the results of Comparative Example 1. In addition, even after 4 weeks at a temperature condition of 40±2°C and a relative humidity condition of 75±5%, the number of insoluble foreign particles measured by MFI in Examples 15-16 is absolutely lower than that of Comparative Example 1, showing that it is a very clean pharmaceutical preparation.

[0164] [Table 29]

[0165] From Table 29, it can be seen that the number of insoluble foreign particles (10.00 μm≦, <100.00 μm) measured by MFI of Examples 15-16 under low-concentration sodium chloride conditions after 4 weeks at a temperature of 5±3° C. is similar to the result of Comparative Example 1, indicating that the pharmaceutical preparations are very clean. In addition, even after 4 weeks at a temperature of 40±2° C. and a relative humidity of 75±5%, the number of insoluble foreign particles measured by MFI of Examples 15-16 under low-concentration sodium chloride conditions is similar to the result of Comparative Example 1. The number of insoluble foreign particles (10.00 μm≦, <100.00 μm) measured with an MFI of 16 was absolutely lower than that of Comparative Example 1, indicating that this is a very clean pharmaceutical preparation.

[0166] [Table 30]

[0167] From Table 30, it can be seen that after 4 weeks at a temperature of 5±3°C, the number of insoluble foreign particles measured by MFI in Examples 15-16 under low-concentration sodium chloride conditions (25.00 μm≦, <100.00 μm) is a very clean pharmaceutical preparation, similar to the results of Comparative Example 1. In addition, after 4 weeks at a temperature of 40±2°C and a relative humidity of 75±5%, the number of insoluble foreign particles measured by MFI in Examples 15-16 is a very clean pharmaceutical preparation, similar to the results of Comparative Example 1.

[0168] [Table 31]

[0169] From Table 31, it can be seen that after 4 weeks at 5±3°C, the VEGF binding affinity of Examples 15-16 under low sodium chloride conditions is similar to that of Comparative Example 1. It can also be seen that after 4 weeks at 40±2°C and 75±5% relative humidity, the VEGF binding affinity of Examples 15-16 is similar to that of Comparative Example 1.

[0170] Experimental Example 4: Confirmation of the superiority of the formulation of the present invention through long-term stability evaluation of Example 13 The long-term stability evaluation of Example 13 was carried out in accordance with the guidelines provided by the International Conference on Harmonisation (ICH) (Guideline Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological or Biological Products and ICH Guideline Q1A(R2): Stability Testing of New Drug Substances and Drug Products). 0.278 mL of the stable liquid pharmaceutical formulation of Example 13 prepared by the method of Experimental Example 1 was stored in a sealed container at 5±3° C. / ambient relative humidity. The stability was confirmed after 0 months, 3 months, 6 months, and 9 months at the above temperature and humidity. However, the protein concentration was 40 mg / mL. To confirm the long-term stability of Example 13, visual analysis and SoloVPE were performed. The protein concentration was measured, the contents of the main component, high molecular weight, and low molecular weight components were measured by SEC-HPLC, the contents of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a part of the Fc of recombinant human IgG (1VEGF + 1VEGF2 + Fc) were measured by reduced CE-SDS, the charge variants (the relative content of peak 11 in peak 4 out of the total 12 peaks) were measured by cIEF, and the VEGF binding affinity and the number of insoluble foreign particles (sub-visible particles) were measured. The results are shown in Tables 32 to 41.

[0171] [Table 32]

[0172] From Table 32, it can be seen that Example 13 is a stable pharmaceutical preparation with no visible changes even after 9 months under long-term storage temperature conditions.

[0173] [Table 33]

[0174] From Table 33, it can be seen that Example 13 is a stable pharmaceutical preparation with no change in protein concentration even after 9 months under long-term storage temperature conditions.

[0175] [Table 34]

[0176] From Table 34, it can be seen that Example 13 is a stable pharmaceutical preparation with no change in the content of high molecular weight components even after 9 months under long-term storage temperature conditions.

[0177] [Table 35]

[0178] From Table 35, it can be seen that Example 13 is a stable pharmaceutical preparation with no change in the content of low molecular weight components even after 9 months under long-term storage temperature conditions.

[0179] [Table 36]

[0180] Table 36 shows that Example 13 is a stable pharmaceutical formulation with no change in the content of one recombinant human VEGF receptor extracellular domain 1, one recombinant human VEGF receptor extracellular domain 2, and a portion of the Fc of recombinant human IgG (1VEGF1+1VEGF2+Fc) even after 9 months under long-term storage temperature conditions.

