Stable liquid formulation of fusion protein having igg fc domain

Abstract

The present invention relates to a stable liquid formulation of a fusion protein having an Fc domain of human immunoglobulin G (IgG) (in particular, a protein in which a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain of human immunoglobulin G (IgG) are fused (e.g. Aflibercept)). The present invention also relates to a composition for stabilizing a protein in which a soluble extracellular domain of a VEGF receptor and an Fc domain of IgG are fused, and a method for stabilizing a protein in which a soluble extracellular domain of a VEGF receptor and an Fc domain of IgG are fused. The present invention can promote stabilization of physiological activity through a stable liquid formulation suitable for intravitreal injection of anti-VEGF-Fc fusion proteins including Aflibercept, and improve the therapeutic effect on various ophthalmic diseases caused by abnormal angiogenesis (e.g., retinal vein occlusion, diabetic macular edema, choroidal neovascularization, and wet age-related macular degeneration, etc.).

Description

Stable liquid formulation of a fusion protein having an IGG FC domain

[0001] The present invention relates to a stable liquid formulation of a fusion protein having the Fc domain of human immunoglobulin G (IgG). More specifically, the present invention relates to a liquid formulation in which a protein is fused with the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG), e.g., aflibercept, is stabilized, a composition for stabilizing a protein fused with the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG, and a method for stabilizing a protein fused with the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG.

[0002] A type of cell-induced dimeric mitogen with selectivity for vascular endothelial cells has been identified and named vascular endothelial growth factor (VEGF). VEGF is an important factor that promotes angiogenesis and increases vascular permeability.

[0003] VEGF is known to activate VEGF receptors (VEGFR-1, VEGFR-2, VEGFR-3), which are membrane-spanning tyrosine kinase receptors. Among VEGF receptors, VEGFR-1 and VEGFR-2 possess seven Ig-like sequences, a single transmembrane region, and a consensus tyrosine kinase region in their extracellular domains for VEGF binding. These characteristics are used for sequences for anti-VEGF agents, and aflibercept, an ophthalmic therapeutic agent, is a soluble decoy receptor of approximately 115 kDa (including glycosylation) with a structure in which the second binding domain of VEGFR-1 and the third binding domain of VEGFR-2 are bound to the Fc region of human IgG1 (see [Drug Design Development and Therapy (2013), 3(7), 711-722]).

[0004] Abnormal neovascularization via VEGF mechanisms is associated with ophthalmic diseases such as wet age-related macular degeneration, diabetic macular edema, and macular edema in retinal vein occlusion (see reference [J. Korean Med. Assoc. (2014), 57(7), 614-623]).

[0005] Treatments for these ophthalmic diseases include Pegaptanib (brand name: Macugen), Ranibizumab (brand name: Lucentis), Bevacizumab (brand name: Avastin), and Aflibercept (brand name: Eylea), with Aflibercept being approved in 2011 as a treatment for wet age-related macular degeneration (see [Biol. Ther. (2012), 2(3) 1-22], [Drug Design Development and Therapy (2013), 3(7), 711-722], etc.). Among the above treatments, Aflibercept has been reported to have the best therapeutic effect for patients with diabetic macular edema who have experienced significant vision loss (see [NEJM (2015), 372(13) 1193-1203]). Aflibercept was marketed as a treatment for metastatic colorectal cancer [brand name: Zaltrap] and as a treatment for retinal vein occlusion, diabetic macular edema, choroidal neovascularization, and wet age-related macular degeneration [brand name: Eylea].

[0006] Protein drugs, including antibody drugs, undergo physicochemical denaturation under suboptimal conditions. In particular, factors such as temperature, pH, salt concentration, contact with air, protein concentration, and the type of buffer significantly influence protein oxidation, deamidation, isomerization, and polymerization. Such denaturation can lead to protein aggregation, fragmentation, and isomerization, thereby reducing physiological activity. These characteristics vary depending on the protein; in the case of Fc fusion proteins, folding issues result in the separation into three peaks during Hydrophobic Interaction Chromatography (see [Antibodies (2013), 2,452-500]).

[0007] Meanwhile, the following prior art patent document 1 (International Publication WO 2007 / 149334) describes an "ophthalmic formulation comprising 1–100 mg / ml of aflibercept, 0.01–5% of an organic co-solvent (e.g., polysorbate, polyethylene glycol, propylene glycol, etc.), 30–150 mM of an isotonic agent (e.g., NaCl, KCl, etc.), 5–40 mM of a sodium phosphate buffer, and 1.0–7.5% of a stabilizer (e.g., sucrose, sorbitol, glycerol, trehalose, mannitol, etc.)" and "5–50 mg / ml of aflibercept, 5–25 mM of a sodium phosphate buffer, 0.01–0.15% of an organic co-solvent, 1–10% of a stabilizer, and 20–150 mM of A “lyophilizable formulation” containing an isotonic agent is disclosed. The formulation described in the following prior art document 1 can be applied to a prefilled syringe suitable for intravitreal administration.

[0008] In the case of the ophthalmic formulation and freeze-dryable formulation described in the prior art document 1 below, it has been reported that they have an effect of suppressing the generation of impurities or by-products caused by aggregation, fragmentation, and isomerization of Aflibercept. However, the formulation of prior art document 1 has a problem in that the stabilization effect of Aflibercept is significantly reduced when harsh conditions are applied, for example, high temperature conditions of 40°C or higher or stirring conditions.

[0009] Accordingly, the inventors completed the present invention by developing a liquid formulation of a fusion protein having an IgG Fc domain, such as Aflibercept, which not only remains stable for a long period under storage conditions but also has improved stability even under harsh conditions.

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] (Patent Document 1) Prior Art Patent Document 1: International Publication WO 2007 / 149334

[0013] Prior Art Patent Document 2: International Publication WO 2016 / 208989

[0014] Prior Art Patent Document 3: International Publication WO 2019 / 217927

[0015]

[0016] The present invention was devised to solve the aforementioned problems and provides a formulation in which a protein is stabilized by fusing the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor with the Fc domain of human immunoglobulin G (IgG).

[0017] Another objective of the present invention is to provide a composition for stabilizing a protein in which the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG are fused.

[0018] The present invention provides a liquid formulation comprising: a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG) are fused; methionine and / or cysteine; and a buffer having a pH range of 5 to 7.

[0019] According to a specific embodiment, the soluble extracellular domain of the VEGF receptor may include an immunoglobulin-like domain 2 of the first VEGF receptor and an immunoglobulin-like domain 3 of the second VEGF receptor.

[0020] According to a specific embodiment, the fusion protein may be present in an amount of 90 to 200 mg / mL.

[0021] According to a specific embodiment, the buffer solution may contain histidine.

[0022] According to a specific embodiment, the concentration of the methionine may be 0.001 mM to 50 mM.

[0023] According to a specific embodiment, the concentration of the cysteine ​​may be 0.001 mM to 10 mM.

[0024] According to a specific embodiment, the concentration of the histidine may be 0.001 mM to 20 mM.

[0025] According to a specific embodiment, the above-described liquid formulation may further include one or more stabilizers selected from the group consisting of sugars and surfactants.

[0026] According to a specific embodiment, the sugar may be any one selected from the group consisting of sucrose, trehalose, mannitol, and glucose in an amount of 2.5 to 10% (w / v).

[0027] According to a specific embodiment, the sugar may be 5 to 10% (w / v) of sucrose.

[0028] According to a specific embodiment, the surfactant may be 0.01 to 1% (w / v) of polysorbate 20 or polysorbate 80.

[0029] According to a specific embodiment, the liquid formulation may be suitable for intravitreal injection.

[0030] The present invention comprises methionine and / or cysteine; a buffer having a pH range of 5 to 7; and one or more stabilizers selected from the group consisting of sugars and surfactants, and

[0031] The above buffer comprises 0.001 mM to 20 mM of histidine, the sugar is one or more selected from the group consisting of sucrose, trehalose, mannitol, and glucose in an amount of 2.5% (w / v) to 10% (w / v), and the surfactant is 0.01 to 1% (w / v) of polysorbate 20 or polysorbate 80, and the composition for stabilizing a protein in which the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG are fused is provided.

[0032] According to a specific embodiment, the concentration of the methionine may be 0.001 mM to 50 mM.

[0033] According to a specific embodiment, the concentration of the cysteine ​​may be 0.001 mM to 10 mM.

[0034] According to a specific embodiment, the protein in which the soluble extracellular domain of the VEGF receptor and the Fc domain of IgG are fused may be Aflibercept.

