VEGFR-Fc fusion protein preparation

Abstract

To provide protein formulations, and to provide methods of making and using such formulations.SOLUTION: The formulation can be an ophthalmic formulation, such as for intravitreal administration. In some embodiments, the formulation comprises a VEGFR-Fc fusion protein, such as aflibercept.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 587,733, filed November 17, 2017, and U.S. Provisional Patent Application No. 62 / 618,904, filed January 18, 2018, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application has been filed with a sequence listing in electronic format. The sequence listing is provided as a file named A-2208-WO-PCT_SeqList_ST25.txt, created on November 15, 2018, and having a size of 7.78 kb. The information in the electronic format sequence listing is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to VEGFR-Fc fusion protein formulations and methods of making and using such formulations, for example, formulations without a buffer or formulations with a buffer having a pH outside the buffering capacity of the buffer.

Background Art

[0004] Vascular endothelial growth factor (VEGF), also referred to as VEGF-A, is a signaling protein that promotes the growth of new blood vessels and binds to VEGFR-1 and VEGFR-2. VEGF has been found to be elevated in many tumors and has a role in angiogenesis. VEGF has also been found to have a role in intraocular angiogenesis such as choroidal neovascularization (CNV), a significant aspect of exudative age-related macular degeneration (AMD).

[0005] VEGF inhibitors, such as anti-VEGF antibodies and fragments, as well as decoy receptors or chimeric receptors, have been developed as therapeutic agents for various conditions, including cancer and eye disorders. For example, anti-VEGF antibodies and anti-VEGF Fab are both marketed as bevacizumab and ranibizumab, respectively. Aflibercept, VEGFR-Fc fusion proteins, or "VEGF-traps" are also marketed.

[0006] Aflibercept is a fusion protein composed of an IgG1 Fc domain fused to the Ig domain 2 of VEGFR-1 and the Ig domain 3 of VEGFR-2. Aflibercept is marketed as Eylea® (Regeneron, Tarrytown, NY) for the treatment of various ophthalmic conditions, including exudative AMD, and is formulated for intravitreal administration. The fusion protein is also marketed as Zaltrap® (ziv-aflibercept) (Regeneron, Tarrytown, NY) for the treatment of certain types of cancer and is formulated for intravenous administration.

[0007] Ophthalmic formulations, particularly those administered intravitreally, may have more specific requirements due to additional safety concerns compared to other routes of administration. For example, more specific requirements may be necessary because the impact on the target due to inflammation or other adverse reactions may be severe. For example, intravitreal formulations may require a narrower range of acceptable osmotic pressure. Intravitreal formulations may require a lower threshold for acceptable particle size. For example, in the United States... <789> Specification No. and United States <788> Please refer to the specification. Having a formulation that offers improved stability is also advantageous. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] United States <789> No. specification [Patent Document 2] United States <788> No. specification [Overview of the project] [Means for solving the problem]

[0009] This disclosure provides formulations that meet the need for novel protein formulations, such as stable VEGFR-Fc fusion formulations or intravitreous formulations, which have reduced aggregation or related advantages.

[0010] This specification provides protein formulations and methods for manufacturing and using such formulations. In some embodiments, the formulations are suitable for intravitreous administration. In some embodiments, the protein is a VEGFR-Fc fusion protein. In some embodiments, the protein formulation is a buffer-free formulation. In one embodiment, the formulation contains a buffer and has a pH outside the buffering capacity of the buffer. In one embodiment, the formulation does not contain a buffer.

[0011] In one embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain, a stabilizer, and optionally a surfactant and / or an isotonic agent. Therefore, in some embodiments, the formulation is buffer-free or contains a buffer residue such that the residue does not provide buffering capacity to the formulation. The formulation may have a pH of 4.0 to 8.5. In some embodiments, the formulation may have a pH of 5.0 to 7.0 or about 5.0, about 5.2, about 5.5, about 5.8, about 6.0, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.8. In some embodiments, the pH is 5.0±0.3, 5.2±0.3, 5.5±0.3, 5.8±0.3, 6.0±0.3, 6.1±0.3, 6.2±0.3, 6.3±0.3, 6.4±0.3, 6.5±0.3, or 6.8±0.3.

[0012] In another embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain, a stabilizer, a buffer, and optionally a surfactant and / or an isotonic agent, wherein the formulation has a pH outside the buffering capacity of the buffer. In some embodiments, the buffer is acetate. In some embodiments, the acetate concentration is 0.1 mM to 50 mM, e.g., 0.5 mM to 50 mM, 1 mM to 50 mM, or 2.5 mM to 40 mM. In one embodiment, the acetate concentration is about 0.5 mM, about 1 mM, about 2.5 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, or about 40 mM. The formulation may have a pH outside the buffering capacity of the acetate, e.g., a pH greater than 5.8, a pH between 5.8 and 7.0, or a pH of about 6.0, about 6.1, or about 6.2. In some embodiments, the pH is 6.0±0.3, 6.1±0.3, or 6.2±0.3.