[0181] [Table 37]

[0182] From Table 37, it can be seen that Example 13 is a very clean pharmaceutical preparation with a low number of insoluble foreign particles (2.00 μm≦, <400.00 μm) even after 9 months under long-term storage temperature conditions.

[0183] [Table 38]

[0184] From Table 38, it can be seen that Example 13 is a very clean pharmaceutical preparation with a low number of insoluble foreign particles (10.00 μm≦, <400.00 μm) even after 9 months under long-term storage temperature conditions.

[0185] [Table 39]

[0186] From Table 39, it can be seen that Example 13 is a very clean pharmaceutical preparation with a low number of insoluble foreign particles (25.00 μm≦, <400.00 μm) even after 9 months under long-term storage temperature conditions.

[0187] [Table 40]

[0188] From Table 40, it can be seen that Example 13 is a stable pharmaceutical preparation with no change in the content of charge transformants (relative content of peak 11 in peak 4 out of a total of 12 peaks) even after 9 months under long-term storage temperature conditions.

[0189] [Table 41]

[0190] As can be seen from Table 41, Example 13 is a stable pharmaceutical preparation with no change in VEGF binding affinity even after 9 months under long-term storage temperature conditions.

Claims

1. (A) a recombinant fusion protein; (B) a surfactant; (C) a sugar or a derivative thereof; and (D) A stable pharmaceutical formulation comprising a buffer.

2. The stable pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation is in liquid form.

3. The stable pharmaceutical formulation according to claim 1, wherein the recombinant fusion protein (A) is a VEGF (Vascular Endothelial Growth Factor) antagonist.

4. The stable pharmaceutical formulation of claim 1, wherein (A) the recombinant fusion protein comprises the extracellular domain of a human VEGF receptor.

5. The stable pharmaceutical formulation of claim 1, wherein (A) the recombinant fusion protein comprises extracellular domain 1, 2, or a mixture thereof of a human VEGF receptor.

6. 6. The stable pharmaceutical formulation according to claim 4 or 5, characterized in that (A) the recombinant fusion protein comprises the Fc portion of human immunoglobulin G (IgG).

7. (A) The recombinant fusion protein comprises: (1) an extracellular domain 1 element of a VEGF receptor comprising amino acids 27 to 129 of SEQ ID NO:2; (2) an extracellular domain 2 element of a VEGF receptor comprising amino acids 130 to 231 of SEQ ID NO:2; and (3) The stable pharmaceutical formulation according to claim 1, characterized in that it comprises an Fc site element comprising amino acids 232 to 457 of SEQ ID NO:

2.

8. The stable pharmaceutical formulation of claim 1, wherein the recombinant fusion protein (A) comprises Aflibercept.

9. The stable pharmaceutical formulation according to claim 1, wherein the concentration of the recombinant fusion protein (A) is 5-100 mg / ml.

10. 2. The stable pharmaceutical formulation of claim 1, wherein the (B) surfactant comprises a polysorbate, a poloxamer, or a mixture thereof.

11. 11. The stable pharmaceutical formulation of claim 10, wherein the (B) surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture of two or more thereof.

12. 12. The stable pharmaceutical formulation of claim 11, wherein the surfactant (B) comprises polysorbate 20.

13. The stable pharmaceutical formulation according to claim 1, wherein the concentration of the surfactant (B) is 0.01 to 0.1% (w / v).

14. (C) Sugars include monosaccharides, disaccharides, oligosaccharides, polysaccharides, or mixtures of two or more thereof.

2. The stable pharmaceutical formulation of claim 1, wherein (C) the sugar derivative comprises a sugar alcohol, a sugar acid, or a mixture thereof.

15. 2. The stable pharmaceutical formulation of claim 1, wherein (C) the sugar or a derivative thereof comprises sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof.

16. 16. The stable pharmaceutical formulation of claim 15, wherein (C) the sugar or its derivative comprises trehalose.

17. The stable pharmaceutical formulation according to claim 1, wherein the concentration of the sugar or its derivative (C) is 1 to 20% (w / v).

18. 2. The stable pharmaceutical formulation of claim 1, wherein the buffer (D) comprises an amino acid.

19. 2. The stable pharmaceutical formulation of claim 1, wherein (D) the buffering agent comprises a free amino acid, an amino acid salt, or a mixture thereof.

20. 20. The stable pharmaceutical formulation of claim 19, wherein (D) the buffering agent comprises histidine, a histidine salt, or a mixture thereof.