[0035]

[0036] The liquid formulation provided in the present invention can significantly suppress the generation of impurities or byproducts caused by aggregation, fragmentation, and isomerization of a fusion protein having the Fc domain of human immunoglobulin G (IgG) [in particular, a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG) are fused (e.g., aflibercept)] under not only general storage conditions but also harsh conditions, e.g., high temperature conditions of 40°C or higher or stirring conditions, thereby improving stability during long-term storage.

[0037] In addition, the present invention can improve the therapeutic effect of various ophthalmic diseases caused by abnormal angiogenesis (e.g., retinal vein occlusion, diabetic macular edema, choroidal neovascularization, wet age-related macular degeneration, etc.) by promoting the stabilization of physiological activity through a stable liquid formulation suitable for intravitreal injection of anti-VEGF-Fc fusion proteins, including aflibercept.

[0038]

[0039] Figures 1a and 1b show the results of SE-HPLC analysis of the stability of samples stored for up to 67 days at 25±2℃ and 60±5% RH in the presence of various types of amino acids (methionine, not added) and sugars (sucrose, trehalose) in histidine buffer containing 90 mg / ml of aflibercept.

[0040] Figure 1a shows the results of SE-HPLC analysis of the change in monomer content (%) over time for 90 mg / ml of aflibercept.

[0041] Figure 1b shows the results of SE-HPLC analysis of the change in aggregate content (%) over time for 90 mg / ml of aflibercept.

[0042] Figures 1c and 1d show the results of SE-HPLC analysis of the stability of samples stored for up to 67 days at 25±2℃ and 60±5% RH in the presence of various types of amino acids (arginine, methionine, and no added) and sugars (sucrose, trehalose) in histidine buffer containing 120 mg / mL of aflibercept.

[0043] Figure 1c shows the results of SE-HPLC analysis of the change in monomer content (%) over time for 120 mg / ml of aflibercept.

[0044] Figure 1d shows the results of SE-HPLC analysis of the change in aggregate content (%) over time for 120 mg / ml of aflibercept.

[0045] Figure 2 shows the results of SE-HPLC analysis of the stability of samples stored for up to 28 days at 25±2℃ and 60±5% RH in a histidine buffer containing 100 mg / ml to 120 mg / mL of aflibercept, in formulations containing various amino acids (methionine, proline, cysteine).

[0046] Figure 2a shows the results of SE-HPLC analysis of the change in monomer content (%) over time for 100 mg / ml of aflibercept.

[0047] Figure 2b shows the results of SE-HPLC analysis of the change in aggregate content (%) over time for 100 mg / ml of aflibercept.

[0048] Figure 2c shows the results of SE-HPLC analysis of the change in monomer content (%) over time for 120 mg / ml of aflibercept.

[0049] Figure 2d shows the results of SE-HPLC analysis of the change in aggregate content (%) over time for 120 mg / ml of aflibercept.

[0050] Figure 3 shows the results of SE-HPLC analysis of a sample containing formulations containing various concentrations of methionine (10 mM, 30 mM) and cysteine ​​(0 mM, 0.3 mM, 1 mM, 3 mM) in a histidine buffer containing 120 mg / mL of aflibercept, stored at 25±2℃ and 60±5% RH for up to 112 days.

[0051] Figure 3a shows the results of SE-HPLC analysis of the change in monomer content (%) over time for 120 mg / ml of aflibercept.

[0052] Figure 3b shows the results of SE-HPLC analysis of the change in aggregate content (%) over time for 120 mg / ml of aflibercept.

[0053] Figure 4 is a graph showing the stability of seven formulations (F31–F37) containing 130 mg / mL of aflibercept. The results are based on SE-HPLC analysis of samples stored at 37±2℃ for up to 14 days, including formulations containing and not containing 10 mM methionine and cysteine ​​(0.3 mM, 1 mM, 3 mM) or N-acetylcysteine ​​(1 mM, 10 mM) in histidine buffer.

[0054] Figure 5 is a graph showing the stability of five formulations (F38–F42) containing 140 mg / mL of aflibercept. The results are based on SE-HPLC analysis of samples stored at 37±2℃ for up to 28 days, including formulations containing 50 mM arginine or 50 mM arginine-hydrochloride in histidine buffer, or containing and not containing 10 mM methionine and cysteine ​​(0.3 mM, 1 mM).

[0055] Figure 5a shows the results of SE-HPLC analysis of the change in monomer content (%) over time for 140 mg / ml of aflibercept.

[0056] Figure 5b shows the results of SE-HPLC analysis of the change in aggregate content (%) over time for 140 mg / ml of aflibercept.

[0057] Figure 6 is a graph showing the stability of seven formulations (F43–F49) containing 140 mg / mL of aflibercept. The results are SE-HPLC analysis of the stability of samples stored for up to 28 days at 37±2℃ in 10 mM histidine buffer containing 50 mM arginine-hydrochloride as an additive, or containing and not containing 10 mM methionine and 0.3 mM cysteine, under three conditions of pH 5.5, pH 6.0, and pH 6.5.

[0058] Figure 6a is a graph showing the change in monomer content (%) over time for each amino acid additive under each pH condition.

[0059] Figure 6b is a graph showing the change in monomer content (%) over time for seven formulations (F43 to F49).

[0060] Figure 7 shows the results of deriving key variables for formulation stability through a 2-level 5-factor Fractional Factorial Design (FFD). The stability of nine formulations (F50–F58) stored at 37±2℃ for up to 7 weeks was analyzed using SE-UPLC, and Minitab ® Regression analysis of a partial factorial design was performed using software.

[0061] Figure 7a is Minitab ® This is a graph showing the importance of each factor, based on a 'Pareto chart of effect' obtained by performing regression analysis using software. Nine formulations (F50 to F58) containing 150 mg / ml of aflibercept were stored at 37±2℃ for 7 weeks, and regression analysis was performed based on the monomer content (%) results obtained by SE-UPLC analysis.

[0062] Figure 7b is Minitab ®This is a graph of the 'interaction plot for Response' obtained by performing regression analysis through software, showing the factors that interact with each other among the five factors. Regression analysis was performed based on the monomer content (%) results obtained by analyzing nine formulations (F50 to F58) containing 150 mg / mL of aflibercept at 37±2℃ for 7 weeks using SE-UPLC.

[0063] Figure 7c shows the results of analyzing the stability of nine formulations (F50 to F58) containing 150 mg / ml of aflibercept stored at 37±2℃ for 3 weeks using SE-UPLC.

[0064] Figure 8 shows the results of analyzing the stability of 11 formulations (F59 to F69) containing 140 mg / ml of aflibercept stored at 37±2℃ for 4 weeks using SE-UPLC.

[0065] Figure 9 is a graph showing the stability of five formulations (F64 to F68) containing 140 mg / ml of aflibercept. The results are based on SE-UPLC analysis of samples stored at 37±2℃ for up to 28 days, including formulations containing 50 mM arginine-hydrochloride in histidine buffer or containing and not containing 15 mM methionine and cysteine ​​(0.2 mM, 0.4 mM, 0.8 mM).

[0066] FIG. 9a is a graph showing the change in monomer content (%) over time for five formulations (F64 to F68) containing aflibercept.

[0067] FIG. 9b is a graph showing the change in aggregate content (%) over time for five formulations (F64 to F68) containing aflibercept.

[0068]

[0069] The present invention will be described in more detail below.

[0070] As mentioned above, existing formulations stabilized with fusion proteins having an IgG Fc domain, such as Aflibercept, exhibit a problem of significantly reduced stability under harsh conditions; therefore, there is a need to develop a formulation in which the fusion protein can be maintained stably not only under general storage conditions but also under harsh conditions.

[0071] Accordingly, the present invention seeks a solution to the aforementioned problem by providing a liquid formulation comprising: a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG) are fused; methionine and / or cysteine; and a buffer having a pH range of 5 to 7. The liquid formulation of the present invention not only allows the fusion protein to be stored stably for a long period under general storage conditions, but also significantly suppresses the generation of impurities or byproducts even under harsh conditions, thereby making it possible to provide a formulation with significantly improved stability compared to known formulations of the fusion protein, even in unavoidable environments where optimal storage conditions cannot be provided.

[0072] The present invention provides a liquid formulation comprising: a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG) are fused; methionine and / or cysteine; and a buffer having a pH range of 5 to 7.