[0013] In some embodiments, the formulation includes a stabilizer, which is an amino acid or a sugar. In one embodiment, the formulation includes two different stabilizers, for example, two different sugars.

[0014] In some embodiments, the formulation includes a surfactant, such as polysorbate 20, polysorbate 80, or Pluronic® F68. In some embodiments, the formulation does not contain a surfactant.

[0015] In some embodiments, the formulation includes an isotonic agent, such as sodium chloride or potassium chloride. In other embodiments, the formulation does not include an isotonic agent.

[0016] In some embodiments, the fusion protein is aflibercept. In some embodiments, the concentration of aflibercept is approximately 40 mg / ml. [Brief explanation of the drawing]

[0017] [Figure 1]As shown by SE-UHPLC for the formulation described in Example 1, a correlation is observed between acetate concentration and aflibercept aggregation level. [Figure 2] This shows the correlation between the target pH and the difference between the pH before buffer exchange (starting pH) and the pH after buffer exchange. (Figure 2A shows the correlation with the target pH. Figure 2B shows the correlation with the titration pH.) [Figure 3] As described in Example 3, a correlation is shown between the salt concentration and the percentage main peak of SEC-UHPLC. [Modes for carrying out the invention]

[0018] This disclosure provides VEGFR-Fc fusion protein formulations and methods for producing and using such formulations. In some embodiments, the formulation is buffer-free. In some embodiments, the formulation contains a buffer, and the formulation has a pH outside the buffering capacity of the buffer. In some embodiments, the formulation does not contain a buffer. In some embodiments, the formulation contains a residual amount of buffer such that the residual amount does not provide buffering capacity for the formulation.

[0019] In some embodiments, formulations without buffers can maintain a stable pH. For example, after storage at approximately 40°C for one or two weeks, the formulation has a pH within approximately 0.1 or 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein exhibits improved stability compared to formulations containing the corresponding buffer. The stability of the VEGFR-Fc fusion protein can be demonstrated by a reduction in aggregation levels, for example, by size exclusion ultrahigh performance liquid chromatography (SEC-UHPLC). The VEGFR-Fc fusion protein may be aflibercept. In some embodiments, the protein concentration is approximately 40 mg / ml.

[0020] In some embodiments, a formulation that includes a buffering agent and has a pH outside the buffering capacity of the buffering agent can maintain a stable pH. For example, after storage at about 40 °C for 1 or 2 weeks, the formulation has a pH within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to a corresponding formulation (same or different buffering agent) having a pH within the buffering capacity of the buffering agent. The stability of the VEGFR-Fc fusion protein can be shown by a reduction in the level of aggregation, for example, by size exclusion ultra-high performance liquid chromatography (SEC-UHPLC). The VEGFR-Fc fusion protein can be aflibercept. In some embodiments, the concentration of the protein is about 40 mg / ml.

[0021] In one embodiment, the formulation includes a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a stabilizer, and optionally a surfactant and / or an isotonic agent. In such embodiments, the formulation does not include a buffering agent. In some embodiments, the formulation includes a residual amount of a buffering agent such that the residual amount lacks buffering capacity in the formulation. In some embodiments, the formulation can maintain a stable pH. For example, after storage at about 40 °C for 1 or 2 weeks, the formulation has a pH within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to a formulation of the same formulation that includes a buffering agent. The stability of the protein can be shown by a reduction in the level of aggregation, for example, by SEC-UHPLC.

[0022] In another embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a buffer, a stabilizer, and optionally a surfactant and / or an isotonic agent, and the formulation has a pH outside the buffering capacity of the buffer. In some embodiments, the formulation can maintain a stable pH. For example, after storage at about 40 °C for 1 or 2 weeks, the formulation has a pH within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to a formulation that is the same but contains a buffer. The stability of the protein can be shown by a reduction in the level of aggregation, such as by SEC-UHPLC and the like.

[0023] In some embodiments, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a stabilizer, and optionally a surfactant and / or an isotonic agent, and the pH is 5.0 - 7.0, 5.5 - 6.5 or 5.5 - 5.6. In some embodiments, the pH is about 7.0, about 6.9, about 6.8, about 6.7, about 6.6, about 6.5, about 6.4, about 6.3, 6.2, about 6.1, about 6.0, about 5.9, about 5.8, about 5.7, about 5.6, about 5.5, about 5.4, about 5.3, about 5.2, about 5.1 or about 5.0. In some embodiments, the pH is about 6.2. In some embodiments, the pH is 7.0 ± 0.3, 6.9 ± 0.3, 6.8 ± 0.3, 6.7 ± 0.3, 6.6 ± 0.3, 6.5 ± 0.3, 6.4 ± 0.3, 6.3 ± 0.3, 6.2 ± 0.3, 6.1 ± 0.3, 6.0 ± 0.3, 5.9 ± 0.3, 5.8 ± 0.3, 5.7 ± 0.3, 5.6 ± 0.3, 5.5 ± 0.3, 5.4 ± 0.3, 5.3 ± 0.3, 5.2 ± 0.3, 5.1 ± 0.3 or 5.0 ± 0.3. In some embodiments, the pH is 6.2 ± 0.3.