21. The stable pharmaceutical formulation according to claim 1, wherein the content of the buffer (D) is 1 to 20 mM.

22. 2. The stable pharmaceutical formulation of claim 1, characterized in that it does not contain acetic acid, citric acid, phosphoric acid, or mixtures thereof.

23. 2. The stable pharmaceutical formulation according to claim 1, further comprising (E) an isotonicity agent.

24. 24. The stable pharmaceutical formulation of claim 23, wherein (E) the isotonicity agent comprises sodium chloride, potassium chloride, calcium chloride, or a mixture of two or more thereof.

25. 24. The stable pharmaceutical formulation according to claim 23, wherein the content of the isotonicity agent (E) is 30 mM or less.

26. The stable pharmaceutical formulation according to claim 1, characterized in that the pH is 5.0 to 7.

0.

27. NaF, KBr, NaBr, Na ２ SO ４ , NaSCN, K ２ SO ４ 10. The stable pharmaceutical formulation according to claim 1, characterized in that it does not contain any of the above or a mixture thereof.

28. 2. The stable pharmaceutical formulation of claim 1, characterized in that it does not contain a chelating agent.

29. (A) 5-100 mg / ml of a recombinant fusion protein comprising (1) an extracellular domain 1 element of a VEGF receptor comprising amino acids 27-129 of SEQ ID NO:2, (2) an extracellular domain 2 element of a VEGF receptor comprising amino acids 130-231 of SEQ ID NO:2, and (3) an Fc portion element comprising amino acids 232-457 of SEQ ID NO:2; (B) surfactant 0.01-0.1% (w / v); (C) 1-20% (w / v) of a sugar or its derivative; and (D) Buffer 1 to 20 mM 1. A stable pharmaceutical formulation comprising:

30. 30. The stable pharmaceutical formulation of claim 29, further comprising: (E) 30 mM or less of an isotonicity agent.

31. (A) VEGF (Vascular Endothelial Growth Factor) antagonist; (B) polysorbate; (C) a sugar or a derivative thereof; and (D) histidine 1. A stable pharmaceutical formulation comprising:

32. 32. The stable pharmaceutical formulation of claim 31, further comprising: (E) sodium chloride.

33. (A) 5-100 mg / ml of a recombinant fusion protein comprising (1) an extracellular domain 1 element of a VEGF receptor comprising amino acids 27-129 of SEQ ID NO:2, (2) an extracellular domain 2 element of a VEGF receptor comprising amino acids 130-231 of SEQ ID NO:2, and (3) an Fc portion element comprising amino acids 232-457 of SEQ ID NO:2; (B) Polysorbate 0.01-0.1% (w / v); (C) 1-20% (w / v) of a sugar or its derivative; and (D) histidine 1 to 20 mM 1. A stable pharmaceutical formulation comprising:

34. 34. The stable pharmaceutical formulation of claim 33, further comprising (E) 30 mM or less sodium chloride.

35. 35. The stable pharmaceutical formulation according to any one of claims 1 to 34, wherein the number of insoluble foreign particles measuring 10.00 μm or more and less than 400.00 μm is 50 or less when measured by HIAC after storage at 5±3° C. for 9 months.

36. 35. The stable pharmaceutical formulation according to any one of claims 1 to 34, which exhibits a content of the main ingredient of 98% or more after storage at 5±3°C for 10 days.

37. 35. The stable pharmaceutical formulation according to any one of claims 1 to 34, which exhibits a content of high molecular weight components of 1% or less after storage at 5±3°C for 10 days.

38. 35. The stable pharmaceutical formulation according to any one of claims 1 to 34, which exhibits a content of low molecular weight components of 0.05% or less after storage at 5±3°C for 10 days.

39. The stable pharmaceutical formulation according to any one of claims 1 to 34, which exhibits a content of charge variants of 87% or more after storage at 5±3°C for 9 months.

40. The stable pharmaceutical formulation according to any one of claims 1 to 34, which exhibits a VEGF binding affinity of 90% or more when stored at 5±3°C for 9 months.

41. A stable pharmaceutical formulation according to any one of claims 1 to 34, characterized in that it is for intraocular administration.

42. A stable pharmaceutical formulation according to any one of claims 1 to 34, characterized in that it does not undergo a reconstitution step, a dilution step, or both, before use.

43. A glass vial filled with the stable pharmaceutical formulation according to any one of claims 1 to 34.

44. A pre-filled syringe containing the stable pharmaceutical formulation according to any one of claims 1 to 34.

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