[0073] In the fusion protein which is the active ingredient of the above liquid formulation, the soluble extracellular domain of vascular endothelial growth factor (VEGF) may include immunoglobulin-like domain 2 of the first VEGF receptor and immunoglobulin-like domain 3 of the second VEGF receptor, and the Fc domain of human immunoglobulin G (IgG) may be the Fc domain of IgG1, IgG2, IgG3, or IgG4, and preferably may be the Fc domain of IgG1. Here, the Fc region is composed of hinge-CH1-CH2-CH3 and may be in a form in which CH1 is deleted. In the present invention, the fusion protein may be aflibercept, but is not limited thereto.

[0074] In the liquid formulation of the present invention, the fusion protein may be included in an amount of 90 to 200 mg / ml. Specifically, the fusion protein may be included in an amount of 90 mg / ml or more, 100 mg / ml or more, 110 mg / ml or more, 120 mg / ml or more, 122 mg / ml or more, 124 mg / ml or more, 126 mg / ml or more, 128 mg / ml or more, 130 mg / ml or more, 132 mg / ml or more, 134 mg / ml or more, 136 mg / ml or more, 138 mg / ml or more, and 140 mg / ml or more, and may be included in an amount of 200 mg / ml or less, 180 mg / ml or less, 160 mg / ml or less, 150 mg / ml or less, 148 mg / ml or less, 146 mg / ml or less, 144 mg / ml or less, 142 mg / ml or less, and 140 mg / ml or less, but is not limited thereto.

[0075] Specifically, the fusion protein is 110 mg / ml to 160 mg / ml, 115 mg / ml to 160 mg / ml, 120 mg / ml to 160 mg / ml, 122 mg / ml to 160 mg / ml, 124 mg / ml to 160 mg / ml, 126 mg / ml to 160 mg / ml, 128 mg / ml to 160 mg / ml, 129 mg / ml to 160 mg / ml, 120 mg / ml to 155 mg / ml, 120 mg / ml to 150 mg / ml, 120 mg / ml to 148 mg / ml, 120 mg / ml to 146 mg / ml, 120 mg / ml to 144 mg / ml, 120 mg / ml to 142 mg / ml, 120 mg / ml to 141 mg / ml, It may be included in 128 mg / ml to 142 mg / ml or 130 mg / ml to 140 mg / ml, but is not limited thereto.

[0076] In the liquid formulation according to the present invention, the "buffer" refers to a buffered solution that withstands changes in pH caused by the action of its acid-base conjugate component. In one embodiment of the present invention, a buffer containing histidine and having a pH range of 5 to 7 was used. The histidine included in the buffer of the present invention may refer to histidine ions.

[0077] The concentration of the above histidine may be from 0.001 mM to 20 mM. In the present invention, the term “concentration of histidine” refers to the total sum concentration of histidine species or histidine ions present in the buffer solution based on the total volume of the buffer solution. In the present invention, “concentration of histidine,” “final concentration of histidine,” and “final concentration of histidine ions” may be used interchangeably.

[0078] Specifically, the concentration of the histidine may be 0.001 mM or more, 0.1 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, or 10 mM or more, and may be 20 mM or less, 19 mM or less, 18 mM or less, 17 mM or less, 16 mM or less, 15 mM or less, 14 mM or less, 13 mM or less, 12 mM or less, or 11 mM or less, but is not limited thereto. Specifically, the concentration of the histidine may be 1 mM to 20 mM, 2 mM to 20 mM, 4 mM to 20 mM, 8 mM to 20 mM, 9 mM to 20 mM, 5 mM to 18 mM, 5 mM to 16 mM, 5 mM to 14 mM, 5 mM to 12 mM, 5 mM to 11 mM, 7 mM to 13 mM, 9 mM to 13 mM, or 9 mM to 11 mM, but is not limited thereto. Additionally, the concentration of the histidine may be 10 mM.

[0079]

[0080] In the liquid formulation according to the present invention, the liquid formulation may contain methionine and / or cysteine. Oxidation is one of the major chemical degradation pathways of protein pharmaceuticals. During the production and preservation of proteins, amino acid residues such as methionine, cysteine, histidine, tryptophan, and tyrosine are oxidized by various types of reactive oxygen species (see [Biotechnol Bioeng. 1995 Dec 5;48(5):490-500]). By adding a suitable antioxidant to inhibit oxidation, the structure and function of the protein can be stabilized.

[0081] The present invention can stabilize a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG) are fused using sulfur-containing methionine and / or cysteine ​​as an antioxidant. In the liquid formulation according to the present invention, the concentration of the methionine may be 0.001 to 50 mM. Specifically, the concentration of the methionine may be 0.001 mM or more, 0.005 mM or more, 0.01 mM or more, 0.05 mM or more, 0.1 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, 3.2 mM or more, 3.4 mM or more, 3.6 mM or more, 3.8 mM or more, 4 mM or more, 4.2 mM or more, 4.4 mM or more, 4.6 mM or more, 4.8 mM or more, or 5 mM or more, and may be 50 mM or less, 40 mM or less, 30 mM or less, 29 mM or less, 28 mM or less, 27 mM or less, 26 mM or less, 25 mM or less, 24 mM or less, 23 mM or less, 22 mM or less, 21 mM or less, 20 mM or less, 15 mM or less, or 10 mM or less, but is not limited thereto.

[0082] Specifically, the concentration of the methionine may be 1 to 40 mM, 2 to 40 mM, 3 to 40 mM, 4 to 40 mM, 5 to 40 mM, 1 to 30 mM, 1 to 25 mM, 1 to 24 mM, 1 to 23 mM, 1 to 22 mM, 1 to 21 mM, 3 to 23 mM, 4 to 21 mM, 5 to 20 mM, and 5 to 15 mM, but is not limited thereto.

[0083] In the liquid formulation according to the present invention, the concentration of cysteine ​​may be 0 to 10 mM or 0.001 to 10 mM.

[0084] Specifically, the concentration of the cysteine ​​may be 0 mM or more, 0.001 mM or more, 0.005 mM or more, 0.01 mM or more, 0.05 mM or more, 0.1 mM or more, 0.12 mM or more, 0.14 mM or more, 0.16 mM or more, 0.18 mM or more, 0.19 mM or more, 0.2 mM or more, 0.22 mM or more, 0.24 mM or more, 0.26 mM or more, 0.28 mM or more, 0.29 mM or more, 0.3 mM or more, and 10 mM or less, 6 mM or less, 2 mM or less, 1 mM or less, 0.8 mM or less, 0.6 mM or less, 0.58 mM or less, 0.56 mM or less, 0.54 mM or less, 0.52 mM or less, 0.5 mM or less, 0.48 mM or less, 0.46 mM or less, 0.44 It may be mM or less, 0.42 mM or less, 0.4 mM or less, 0.38 mM or less, 0.36 mM or less, 0.34 mM or less, or 0.32 mM or less, but is not limited thereto.

[0085] Specifically, the concentration of the cysteine ​​is 0 to 8 mM, 0 to 6 mM, 0 to 4 mM, 0 to 2 mM, 0 to 1 mM, 0 to 0.8 mM, 0 to 0.6 mM, 0 to 0.5 mM, 0 to 0.4 mM, 0 to 0.35 mM, 0 to 0.3 mM, 0.001 to 1 mM, 0.001 to 0.5 mM, 0.001 to 0.4 mM, 0.001 to 0.3 mM, 0.01 to 1 mM, 0.01 to 0.5 mM, 0.01 to 0.4 mM, 0.01 to 0.3 mM, 0.05 to 1 mM, 0.05 to 0.5 mM, 0.05 to 0.4 mM, 0.01 to 0.3 mM, 0.05 to 1 mM, 0.05 to 0.4 mM, 0.05 to 0.3 mM, 0.08 to It may be 0.32 mM, but is not limited to this.

[0086]

[0087] The liquid formulation of the present invention may further include one or more stabilizers selected from the group consisting of sugars and surfactants, in addition to the fusion protein and histidine buffer, which are active ingredients of the liquid formulation.

[0088] In the liquid formulation of the present invention, the sugar may be any one selected from the group consisting of trehalose, sucrose, mannitol, sorbitol, glucose, lactose, xylitol, inositol, glycerol, and hydroxypropyl cyclodextrin, and specifically, may be any one selected from the group consisting of trehalose, sucrose, mannitol, and glucose.