[0024] In another embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain, a buffer, a stabilizer, and optionally a surfactant and / or isotonic agent, wherein the formulation has a pH outside the buffering capacity of the buffer. The buffer typically has a buffering capacity in a range of ±1 pH units centered on its pKa value. In one embodiment, the formulation containing the buffer has a pH greater than 1 pH unit from the pKa of the buffer. In some embodiments, the formulation has a pH of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pH units from the pKa of the buffer. In some embodiments, the formulation has a pH of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 pH units from the pKa of the buffer. In one embodiment, the formulation has a pH of approximately 1.4 pH units from the pKa of the buffering agent.

[0025] In one embodiment, a formulation comprising a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain, a buffer, a stabilizer, and optionally a surfactant and / or an isotonic agent, wherein the formulation has a pH outside the buffering capacity range of the buffer, the buffer comprises acetate. The pKa value of acetate is 4.8, and therefore its buffering capacity is in the pH range of 3.8 to 5.8. Therefore, in one embodiment, the formulation has a pH less than 3.8 or greater than 5.8. In one embodiment, the pH of the formulation is greater than 5.8. In one embodiment, the pH is 4.0 to 8.5, 5.0 to 8.5, 5.8 to 8.0, 5.8 to 7.5, 5.8 to 7.0, 5.9 to 7.0, 6.0 to 7.0, 6.0 to 6.8, 6.0 to 6.5, or 6.1 to 6.5. In some embodiments, the pH is about 7.0, about 6.9, about 6.8, about 6.7, about 6.6, about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 6.0, or about 5.9. In some embodiments, the pH is about 6.2. In some embodiments, the pH is 8.5±0.3, 8.4±0.3, 8.3±0.3, 8.2±0.3, 8.1±0.3, 8.0±0.3, 7.9±0.3, 7.8±0.3, 7.7±0.3, 7.6±0.3, 7.5±0.3, 7.4±0.3, 7.3±0.3, 7.2±0.3, 7.1±0.3, 7.0±0.3, 6.9±0.3, 6.8±0.3, 6.7±0.3, 6.6±0.3, 6.5±0.3, 6.4±0.3, 6.3±0.3, 6.2±0.3, 6.1±0.3, 6.0±0.3, 5.9±0.3, or 5.8±0.3. In some embodiments, the pH is 6.2±0.3.

[0026] In one embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain, acetate, a stabilizer, and optionally a surfactant and / or an isotonic agent, and has a pH outside the buffering capacity range of the acetate (e.g., greater than 5.8, e.g., about 6.2), and the acetate is derived from a non-salt form of acetate. In one embodiment, the acetate is derived from a salt form of acetate. The buffer may be an acetate salt or acetate derived from acetic acid, e.g., glacial acetic acid. In one embodiment, the acetate is derived from sodium acetate. In one embodiment, the concentration of acetate or acetate buffer is 0.1mM to 50mM, 0.5mM to 50mM, 1mM to 50mM, 1mM to 40mM, 2.5mM to 40mM, 1mM to 30mM, 1mM to 20mM, 1mM to 10mM, or 1mM to 5mM. In one embodiment, the concentration of acetate or acetate buffer is approximately 0.5mM, approximately 1mM, approximately 2.5mM, approximately 5mM, approximately 10mM, approximately 20mM, approximately 30mM, approximately 40mM, or approximately 50mM.

[0027] In some embodiments, a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain is present in the formulations disclosed herein at a concentration of 1 to 300 mg / ml. In some embodiments, the formulations described herein contain fusion proteins at concentrations of 1 to 50 mg / ml, 1 to 300 mg / ml, 1 to 250 mg / ml, 1 to 200 mg / ml, and 1 to 100 mg / ml. In one embodiment, the formulation contains a fusion protein at a concentration of 10 to 50 mg / ml. In one embodiment, the formulation contains a fusion protein at concentrations of less than 300 mg / ml, less than 250 mg / ml, less than 200 mg / ml, less than 100 mg / ml, less than 50 mg / ml, less than 45 mg / ml, less than 40 mg / ml, less than 30 mg / ml, or less than 25 mg / ml. In one embodiment, the formulation contains a fusion protein in amounts of approximately 300 mg / ml, approximately 250 mg / ml, approximately 200 mg / ml, approximately 100 mg / ml, approximately 50 mg / ml, approximately 45 mg / ml, approximately 40 mg / ml, approximately 30 mg / ml, or approximately 25 mg / ml. In another embodiment, the formulation contains a fusion protein in amounts of approximately 40 mg / ml.

[0028] In some embodiments, the fusion protein includes a domain of VEGFR1, a domain of VEGFR2, or a combination thereof. In some embodiments, the fusion protein includes a domain of VEGFR1 and a domain of VEGFR2. In one embodiment, the fusion protein includes Ig domain 2 of VEGFR1 and Ig domain 3 of VEGFR2. In one embodiment, the fusion protein includes Ig domain 2 of VEGFR1, Ig domain 3 of VEGFR2, and Fc domain of IgG1. In one embodiment, the fusion protein is VEGF Trap. In another embodiment, the fusion protein is aflibercept. In another embodiment, the fusion protein includes the amino acid sequence of SEQ ID NO: 1. In another embodiment, the fusion protein includes the amino acid sequence of SEQ ID NO: 2. In one embodiment, the formulation contains about 40 mg / ml of aflibercept. In one embodiment, the formulation contains about 40 mg / ml of fusion protein containing a protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the formulation comprises a fusion protein containing approximately 40 mg / ml of a protein having the amino acid sequence of SEQ ID NO: 1 and a fusion protein containing a protein having the amino acid sequence of SEQ ID NO: 2.

[0029] [ka]

[0030] [ka]

[0031] In one embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer, and optionally a surfactant, wherein the pH is outside the buffering capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, if the buffer is acetate), and the stabilizer is an amino acid or a sugar.

[0032] In one embodiment, the stabilizer is an amino acid. In one embodiment, the amino acid is proline. In another embodiment, the amino acid is glycine. In some embodiments, the amino acid is a basic amino acid, such as arginine or lysine. In other embodiments, the amino acid is an acidic amino acid, such as aspartic acid. In yet another embodiment, the amino acid is a hydrophobic amino acid, such as alanine. In some embodiments, the formulation contains two different amino acids. In one embodiment, the stabilizer is a sugar. The sugar may be sucrose, sorbitol, glycerol, trehalose (e.g., α,α-trehalose or trehalose dihydrate), mannitol, dextrose, dextran, glucose, or any combination thereof. In one embodiment, the stabilizer is sucrose. In another embodiment, the stabilizer is trehalose. In another embodiment, the stabilizer is cyclodextrin, such as hydroxypropyl-β-cyclodextrin (HPBCD). In yet another embodiment, the formulation contains two different sugars, such as sucrose and trehalose. In another embodiment, the stabilizer is cyclodextrin. In yet another embodiment, the formulation comprises one or more sugars and one or more amino acids.

[0033] The stabilizer concentration may be 1 mM to 300 mM, 10 mM to 300 mM, 100 mM to 300 mM, 200 mM to 300 mM, and 200 mM to 280 mM. In one embodiment, the stabilizer concentration is about 200 mM, for example, about 200 mM proline. In another embodiment, the stabilizer concentration is about 280 mM, for example, about 280 mM glycine.

[0034] In further embodiments, the formulation contains 0-50% (w / v) of a stabilizer. In some embodiments, the formulation contains 0-20% (w / v) of a stabilizer. In some embodiments, the formulation contains 0-10% (w / v), 5-10% (w / v), or 2-10% (w / v) of a stabilizer. In some embodiments, the formulation contains about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, or about 10% (w / v) of a stabilizer, such as a sugar. The sugar may be sucrose or trehalose. In one embodiment, the formulation contains about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% (w / v) of sucrose. In one embodiment, the formulation contains about 5% of sucrose. In yet another embodiment, the formulation contains about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% (w / v) of trehalose. In one embodiment, the formulation contains about 3% (w / v) trehalose. In one embodiment, the formulation contains about 3.5% (w / v) trehalose. In one embodiment, the formulation contains about 4% (w / v) trehalose. In one embodiment, the formulation contains about 4.5% (w / v) trehalose. In one embodiment, the formulation contains about 5% (w / v) trehalose. In another embodiment, the formulation contains about 6.5% (w / v) trehalose.

[0035] In one embodiment, the formulation contains two different sugars. In one embodiment, the concentrations of the first sugar and the second sugar are 0-10% (w / v), 5-10% (w / v), or 2-10% (w / v), respectively. In one embodiment, the total concentration of the first sugar and the second sugar is 0-10% (w / v), 5-10% (w / v), or 2-10% (w / v). In another embodiment, the concentration of the first sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, or about 5% (w / v). In another embodiment, the concentration of the second sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 4%, or about 5% (w / v). In one embodiment, the first sugar is sucrose, and the second sugar is trehalose. In yet another embodiment, the formulation contains about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, or about 10% (w / v) of sucrose and trehalose, for example, about 1% (w / v) of sucrose and about 7% (w / v) of trehalose, about 2% (w / v) of sucrose and about 6% (w / v) of trehalose. Halos contains approximately 3% (w / v) sucrose and approximately 5% (w / v) trehalose, approximately 4% (w / v) sucrose and approximately 4% (w / v) trehalose, approximately 5% (w / v) sucrose and approximately 3% (w / v) trehalose, approximately 6% (w / v) sucrose and approximately 2% (w / v) trehalose, or approximately 7% (w / v) sucrose and approximately 1% (w / v) trehalose. In another embodiment, the formulation comprises about 5% (w / v) sucrose and about 3.5% (w / v) trehalose, about 5% (w / v) sucrose and about 4% (w / v) trehalose, about 5% (w / v) sucrose and about 2.5% (w / v) trehalose, about 5% (w / v) sucrose and about 2% (w / v) trehalose, about 5% (w / v) sucrose and about 1.5% (w / v) trehalose, or about 4% (w / v) sucrose and about 2.5% (w / v) trehalose.