[0089] In the liquid formulation of the present invention, the sugar may be sucrose. Specifically, the concentration of the sugar may be 2.5 to 10% (w / v). Specifically, the concentration of the sugar may be 2.5% (w / v) or more, 3% (w / v) or more, 4% (w / v) or more, 4.2% (w / v) or more, 4.4% (w / v) or more, 4.6% (w / v) or more, 4.8% (w / v) or more, or 5% (w / v) or more, and may be 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7.8% (w / v) or less, 7.6% (w / v) or less, 7.4% (w / v) or less, 7.2% (w / v) or less, or 7% (w / v) or less, but is not limited thereto. Specifically, the concentration of the sugar may be 3 to 10% (w / v), 4 to 10% (w / v), 5 to 10% (w / v), 3 to 9% (w / v), 3 to 8% (w / v), 3 to 7% (w / v), 4 to 9% (w / v), 4 to 8% (w / v), 4 to 7% (w / v), 5 to 9% (w / v), 5 to 8% (w / v), and 5 to 7% (w / v), but is not limited thereto.

[0090] In the liquid formulation of the present invention, the surfactant may be used for ophthalmic drug delivery, and there are no limitations on the type thereof, such as anionic surfactants, cationic surfactants, nonionic surfactants, and zwitterionic surfactants.

[0091] Examples of the above anionic surfactants include sulfates, sulfonates, phosphates, carboxylates, etc., but are not limited to these.

[0092] Examples of the above-mentioned cationic surfactants include octenidine dihydrochloride, cetyl trimethylammonium bromide (CTAB), hexadecyl trimethylammonium bromide, cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride, 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyl dimethylammonium bromide (DODAB), etc.

[0093] Examples of the above non-ionic surfactants include polyoxyethylene glycol alkyl ethers, polyoxypropylene glycol alkyl ethers, polyoxyethylene glycol octylphenol ethers (e.g., Triton X-100, etc.), polyoxyethylene glycol alkylphenol ethers, glycerol alkyl esters, polyoxyethylene glycol sorbitan alkyl esters (e.g., polysorbate 20, polysorbate 80, etc.), cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, copolymers of polyethylene glycol and polypropylene glycol, polyethoxylated tallow amine (POEA), etc.

[0094] Examples of the above zwitter-ionic surfactants include CHAPS (3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfate), cocaamidopropyl hydroxysulsteine, etc.

[0095] In addition, alkyl sulfates such as ammonium lauryl sulfate, sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS), sodium lauryl ether sulfate (SLES), sodium myreth sulfate, etc.; docusates such as dioctyl sodium sulfosuccinate, perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, linear alkylbenzene sulfonate (LAB), etc.; phospholipids such as phosphatidylserine, phosphatidylethanolamine, phosphatidine choline, etc.; sphingomyelin, etc. may be used as surfactants.

[0096] In the liquid formulation of the present invention, the surfactant may be polysorbate 20 or polysorbate 80.

[0097] In the liquid formulation of the present invention, the concentration of the surfactant may be 0.01 to 1% (w / v). Specifically, the concentration of the surfactant is 0.01 to 1% (w / v), 0.01 to 0.8% (w / v), 0.01 to 0.6% (w / v), 0.01 to 0.4% (w / v), 0.01 to 0.2% (w / v), 0.01 to 0.1% (w / v), 0.01 to 0.08% (w / v), 0.01 to 0.06% (w / v), 0.01 to 0.05% (w / v), 0.01 to 0.04% (w / v), 0.01 to 0.03 (w / v), 0.015 to 0.05% (w / v), 0.02 to 0.05% (w / v), 0.022 to 0.05% (w / v), and 0.024 The concentration may be up to 0.05% (w / v), 0.026 to 0.05% (w / v), 0.028 to 0.05% (w / v), 0.015 to 0.03% (w / v), 0.02 to 0.03% (w / v), 0.022 to 0.03% (w / v), 0.024 to 0.03% (w / v), 0.026 to 0.03% (w / v), or 0.028 to 0.03% (w / v), but is not limited thereto. Additionally, the concentration of the surfactant may be 0.03% (w / v).

[0098] The liquid formulation according to the present invention maintains the stability of the fusion protein for a long period under general storage conditions and exhibits excellent inhibition of the formation of impurities or byproducts of the fusion protein even under harsh conditions, thereby improving the stability of the fusion protein compared to existing liquid formulations. In the present invention, "harsh conditions" refer to chemically and physically undesirable environments for the fusion protein, which may result in unacceptable protein stability. Such harsh conditions include, for example, high temperatures or stirring conditions.

[0099] In the present invention, the term "stable" refers to a protein essentially maintaining its physical and / or chemical stability and / or biological activity during storage. Various analytical techniques for measuring protein stability are available in the art, and stability can be measured at a selected temperature for a selected period.

[0100] A protein can be said to "maintain its physical stability" in a formulation if it shows little or no change in aggregation, precipitation, and / or denaturation when observed by visual inspection of color and / or transparency, or when measured by UV light scattering (measuring visible aggregates) or size exclusion chromatography (SEC).

[0101] The above liquid formulation can be used for medicinal purposes without special limitations for the prevention or treatment of various diseases, provided that the disease can be treated by treatment with the fusion protein, and preferably can be used to prevent or treat various ophthalmic diseases such as retinal vein occlusion, diabetic macular edema, choroidal neovascularization, and wet age-related macular degeneration.

[0102] The liquid formulation according to the present invention may further comprise arginine, arginine hydrochloride (Arginine-HCl), proline, and N-acetylcysteine ​​(NAC). More specifically, the liquid formulation according to the present invention may further comprise arginine or proline.

[0103]

[0104] The liquid formulation according to the present invention may further include a pharmaceutically acceptable carrier, a diluent, an excipient, etc.

[0105] Pharmaceutically acceptable carriers are those commonly used in formulations and may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0106] The liquid formulation of the present invention may additionally include lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0107] As protein concentration increases, the viscosity of the solution tends to increase exponentially. When Connolly et al. measured the viscosity of 29 types of antibody solutions at a concentration of 175 mg / ml, the viscosity was distributed over a wide range of about 5 to 200 cP.

[0108]

[0109] Generally, interactions between protein molecules, namely electrostatic and hydrophobic interactions, act in combination to increase viscosity, and high viscosity is a major cause of difficulty in administering protein drugs. Research is actively being conducted to develop additives to lower the viscosity of solutions in high-concentration protein drugs.

[0110] The viscosity of a protein solution used as an injectable is generally considered to be within 20 to 25 cP as having no issues in terms of ease of injection, and Berteau et al. reported that a solution with a viscosity of 15 to 20 cP is injected without pain (see [Medical Devices: Evidence and Research 2015:8 473-484]). Despite being a high-concentration protein solution, the formulation of the present invention maintains a viscosity within the range of 5 to 20 cP, thereby having a viscosity suitable for use as an injectable.

[0111] Specifically, the liquid formulation of the present invention may have a viscosity of 15 to 20 cP, but is not limited thereto.

[0112] The liquid formulation of the present invention can be administered via various routes depending on the purpose, and preferably, it is administered by intravitreal injection for the treatment of ophthalmic diseases, and can be applied to a pre-filled syringe suitable for intravitreal injection.

[0113] The suitable dosage of the liquid formulation of the present invention varies depending on factors such as the formulation method, mode of administration, patient's age, weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician who is normally skilled can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0114]

[0115] The present invention comprises a fusion protein of 90 to 200 mg / mL;

[0116] 0.001 to 50 mM methionine;

[0117] 0.001 to 10 mM cysteine;

[0118] 0.001 to 20 mM histidine buffer;

[0119] 2.5 to 10% (w / v) of sucrose; and

[0120] It may be a liquid formulation comprising 0.01 to 1% (w / v) of polysorbate 20.

[0121] The present invention comprises a fusion protein of 100 to 190 mg / mL;

[0122] 0.01 to 40 mM methionine;

[0123] 0.01 to 5 mM cysteine;

[0124] 0.01 to 15 mM histidine buffer;

[0125] 3 to 10% (w / v) of sucrose; and

[0126] It may be a liquid formulation comprising 0.01 to 0.9% (w / v) of polysorbate 20.

[0127] The present invention comprises a fusion protein of 110 to 180 mg / mL;

[0128] 0.1 to 30 mM methionine;

[0129] 0.01 to 3 mM cysteine;

[0130] 0.1 to 15 mM histidine buffer;

[0131] 3 to 9% (w / v) of sucrose; and

[0132] It may be a liquid formulation comprising 0.01 to 0.8% (w / v) of polysorbate 20.

[0133] The present invention comprises a fusion protein of 120 to 170 mg / mL;

[0134] 0.1 to 25 mM methionine;

[0135] 0.01 to 2 mM cysteine;

[0136] 1 to 15 mM histidine buffer;

[0137] 4 to 9% (w / v) of sucrose; and

[0138] It may be a liquid formulation comprising 0.01 to 0.5% (w / v) of polysorbate 20.