[0036] In one embodiment, the formulation does not contain a surfactant. In another embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer (e.g., sucrose and / or trehalose), and a surfactant, wherein the pH is outside the buffering capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, if the buffer is acetate).

[0037] In one embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a stabilizer (e.g., sucrose and / or trehalose), and a surfactant. The surfactant may be polyoxyethylene glycol alkyl ether, polyoxypropylene glycol alkyl ether, glucoside alkyl ether, polyoxyethylene glycol octylphenol ether, polyoxyethylene glycol alkylphenol ether, glycerol alkyl ester, polyoxyethylene glycol sorbitan alkyl ester, sorbitan alkyl ester, cocamide MEA, cocamide DEA, dodecyldimethylamine oxide, poloxamer, polyethoxylated tallowamine (POEA), or a combination thereof. In one embodiment, the surfactant is polysorbate. In one embodiment, the surfactant is polysorbate 20. In another embodiment, the surfactant is polysorbate 80. In yet another embodiment, the surfactant is poloxamer, e.g., poloxamer 188. In one embodiment, the surfactant is Pluronic® F-68. In some embodiments, the formulation contains 0.001-3% (w / v), 0.001-2% (w / v), 0.001-1% (w / v), 0.001-0.5% (w / v), or 0.01-0.1% (w / v) of the surfactant. In some embodiments, the formulation contains about 0.03% (w / v) of the surfactant, such as polysorbate 80. In some embodiments, the formulation contains about 0.02% (w / v) of the surfactant, such as polysorbate 20. In some embodiments, the formulation contains about 0.01% (w / v) of the surfactant, such as polysorbate 80. In some embodiments, the formulation contains about 0.005% (w / v) of the surfactant, such as polysorbate 80. In some embodiments, the formulation contains about 0.03% (w / v) of a surfactant, such as polysorbate 20. In some embodiments, the formulation contains about 0.02% (w / v) of a surfactant, such as polysorbate 20.In some embodiments, the formulation contains about 0.01% (w / v) of a surfactant, such as polysorbate 20. In some embodiments, the formulation contains about 0.1% (w / v) of a surfactant, such as Pluronic® F-68.

[0038] In one embodiment, the formulation comprises acetate, sucrose, trehalose, and a surfactant. In one embodiment, the formulation comprises about 2.5 mM acetate, about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.01% (w / v) polysorbate 80 at a pH of about 6.2. In one embodiment, the formulation comprises about 2.5 mM acetate, about 5% (w / v) sucrose, about 3% (w / v) trehalose, about 5 mM NaCl, and about 0.01% (w / v) polysorbate 80 at a pH of about 6.2.

[0039] In one embodiment, the formulation does not contain a buffer. In some embodiments, the formulation contains a buffer residue such that the residue does not provide buffering capacity to the formulation. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.01% (w / v) a surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.02% (w / v) a surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.03% (w / v) a surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.01% (w / v) polysorbate 80 at a pH of about 6.2. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.02% (w / v) polysorbate 80 at a pH of about 6.2. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3.5% (w / v) trehalose, and about 0.03% (w / v) polysorbate 20 at a pH of about 6.2. In one embodiment, the formulation contains about 5% (w / v) sucrose, about 3% (w / v) trehalose, about 5 mM sodium chloride, and about 0.01% (w / v) a surfactant (e.g., polysorbate) at a pH of about 6.2. In another embodiment, the formulation contains about 5% (w / v) sucrose, about 3% (w / v) trehalose, about 5 mM sodium chloride, and about 0.01% (w / v) polysorbate 80 at a pH of about 6.2.

[0040] In one embodiment, the formulation does not contain an isotonic agent. In another embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer (e.g., sucrose and / or trehalose), and an isotonic agent, wherein the pH is outside the buffering capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, if the buffer is acetate). In another embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM of acetate), a stabilizer (e.g., sucrose and / or trehalose), an isotonic agent, and a surfactant, wherein the pH is outside the buffering capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, if the buffer is acetate). In one embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a stabilizer (e.g., sucrose and / or trehalose), and an isotonic agent. In another embodiment, the formulation comprises a fusion protein containing a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain (e.g., aflibercept, e.g., about 40 mg / ml of aflibercept), a stabilizer (e.g., sucrose and / or trehalose), an isotonic agent, and a surfactant (e.g., polysorbate).