[0139] The present invention comprises a fusion protein of 120 to 170 mg / mL;

[0140] 1 to 25 mM methionine;

[0141] 0.01 to 1 mM cysteine;

[0142] 3 to 15 mM histidine buffer;

[0143] 4 to 9% (w / v) of sucrose; and

[0144] It may be a liquid formulation comprising 0.01 to 0.4% (w / v) of polysorbate 20.

[0145] The present invention comprises a fusion protein of 120 to 160 mg / mL;

[0146] 2 to 25 mM methionine;

[0147] 0.01 to 0.9 mM cysteine;

[0148] 5 to 15 mM histidine buffer;

[0149] 4 to 9% (w / v) of sucrose; and

[0150] It may be a liquid formulation comprising 0.01 to 0.3% (w / v) of polysorbate 20.

[0151] The present invention comprises a fusion protein of 120 to 150 mg / mL;

[0152] 3 to 25 mM methionine;

[0153] 0.01 to 0.7 mM cysteine;

[0154] 6 to 15 mM histidine buffer;

[0155] 4 to 8% (w / v) of sucrose; and

[0156] It may be a liquid formulation comprising 0.01 to 0.2% (w / v) of polysorbate 20.

[0157] The present invention comprises a fusion protein of 120 to 140 mg / mL;

[0158] 3 to 25 mM methionine;

[0159] 0.01 to 0.7 mM cysteine;

[0160] 7 to 15 mM histidine buffer;

[0161] 4 to 8% (w / v) of sucrose; and

[0162] It may be a liquid formulation comprising 0.01 to 0.1% (w / v) of polysorbate 20.

[0163] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0164] 3 to 25 mM methionine;

[0165] 0.01 to 0.7 mM cysteine;

[0166] 7 to 15 mM histidine buffer;

[0167] 4 to 8% (w / v) of sucrose; and

[0168] It may be a liquid formulation comprising 0.01 to 0.1% (w / v) of polysorbate 20.

[0169] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0170] 4 to 25 mM methionine;

[0171] 0.01 to 0.5 mM cysteine;

[0172] 7 to 14 mM histidine buffer;

[0173] 4 to 8% (w / v) of sucrose; and

[0174] It may be a liquid formulation comprising 0.01 to 0.09% (w / v) of polysorbate 20.

[0175] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0176] 4 to 25 mM methionine;

[0177] 0.01 to 0.5 mM cysteine;

[0178] 7 to 14 mM histidine buffer;

[0179] 4 to 8% (w / v) of sucrose; and

[0180] It may be a liquid formulation comprising 0.01 to 0.09% (w / v) of polysorbate 20.

[0181] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0182] 4 to 23 mM methionine;

[0183] 0.01 to 0.5 mM cysteine;

[0184] 7 to 12 mM histidine buffer;

[0185] 4 to 8% (w / v) of sucrose; and

[0186] It may be a liquid formulation comprising 0.01 to 0.08% (w / v) of polysorbate 20.

[0187] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0188] 5 to 20 mM methionine;

[0189] 0.01 to 0.5 mM cysteine;

[0190] 9 to 12 mM histidine buffer;

[0191] 4 to 8% (w / v) of sucrose; and

[0192] It may be a liquid formulation comprising 0.01 to 0.08% (w / v) of polysorbate 20.

[0193] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0194] 5 to 20 mM methionine;

[0195] 0.01 to 0.3 mM cysteine;

[0196] 10 mM histidine buffer;

[0197] 5 to 7% (w / v) of sucrose; and

[0198] It may be a liquid formulation containing 0.03% (w / v) of polysorbate 20.

[0199]

[0200] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0201] 5 to 20 mM methionine;

[0202] 0 to 0.3 mM cysteine;

[0203] 10 mM histidine buffer;

[0204] 5 to 7% (w / v) of sucrose; and

[0205] It may be a liquid formulation containing 0.03% (w / v) of polysorbate 20.

[0206]

[0207] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0208] 5 to 20 mM methionine;

[0209] 10 mM histidine buffer;

[0210] 5 to 7% (w / v) of sucrose; and

[0211] It may be a liquid formulation containing 0.03% (w / v) of polysorbate 20.

[0212]

[0213] The present invention comprises a fusion protein of 130 to 140 mg / mL;

[0214] 5 to 20 mM methionine;

[0215] 0.3 mM cysteine;

[0216] 10 mM histidine buffer;

[0217] 5 to 7% (w / v) of sucrose; and

[0218] It may be a liquid formulation containing 0.03% (w / v) of polysorbate 20.

[0219]

[0220]

[0221] The present invention also provides a composition for stabilizing a protein in which a soluble extracellular domain of a VEGF receptor and an Fc domain of IgG are fused, comprising methionine and / or cysteine; a buffer having a pH range of 5 to 7; and one or more stabilizers selected from the group consisting of sugars and surfactants.

[0222] Since the description of the composition of the above-mentioned stabilization composition is the same as previously described, redundant details are omitted to avoid excessive complexity in this specification.

[0223] The protein in which the soluble extracellular domain of the VEGF receptor of the present invention and the Fc domain of IgG are fused may be aflibercept.

[0224] The present invention also provides a method for stabilizing a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the Fc domain of human immunoglobulin G (IgG) are fused, using a buffer containing histidine and having a pH range of 5 to 7.

[0225]

[0226] The scope of the present invention shall not be interpreted as being limited to the following examples, and all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains ('person skilled in the art').

[0227] [Example 1]

[0228] Evaluation of the effects of amino acid additives

[0229] In this example, a sample was prepared as follows to identify the optimal amino acid additive for a liquid formulation containing aflibercept, an anti-VEGF-Fc fusion protein, as an active ingredient.

[0230] Specifically, formulations containing and not containing 50 mM methionine based on a final concentration of aflibercept of 90 mg / ml, 10 mM histidine buffer at pH 6.0, 0.03% (w / v) polysorbate 20, and 7% (w / v) sugars (sucrose or trehalose) were tested.

[0231] In addition, specifically, a final concentration of aflibercept was set to 120 mg / mL, and formulations containing 50 mM arginine or 50 mM methionine, respectively, as amino acid additives based on 10 mM histidine buffer at pH 6.0 and 0.03% (w / v) polysorbate 20, a control formulation without amino acid additives, and conditions containing 7% (w / v) sucrose or 7% (w / v) trehalose as sugars were tested.

[0232] The list of ingredients for 10 formulations F1 to F10 is summarized in Table 1. Monomers, aggregates, and modified products generated over time were analyzed by SE-HPLC (size exclusion high-performance liquid chromatography) after storage for 7 to 67 days at a temperature of 25±2℃ and 60±5% relative humidity (RH).

[0233] In SE-HPLC analysis, a TSK-gel G3000SWXL (7.8 × 300 mm) column (TOSOH, Japan) was used, and 0.2 M sodium phosphate (pH 6.2) and 0.25 M NaCl buffer were flowed at a rate of 0.5 ml / min, and the peak of the anti-VEGF-Fc fusion protein was confirmed at an absorbance of 280 nm.

[0234] The monomer% and aggregate% evaluated by SE-HPLC of 90 mg / mL anti-VEGF-Fc fusion protein stored at 25±2℃ and 60±5% RH are shown in Figures 1a and 1b, and the monomer% and aggregate% SE-HPLC results of 120 mg / mL anti-VEGF-Fc fusion protein are shown in Figures 1c and 1d.

[0235]

[0236] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 1 Formulation Protein (mg / mL) Histidine (mM) Arginine (mM) Methionine (mM) Sucrose (w / v) Trehalose (w / v) Polysorbate 20 (w / v)pHF19010N / AN / A7N / A0.036.0F29010N / AN / AN / A70.036.0F39010N / A507N / A0.036.0F49010N / A50N / A70.036.0F512010N / AN / A7N / A0.036 .0F612010N / AN / AN / A70.036.0F71201050N / A7N / A0.036.0F81201050N / AN / A70.036.0F912010N / A507N / A0.036.0F1012010N / A50N / A70.036.0

[0237]

[0238] The results show that a formulation containing 7% (w / v) sugars and methionine at an anti-VEGF-Fc fusion protein concentration of 90 mg / ml is stable with reduced aggregate formation compared to a control formulation without added ingredients.