[0041] The concentration of the isotonic agent may be 1 mM to 250 mM, 5 mM to 200 mM, 40 mM to 200 mM, or 40 mM to 150 mM. In one embodiment, the concentration of the isotonic agent is about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM, about 140 mM, or about 150 mM. The isotonic agent may be a salt, such as a chloride salt. In one embodiment, the isotonic agent is sodium chloride. In another embodiment, the isotonic agent is potassium chloride.

[0042] In some embodiments, the formulations disclosed herein may further comprise excipients. In one embodiment, the formulation may further comprise polymer excipients, such as hyaluronic acid, sodium carboxymethylcellulose (CMC), or poly(lactic acid-coglycolic acid) (PLGA).

[0043] In some embodiments, the formulations disclosed herein are used for intravitreal administration to treat ocular conditions such as exudative age-related macular degeneration (AMD). In some embodiments, the condition is macular edema following retinal vein occlusion (RVO) or diabetic retinopathy (DR). In yet other embodiments, the condition causes blindness. In one embodiment, the formulation can be used in a pre-filled syringe. In one embodiment, the pre-filled syringe is for intravitreal administration of the formulation.

[0044] In some embodiments, the formulations disclosed herein have a particle count (e.g., invisible particle concentration or number) of less than 100, less than 75, less than 50, less than 25, less than 20, less than 15, less than 10, less than 5, or less than 2 particles per milliliter for particle sizes of 10 μm or larger. In some embodiments, the formulations disclosed herein have a particle count of less than 100, less than 75, less than 50, less than 25, less than 20, less than 15, less than 10, less than 5, or less than 2 particles per milliliter for particle sizes of 25 μm or larger. In some embodiments, the formulations disclosed herein have a particle count of less than 100, less than 75, less than 50, less than 25, less than 20, less than 15, less than 10, less than 5, or less than 2 particles per milliliter for particle sizes of 50 μm or larger. In some embodiments, the formulations disclosed herein have a particle count of less than 50 particles per milliliter for particle sizes of 10 μm or larger. In some embodiments, the formulations disclosed herein have a particle count of less than 5 particles per milliliter for particle sizes of 25 μm or larger. In some embodiments, the formulations disclosed herein have a particle count of less than 2 particles per milliliter for particle sizes of 50 μm or larger. In some embodiments, the formulations disclosed herein have an average particle count of 50 particles or less per milliliter for particle sizes of 10 μm or larger. In some embodiments, the formulations disclosed herein have an average particle count of 5 particles or less per milliliter for particle sizes of 25 μm or larger. In some embodiments, the formulations disclosed herein have an average particle count of 2 particles or less per milliliter for particle sizes of 50 μm or larger.

[0045] In some embodiments, the particle count is measured by light shielding, such as by using a liquid particle counter, such as a commercially available counter developed by HIAC. In some embodiments, the particle count is measured at a temperature of 25°C. In some embodiments, if the first formulation has a lower particle count or fewer invisible particles compared to the second formulation, the first formulation (e.g., a formulation without a buffer or a formulation containing a buffer, where the pH of the formulation is outside the buffering capacity of the buffer) is deemed preferable to the second formulation (e.g., a formulation containing a buffer, where the pH of the formulation is within the buffering capacity of the buffer). In another embodiment, the first formulation has a similar particle count or invisible particle count to the second formulation (e.g., no significant difference).

[0046] In some embodiments, if the fusion protein of the first formulation retains more of its original characteristics or properties than the fusion protein of the second formulation after one or more process stresses and / or after storage for a predetermined period, the first formulation (e.g., a formulation without a buffer or a formulation containing a buffer, where the pH of the formulation is outside the buffering capacity of the buffer) is determined to be more stable than the second formulation (e.g., a formulation containing a buffer, where the pH of the formulation is within the buffering capacity of the buffer). The stability of a formulation can be determined by analyzing the properties and characteristics of proteins known in the art, such as those described in U.S. Patent No. 8,092,803 and No. 9,982,032 and International Publication Nos. 2017129685 and 2018094316.

[0047] In one embodiment, if a first formulation exhibits less aggregation than a second formulation after one or more process stresses or stress conditions known in the art, such as those described in International Publication No. 2017129685, then the first formulation (e.g., a formulation without a buffer or a formulation containing a buffer, where the pH of the formulation is outside the buffering capacity of the buffer) is determined to be more stable than the second formulation (e.g., a formulation containing a buffer, where the pH of the formulation is within the buffering capacity of the buffer). In one embodiment, the stress condition is shaking. In another embodiment, the stress condition is one or more freeze / thaw cycles, e.g., 1, 2, 3, 4, or 5 freeze / thaw cycles. In another embodiment, the stress condition is vibration, pressure, and / or drop impact. In one embodiment, the stress condition is light exposure. In one embodiment, the stress condition is mixing. In one embodiment, the formulation is subjected to any one or more stress conditions. Stress conditions may include shaking, one or more freeze / thaw cycles, filtration, mixing, light exposure, vibration, pressure, drop impact stress, and / or combinations thereof. In one embodiment, the stress process includes shaking (e.g., 300 rpm at 25°C for 7 days); three freeze / thaw cycles at a rate of 1°C / min between 25°C and -20°C, with the temperature kept constant for 10 minutes after each cooling / heating step. In another embodiment, the stress process includes three freeze / thaw cycles at 25°C to -30°C; filtration through a 0.2 μm PVDF filter; optional mixing; holding at 2°C to 8°C, light exposure, and a full transport simulation (for example, over 24 hours, 48 ​​hours, 72 hours, 96 hours, or 110 hours, or 24 to 110 hours, 48 ​​to 96 hours, for example, about 50 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, about 100 hours, or about 100 hours, which includes vibration, pressure, and drop impact stress).