[0239] This indicates that at an anti-VEGF-Fc fusion protein concentration of 120 mg / mL, formulations containing amino acid excipients such as arginine or methionine are more stable, with reduced aggregate formation compared to control formulations without such excipients. Additionally, no significant difference in stability was observed between samples containing sucrose and trehalose among sugars.

[0240] [Example 2]

[0241] Evaluation of combination conditions of proline, methionine, and cysteine

[0242] In this example, to confirm the effect of an additional amino acid additive in a liquid formulation containing aflibercept, an anti-VEGF-Fc fusion protein, as an active ingredient, a sample was prepared as follows.

[0243] Specifically, conditions were tested in which the final concentration of aflibercept was set to 100 mg / ml to 120 mg / mL and based on 10 mM histidine buffer at pH 6.0 and 0.03% (w / v) polysorbate 20, formulations containing 50 mM methionine identified in Example 1, formulations combining methionine and cysteine, and formulations containing 7% sucrose or 200 mM to 250 mM proline as osmotic pressure regulators were added. The ingredient lists for each of the four formulations, F11 to F18, according to protein concentration are summarized in Table 2. The monomers, aggregates, and modified products formed over time were analyzed by SE-HPLC after storage at a temperature of 25±2℃ and 60±5% relative humidity (RH) for 7 to 28 days.

[0244]

[0245] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 2 Formulation Protein (mg / mL) Histidine (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Proline (mM) Polysorbate 20 (w / v)pHF1110010N / AN / AN / A2500.036.0F121001050N / A7N / A0.036.0F131001050N / AN / A2000.036.0F14100105017N / A0.0 36.0F1512010N / AN / AN / A2500.036.0F161201050N / A7N / A0.036.0F171201050N / AN / A2000.036.0F18120105017N / A0.036.0

[0246] SE-HPLC analysis confirmed that under formulation conditions stored at 25±2℃ and 60±5% RH, aggregate formation was inhibited in formulations containing 100 mg / mL of the anti-VEGF-Fc fusion protein of the present invention, either by adding methionine alone or in combination with proline and cysteine, compared to formulations containing proline alone (Figs. 2a, 2b). A similar effect was confirmed in formulation combinations under the same conditions containing 120 mg / mL of the anti-VEGF-Fc fusion protein shown in Figs. 2c and 2d. In particular, it was confirmed that aggregate formation was significantly inhibited in formulations containing 1 mM cysteine ​​along with methionine (F14, F18) (Fig. 2). These results demonstrate that the addition of excipients such as methionine and cysteine ​​in histidine buffer has a positive effect on the stability of the anti-VEGF-Fc fusion protein.

[0247] [Example 3]

[0248] Effects of methionine and cysteine ​​concentrations

[0249] In this example, to determine the optimal methionine and cysteine ​​concentrations in a liquid formulation containing aflibercept, an anti-VEGF-Fc fusion protein, as an active ingredient, a sample was prepared as follows.

[0250] Specifically, conditions were tested in which the final concentration of aflibercept was set to 120 mg / ml and based on 10 mM histidine buffer at pH 6.0 and 0.03% (w / v) polysorbate 20, a formulation containing 50 mM arginine was used as a control, formulations containing 10 mM and 30 mM methionine, formulations containing 0 mM, 0.3 mM, 1 mM, and 3 mM cysteine, and formulations containing 7% (w / v) to 8% (w / v) sucrose or 200 mM proline as osmotic pressure regulators were added. The list of ingredients for a total of 10 formulations, F21 to F30, for the above high concentration of protein (120 mg / mL) is summarized in Table 3. Monomers, aggregates, and modified products generated over time were analyzed by SE-HPLC after storage for 7 to 112 days at a temperature of 25±2℃ and 60±5% relative humidity (RH).

[0251] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 3 Formulation Protein (mg / mL) Histidine (mM) Arginine (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Proline (mM) Polysorbate 20 (w / v) pHF 211 2010 50 N / AN / A 7 N / A 0.03 6.0 F 221 2010 50 N / AN / AN / A 200 0.03 6.0 F 231 2010 N / A 10 N / A 8 N / A 0.03 6.0 F 241 2010 N / A 10 0.38 N / A 0.03 6.0 F 251 2010 N / A 10 18 N / A 0.036.0F2612010N / A1038N / A0.036.0F2712010N / A30N / A7N / A0.036.0F2812010N / A300.37N / A0.036.0F2912010N / A3017N / A0.036.0F3012010N / A3037N / A0.036.0

[0252]

[0253] As a result of SE-HPLC analysis, formulations of the present invention containing 10 mM to 30 mM methionine (F23, F27) exhibited a degree of stability similar to that of the control group (F21) containing 50 mM arginine, and formulations containing 10 mM methionine showed a better effect in inhibiting aggregate formation compared to formulations containing 30 mM methionine (Figs. 3a, 3b). It was confirmed that the best effect in inhibiting aggregate formation was achieved when cysteine ​​was added at a concentration of 0.3 mM to 1 mM along with 10 mM methionine in the formulations of the present invention.

[0254] [Example 4]

[0255] Stability of 130 mg / mL liquid formulation at 37℃

[0256] The stability of seven liquid formulations containing 130 mg / mL of the anti-VEGF-Fc fusion protein aflibercept as an active ingredient was evaluated at 37°C. As for the additive combinations, methionine, cysteine, and N-acetylcysteine ​​were used individually or in combination. The ingredient list for the total of seven formulations, F31 to F37, for high concentrations of protein (130 mg / mL) is summarized in Table 4. The formulations were stored at a temperature of 37±2°C, and stability at 7 and 14 days was evaluated by SE-HPLC.

[0257]

[0258] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 4 Formulation Protein (mg / mL) Histidine (mM) Arginine (mM) Methionine (mM) Cysteine ​​(mM) N-acetylcysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v)pHF311301050N / AN / AN / A50.036.0F3213010N / A10N / AN / A70.036.0F3313010N / A100.3N / A70.036.0F34130 10N / A101N / A70.036.0F3513010N / A103N / A70.036.0F3613010N / A10N / A170.036.0F3713010N / A10N / A1070.036.0

[0259]

[0260] As a result of SE-HPLC analysis of samples on days 7 and 14, formulations (F32, F33) containing methionine alone or 0.3 mM to 1 mM cysteine ​​together, as shown in Fig. 4, maintained higher purity than the control formulation (F31) containing 50 mM arginine.

[0261] It was confirmed that formulations (F36, F37) containing N-acetylcysteine ​​at a concentration of 1 mM or 10 mM rapidly degraded after 7 days of storage and that stability was significantly reduced.

[0262] [Example 5]

[0263] Stability of the 140 mg / mL liquid formulation at 37℃

[0264] The stability of five liquid formulations containing 140 mg / mL of the anti-VEGF-Fc fusion protein aflibercept as an active ingredient was evaluated at 37°C. As for the additive combinations, arginine, arginine-hydrochloride, methionine, and cysteine ​​were used individually or in combination. The ingredient list for the five formulations, F38 to F42, for high concentrations of protein (140 mg / mL) is summarized in Table 5. The formulations were stored at a temperature of 37±2°C, and stability was evaluated from 7 to 28 days using SE-HPLC.

[0265]

[0266] Formulation containing the anti-VEGF-Fc fusion protein aflibercept of Example 5 Formulation Protein (mg / mL) Histidine (mM) Arginine (mM) Arginine-hydrochloride (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v) pHF381401050N / AN / AN / A50.036.0F3914010N / A50N / AN / A50.036.0F4014010N / AN / A10N / A70.036.0F4114010N / AN / A100.370.036.0F4214010N / AN / A10170.036.0

[0267]

[0268] SE-HPLC analysis results showed that the 140 mg / mL formulations stored for 28 days at 37±2℃ exhibited the lowest HMW type formation rate (Fig. 5b) and the highest major peak ratio (Fig. 5a) compared to F38 and F39. These results indicated that the addition of methionine and cysteine ​​improved the stability of anti-VEGF-Fc fusion protein formulations compared to formulations with added arginine and arginine-hydrochloride.

[0269] [Example 6]

[0270] pH-dependent stability and the effect of amino acid additives

[0271] The stability of formulations containing methionine and arginine-hydrochloride as additives, respectively, and formulations containing both methionine and cysteine ​​were compared in the pH range from 5.5 to 6.5. The ingredient list of a total of 7 formulations, F43 to F49, for high concentrations of protein (140 mg / mL) is summarized in Table 6. The formulations were stored at a temperature of 37±2℃, and stability was evaluated from 7 to 28 days using SE-HPLC.