[0048] In another embodiment, if the first formulation exhibits less aggregation than the second formulation after storage for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months, then the first formulation (e.g., a formulation without a buffer or a formulation containing a buffer, where the pH of the formulation is outside the buffering capacity of the buffer) is determined to be more stable than the second formulation (e.g., a formulation containing a buffer, where the pH of the formulation is within the buffering capacity of the buffer). Storage may be carried out at a predetermined temperature, for example, about 40°C, about 30°C, about 25°C, about 5°C, about -20°C, or about -30°C.

[0049] In one embodiment, if the first formulation exhibits less aggregation than the second formulation after one or more process stresses and storage for a predetermined period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months, at about 40°C, about 30°C, about 25°C, about 5°C, about -20°C, or about -30°C), then the first formulation (e.g., a formulation without a buffer or a formulation containing a buffer, where the pH of the formulation is outside the buffering capacity of the buffer) is more stable than the second formulation (e.g., a formulation containing a buffer, where the pH of the formulation is within the buffering capacity of the buffer).

[0050] The stability of a formulation can be determined by any method known in the art, such as, for example, U.S. Patent No. 8,092,803 and No. 9,982,032 and International Publication Nos. 2017129685 and 2018094316. In one embodiment, the stability of a formulation is determined by chromatography, for example, size exclusion chromatography, for example, size exclusion high-performance liquid chromatography (SE-HPLC) or size exclusion ultrahigh-performance liquid chromatography (SE-UHPLC) or hydrophobic high-performance liquid chromatography (HI-HPLC), where the change or difference in the first peak from the first formulation before the stress process and / or storage conditions is smaller than that of the second peak from the same formulation after the stress process and / or storage conditions, and smaller than that of the second formulation where the changes or differences in the first and second peaks before and after the stress process and / or storage conditions are larger, indicating that the first formulation is more stable than the second formulation.

[0051] In another embodiment, the stability of the formulation is determined by the turbidity of the formulation (e.g., OD 405 Stability is measured by the percentage of recovered protein (e.g., determined by size exclusion HPLC (SE-HPLC)) and / or the purity of the protein (e.g., measured by SE-HPLC), where lower turbidity, higher recovery, and higher purity indicate higher stability. In some embodiments, SDS-PAGE (reducing or non-reducing) is used to determine the stability of the formulation. In some embodiments, asymmetric flow field-flow fractionation (AF4) is used. In other embodiments, isoelectric focusing (IEF), e.g., capillary isoelectric focusing (cIEF), is used. An increase in fragmentation and / or a change in IEF in the first formulation compared to the second formulation indicates lower stability of the first formulation. Any one or combination of methods can be used to determine the stability of the formulation.

[0052] The detailed description and the following examples are illustrative of the present invention and should not be construed as limiting the invention thereto. Various modifications and alterations can be made by those skilled in the art based on the description of the present invention, and such modifications and alterations are included in the present invention. [Examples]

[0053] Example 1: Stability of aflibercept acetate formulation at pH 6.2 The stability of aflibercept was tested in acetate formulations with varying concentrations at pH levels outside the ideal buffering capacity range of the acetate. Since buffers have a buffering capacity range of approximately ±1 pH units around their pKa value, the pKa value of the acetate is 4.8, and it has a buffering capacity in the pH range of approximately 3.8 to 5.8. The effect of acetate concentration on the ability to maintain the pH and stability profile of aflibercept was tested by varying the acetate concentration. Eylea®, formulated as 40 mg / mL aflibercept in 10 mM sodium phosphate, 40 mM sodium chloride, 5% sucrose, and 0.03% polysorbate 20 at pH 6.2, was included for comparison (Formulation 8).

[0054] 40 mg / ml of aflibercept was buffer-exchanged with the formulations specified in Table 1. After buffer exchange, surfactants were added to the different formulations. The performance of the buffer exchange was verified by testing osmotic pressure, protein concentration, and pH. Before storage at 40°C under stress conditions for up to two weeks, each formulation was subjected to filtration, three freeze / thaw cycles, and drop impact process stress.