[0272]

[0273] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 6 Formulation Protein (mg / mL) Histidine (mM) Arginine-hydrochloride (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v)pHF431401050N / AN / A50.035.5F441401050N / AN / A50.036.0F451401050N / AN / A50.036.5F4614010 N / A10N / A70.035.5F4714010N / A10N / A70.036.0F4814010N / A10N / A70.036.5F4914010N / A100.370.036.0

[0274]

[0275] It was found that formulations F46, F47, F48, and F49 containing methionine maintained higher stability across all pH ranges than formulations F43, F44, and F45 using arginine hydrochloride as an additive (Figs. 6a, 6b). In particular, under pH 6.0 conditions, formulation F49, which combines methionine and cysteine, was confirmed to have improved stability compared to the methionine-alone formulation when stored at 37°C for 28 days. This demonstrates that the combination of methionine and cysteine ​​has a synergistic stabilizing effect on anti-VEGF-Fc fusion proteins.

[0276] [Example 7]

[0277] Evaluation of the influence of key factors using experimental design

[0278] As a factor analysis experiment for formulation development, a 2-level 5-factor Design of Experiment (DOE) was conducted with pH, ​​methionine, cysteine, histidine, and sucrose as variables. The ingredient list for a total of 9 formulations, F50 to F58, for high-concentration protein (150 mg / mL) is summarized in Table 7. The samples were stored at a temperature of 37±2℃ for 7 to 49 days, and the formation of aggregates and stability over time were evaluated using SE-UPLC (Size Exclusion Ultra-High Performance Liquid Chromatography).

[0279] According to the analysis method of the present invention, by using SE-UPLC, high molecular weight components present in a formulation can be analyzed quantitatively and selectively more effectively than the conventional HPLC method. In SE-UPLC analysis, an ACQUITY UPLC Protein BEH SEC 200 Å (4.6 × 300 mm) column (Waters) was used, and 0.2 M sodium phosphate (pH 6.2) and 0.25 M NaCl buffer were flowed at a rate of 0.3 ml / min, and the peak of the anti-VEGF-Fc fusion protein was confirmed at an absorbance of 280 nm.

[0280] In this example, two levels of conditions were established for each of the five factors. Specifically, pH was set to 5.7 and 6.3, methionine to 5 mM and 15 mM, cysteine ​​to 0 mM and 0.4 mM, histidine to 5 mM and 15 mM, and sucrose to 4% (w / v) and 8% (w / v). The established level conditions for the factors are summarized in Table 8. Based on these conditions, Minitab ® As a result of performing a design of experiment (DOE) using software, a total of eight compositions (F51 to F58) were selected. For excipient comparison, composition F50 containing arginine hydrochloride as an additive was also prepared separately.

[0281] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 7 Formulation Protein (mg / mL) Histidine (mM) Arginine-hydrochloride (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v)pHF501501050N / AN / A50.036.0F5115015N / A15N / A40.036.3F521505N / A5N / A40.036.3F5315015N / A5N / A80.035.7F541505 N / A15N / A80.035.7F5515015N / A50.440.035.7F561505N / A150.440.035.7F5715015N / A150.480.036.3F581505N / A50.480.036.3

[0282]

[0283] Summary of the 2-Level 5-Factor DOE of Example 7 Factor Level 1 Level 2 pH 5.7 6.3 Methionine (mM) 5.15 Cysteine ​​(mM) 0.04 Histidine (mM) 5.15 Sucrose (%) 4.8

[0284]

[0285] The 49-day stability of a total of 9 formulations was evaluated using SE-UPLC, and Minitab ® As a result of regression analysis using [the method], sucrose was found to be the primary factor having the greatest influence on formulation stability, followed by pH and methionine in order of importance (Fig. 7a). As shown in Fig. 7b, cysteine, in particular, showed a distinct effect in improving stability when used in combination with methionine rather than alone. These analysis results can be used as a basis for selecting key factors for formulation optimization. Furthermore, as a result of performing SE-UPLC analysis on the samples recovered at week 3 in this example, it was confirmed through comparison with F50 that the use of methionine or the combination of methionine and cysteine ​​is more beneficial than using arginine-hydrochloride as an excipient in histidine buffer (Fig. 7c).

[0286] [Example 8]

[0287] Optimization of Methionine and Sucrose Concentrations Using Design of Experiments

[0288] In this embodiment, to optimize process variables, a Design of Experiment (DOE) was performed by applying the Central Composite Design (CCD) of Response Surface Methodology (RSM) to two factors. The experimental design was performed using Minitab ® I used software.

[0289] Specifically, to determine the optimal concentrations for the two factors of methionine and sucrose, lower and upper limits of the experimental range for each factor were set, and experimental conditions were configured based on these ranges. Accordingly, the ranges for methionine were set from 5 mM to 20 mM, and for sucrose from 5% (w / v) to 10% (w / v). A total of 10 formulations with different compositions and manufacturing conditions, F60 to F69, were designed and manufactured. For comparison with these, a control formulation F59 containing arginine hydrochloride was also manufactured, and all formulations are shown in Table 9.

[0290] A total of 11 high-concentration protein (140 mg / mL) formulations were stored at 37±2℃ for 7 to 28 days, and stability response evaluations were performed using SE-UPLC. Based on the experimental results, the optimal methionine and sucrose concentrations were determined through response surface analysis.

[0291] As shown in Table 10, Minitab ® As a result of reaction optimization through regression analysis, methionine 15 mM and sucrose 10% (w / v) were determined to be the optimal concentrations. Among these, since the sucrose concentration directly affects osmotic pressure, the optimal concentration was set within a range considering isotonicity, while the concentration of methionine was determined based on stability, reflecting the relatively small influence of osmotic pressure.

[0292]

[0293] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 8 Formulation Protein (mg / mL) Histidine (mM) Arginine-hydrochloride (mM) Methionine (mM) Sucrose (w / v) Polysorbate 20 (w / v) pHF 59 140 10 50 N / A 50.0 36.0 F 60 140 10 N / A 20 50.0 36.0 F 61 140 10 N / A 12.5 7.5 0.0 36.0 F 62 140 10 N / A 55 0.0 36.0 F 63 140 10 N / A 20 100.0 36.0 F 64 140 10 N / A 12.550.036.0F6514010N / A57.50.036.0F6614010N / A12.57.50.036.0F671 4010N / A12.5100.036.0F6814010N / A5100.036.0F6914010N / A207.50.036.0

[0294]

[0295] Reaction Optimization Solution by Design of Experiment for Example 8 Methionine (mM) Sucrose % (w / v) UPLC 4w Fit Value (%) Overall Satisfaction 1 15.30 30 10 9 2.65 111

[0296]

[0297] Furthermore, as a result of performing SE-UPLC analysis on the samples recovered at week 4 in this embodiment, it was confirmed that methionine had a significantly greater effect in stabilizing the formulation than arginine-hydrochloride through comparison with F60, F62, F64, etc., which had the same sucrose concentration as formulation F59 (Fig. 8).

[0298] [Example 9]

[0299] Setting the cysteine ​​concentration range

[0300] To confirm the stabilizing effect of cysteine, used as an antioxidant in the formulation, on the anti-VEGF-Fc fusion protein aflibercept, the stability of formulations containing various concentrations of cysteine ​​was evaluated by storing them at 37±2℃ for 4 weeks. Sucrose was set to 5% (w / v) to 6% (w / v) considering the osmotic pressure effect, and the concentration of methionine was fixed at 15 mM, determined through response optimization in response surface design regression analysis, to design and manufacture five formulations summarized in Table 11.

[0301]

[0302] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 9 Formulation Protein (mg / mL) Histidine (mM) Arginine-hydrochloride (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v) pHF641401050N / AN / A50.036.0F6514010N / A15N / A60.036.0F6614010N / A150.260.036.0F6714010N / A150.460.036.0F6814010N / A150.860.036.0

[0303]

[0304] The stabilizing effect of the formulation was confirmed at all added concentrations of cysteine ​​from 0 mM to 0.8 mM, and in particular, the optimal stability improvement effect was observed at the addition of 0.2 mM (Figs. 9a, 9b), allowing the optimal range for cysteine ​​use to be established based on this concentration. These results can be utilized in a strategy to improve formulation stability as an antioxidant condition.

[0305] [Example 10]

[0306] Stability analysis of non-buffered formulations

[0307] Conditions were established in which only sodium chloride was added as a stabilizer to a composition without histidine buffer, and the stability of the protein was evaluated by comparing it with a condition containing histidine buffer in the same composition. A total of three formulations for high-concentration protein (140 mg / mL) were prepared, and the aggregates formed and stability over time were evaluated by SE-UPLC (Size Exclusion Ultra-High Performance Liquid Chromatography) after storage at a temperature of 37±2℃ for 7 to 14 days.

[0308] As a result, as summarized in Tables 12 and 13, a phenomenon was observed in which the main peak decreased significantly and aggregates increased under conditions of buffer-free formulations and sodium chloride addition, indicating limitations in securing long-term stability and that it is difficult to maintain sufficient stability under buffer-free conditions.

[0309]

[0310] SE-UPLC major peak of Example 10 %37 Incubation period at °C (Note) Buffer-free + Sodium chloride 150 mM, 10 mM histidine, 12.5 mM methionine, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20, pH 6.00 98.8 898.8 598.7 1194.1 191.8 495.7 6288.8 183.4 193.42

[0311]

[0312] HMW% analyzed by SE-UPLC of Example 10 during incubation period at °C (Note) Buffer-free + Sodium Chloride 150 mM, 10 mM Histidine, 12.5 mM Methionine, 5% (w / v) Sucrose, 0.03% (w / v) Polysorbate 20, pH 6.00 1.12 1.15 1.29 15.8 98.16 4.24 21 1.19 16.5 96.58

[0313] [Example 11]

[0314] Viscosity analysis of the formulation

[0315] RheoSense m-VROC viscosity at 20°C depending on the concentration of the anti-VEGF-Fc fusion protein Aflibercept (methionine alone or formulations containing both methionine and cysteine) and the concentrations of various components including sucrose. ® Measurements were taken using a viscometer. Approximately 500 μL of undiluted sample was equilibrated to the target temperature, loaded into a syringe, and connected to the chip for analysis. A total of six formulations, F69 to F74, are summarized in Table 14.

[0316] Table 15 shows that the viscosity of high-concentration formulations of aflibercept at concentrations of 100 mg / mL to 140 mg / mL, with sucrose added at various concentrations and histidine buffer-based methionine and cysteine ​​excipients, ranged from approximately 5 to 20 cP. These results can be used as important design indicators for the development of viscosity-controllable ophthalmic formulations. In particular, F74 was found to have a viscosity of 16.82 cP, confirming its suitability as an injectable.

[0317]

[0318] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 11 Formulation Protein (mg / mL) Histidine (mM) Arginine-hydrochloride (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v) pHF691141050N / AN / A50.036.0F7011410N / A15N / A60.036.0F7111410N / A100.370.036.0F7211410N / A100.380.036.0F7312010N / A15N / A60.036.0F7414010N / A100.370.036.0

[0319]

[0320] Viscosity of the formulation of Example 11 Formulation F69 (114 mg / mL) 50 mM arginine + 5% sucrose F70 (114 mg / mL) 15 mM methionine + 6% sucrose F71 (114 mg / mL) 10 mM methionine + 7% sucrose F72 (114 mg / mL) 10 mM methionine + 8% sucrose F73 (120 mg / mL) 15 mM methionine + 6% sucrose F74 (140 mg / mL) 10 mM methionine + 7% sucrose Viscosity (cP) 8.36 9.43 10.06 10.32 11.95 16.82

[0321]

[0322] [Example 12]

[0323] Osmotic pressure analysis of the formulation

[0324] The osmotic pressure of a total of six different high-concentration (140 mg / mL) aflibercept formulations was measured using a Gonotec Osmomat 3000 Osmometer according to changes in the concentration of the composition containing methionine, sucrose, and cysteine. Approximately 50 μL of undiluted sample was stabilized by leaving it at room temperature, then dispensed into a measuring vessel for measurement. The osmotic pressures of formulations F75 to F80, for which measurements were taken, are summarized in Table 16.

[0325]

[0326] Formulation containing anti-VEGF-Fc fusion protein aflibercept of Example 12 Formulation Protein (mg / mL) Histidine (mM) Arginine-hydrochloride (mM) Methionine (mM) Cysteine ​​(mM) Sucrose (w / v) Polysorbate 20 (w / v) pHF751401050N / AN / A50.036.0F7614010N / A12.5N / A50.036.0F7714010N / A12.5N / A7.50.036.0F7814010N / A12.5N / A100.036.0F7914010N / A15N / A60.036.0F8014010N / A150.460.036.0

[0327]

[0328] Osmolality of the high-concentration formulation (140 mg / mL) of Example 12 Formula F75 50 mM Arginine + 5% Sucrose F76 12.5 mM Methionine + 5% Sucrose F77 12.5 mM Methionine + 7.5% Sucrose F78 12.5 mM Methionine + 10% Sucrose F79 15 mM Methionine + 6% Sucrose F80 15 mM Methionine + 6% Sucrose Osmolality (mOsm / kg) 35 6 28 0 40 85 65 32 6 329

[0329]

[0330] As shown in Table 17, the osmotic pressure of the composition containing methionine, cysteine, and sucrose together could be adjusted to a range of approximately 280–565 mOsm / kg depending on the component concentration. In particular, it was demonstrated that even in high-concentration formulations, osmotic pressure approaching isotonicity can be maintained by adjusting the content of sucrose, thereby enabling the design of formulations that minimize tissue irritation upon intraocular administration. These results demonstrate that physiologically acceptable osmotic pressure characteristics can be secured even in high-dose or high-viscosity formulations.

[0331] As a result, through Examples 1 to 12 above, it was confirmed that aggregation and fragmentation are significantly suppressed in the liquid formulations of the present invention under various conditions.

[0332] Through such formulation stability evaluation, it was confirmed that the liquid formulation of the present invention has an excellent inhibitory effect on the formation of impurities or byproducts caused by aggregation, fragmentation, and isomerization of the fusion protein having an IgG Fc domain, thereby providing enhanced stability to the fusion protein.

[0333] Foregoing, specific parts of the content of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A liquid formulation comprising: a protein fused with a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain of human immunoglobulin G (IgG); methionine and / or cysteine; and a buffer having a pH range of 5 to 7.

2. A liquid formulation according to claim 1, characterized in that the soluble extracellular domain of the VEGF receptor comprises an immunoglobulin-like domain 2 of the first VEGF receptor and an immunoglobulin-like domain 3 of the second VEGF receptor.

3. A liquid formulation according to claim 1, characterized in that the fusion protein is included in an amount of 90 to 200 mg / mL.

4. A liquid formulation according to claim 1, wherein the buffer solution comprises histidine.

5. A liquid formulation according to claim 1, characterized in that the concentration of the methionine is 0.001 mM to 50 mM.

6. A liquid formulation according to claim 1, characterized in that the concentration of the cysteine ​​is 0.001 mM to 10 mM.

7. A liquid formulation according to claim 4, characterized in that the concentration of the histidine buffer is 0.001 mM to 20 mM.

8. A liquid formulation according to claim 1, characterized by further comprising one or more stabilizers selected from the group consisting of sugars and surfactants.

9. A liquid formulation according to claim 8, characterized in that the sugar is selected from the group consisting of sucrose, trehalose, mannitol, and glucose in an amount of 2.5 to 10% (w / v).

10. A liquid formulation according to claim 9, characterized in that the sugar is 5 to 10% (w / v) sucrose.

11. A liquid formulation according to claim 8, characterized in that the surfactant is 0.01 to 1% (w / v) of polysorbate 20 or polysorbate 80.

12. A liquid formulation according to claim 1, characterized in that the liquid formulation is suitable for intravitreal injection.

13. methionine and / or cysteine; a buffer having a pH range of 5 to 7; and one or more stabilizers selected from the group consisting of sugars and surfactants; comprising, A composition for stabilizing a protein in which the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG are fused, wherein the buffer comprises 0.001 mM to 20 mM of histidine, the sugar is one or more selected from the group consisting of sucrose, trehalose, mannitol, and glucose in an amount of 2.5% (w / v) to 10% (w / v), and the surfactant is 0.01 to 1% (w / v) of polysorbate 20 or polysorbate 80.

14. A composition for stabilizing a protein in which the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG are fused, wherein the concentration of methionine is 0.001 mM to 50 mM, in accordance with claim 13.

15. A composition for stabilizing a protein in which the soluble extracellular domain of a VEGF receptor and the Fc domain of IgG are fused, wherein, in claim 13, the concentration of cysteine ​​is 0.001 mM to 10 mM.

16. A composition for stabilizing a protein fused with a soluble extracellular domain of a VEGF receptor and an IgG Fc domain, wherein, in claim 13, the protein fused with a soluble extracellular domain of a VEGF receptor and an IgG Fc domain is aflibercept.

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