[0055] [Table 1]

[0056] The pH for each formulation in Table 1 was measured at 0, 1 week, and 2 weeks, and is shown in Table 2. The pH difference or delta between T=0 and T=1 week, and the pH difference or delta between T=0 and T=2 weeks for each formulation are also shown in Table 2.

[0057] [Table 2]

[0058] As shown in Table 2, the pH difference between T=0 and the subsequent two weeks of storage at 40°C was a maximum of 0.2 pH units, which is within typical manufacturing variability (usually ±0.3 pH units) and is a pH shift not expected to affect the quality attributes of aflibercept. Furthermore, formulation 8, which contains a buffer within its buffering capacity pH, also showed a shift of 0.1 pH units. Therefore, formulations without an active buffer (i.e., formulations containing a buffer that is not an active buffer, because it has a pH outside the buffering capacity range of the buffer) were able to maintain the pH to the same extent as formulations containing an active buffer.

[0059] To determine the protein stability of the formulations listed in Table 1, the formulations were tested by size exclusion ultrahigh performance liquid chromatography (SE-UHPLC), and the aggregation patterns after buffer exchange and during storage were analyzed. SE-UHPLC separates proteins based on differences in hydrodynamic volume. Molecules with larger hydrodynamic volumes elute faster than molecules with smaller volumes. Samples were loaded onto an SE-UHPLC column, separated to uniform concentrations, and the eluent was monitored by UV absorbance. Purity was determined by calculating the percentage of each separated component compared to the integrated total area. A higher main peak value (e.g., percentage) of a formulation measured by SE-UHPLC indicates a lower level of aggregation, thus indicating greater formulation stability. Another indicator of improved stability is the absence of change in the main peak value between the initial and subsequent time points compared to a different formulation.

[0060] The percentage of the main peak for each formulation in Table 1 was measured at 0, 1, and 2 weeks for formulations stored at 40°C and is shown in Table 3. Table 3 also shows the difference or delta value between the main peak percentages at T=0 and T=1 weeks, and the difference or delta value between the main peak percentages at T=0 and T=2 weeks for each formulation.

[0061] [Table 3]

[0062] The improved stability profile is indicated by a reduction in the aggregation level, resulting in a higher main peak value and a lower delta value. Surprisingly, all acetate buffer formulations (Formulations 1-7), which had a pH outside the ideal buffering pH range for acetate, showed a reduction in aggregation levels compared to the phosphate buffer formulation (Formulation 8), which had a pH within the ideal buffering pH range for phosphate. Also surprisingly, the results, as shown in Figure 1, indicate a strong correlation between the reduction in acetate concentration and the reduction in aggregation levels, thus demonstrating improved aflibercept stability.

[0063] Example 2: Stability of aflibercept formulation without buffering agent As determined by the level of aggregation, Example 1 demonstrated that the stability of aflibercept improves with decreasing acetate concentration in formulations with pH levels outside the ideal buffering capacity range of acetate. Therefore, formulations without buffering agents were tested to determine the stability of aflibercept in buffer-free formulations.

[0064] To test the stability of aflibercept and its ability to reach and maintain target pH in formulations without buffering, aflibercept was purified from cell cultures using a CEX column at 3.6 mg / mL in 100 mM Na acetate and 300 mM NaCl to pH 5.0. The target pH values ​​for the compositions were pH 5.0, 5.5, 5.8, 6.2, 6.4, or 6.8, respectively. However, when titrated, the pH values ​​were 5.0, 5.5, 6.0, 6.2, 6.5, and 6.9, respectively (Table 4). The protein was then concentrated to 40 mg / mL and buffer-exchanged with a 5% sucrose and 5% trehalose formulation buffer. The pH and protein concentration after buffer exchange were tested, and the samples were stored under stress conditions at 40°C for up to 2 weeks.

[0065] [Table 4]

[0066] Figure 2 shows the correlation between the target pH and the difference between the pH before buffer exchange (starting pH) and the pH after buffer exchange (Figure 2A shows the correlation with the target pH, and Figure 2B shows the correlation with the titration pH). There was a maximum difference of 0.4 units between the starting pH and the pH after buffer exchange. The difference between the starting pH and the pH after buffer exchange has a linear correlation, as shown in Figure 2B. Therefore, the target pH can be obtained by adjusting the starting pH.

[0067] Table 5 shows the pH values ​​for each formulation in Table 4 before buffer exchange, after buffer exchange and UF / DF, and after 1 week and 2 weeks at 40°C.

[0068] [Table 5]

[0069] pH remained stable throughout the storage period under the stress conditions shown in Table 5. After one week of storage at 40°C, the pH of the formulations remained the same, except for a 0.1 unit decrease in formulation VI. After two weeks at 40°C, the pH decreased by 0.1–0.2 units, which is within the typical manufacturing variability (usually ±0.3 pH units) and is not expected to affect the quality attributes of aflibercept.

[0070] To determine the stability of the formulations listed in Table 4, the formulations were tested by SEC-UHPLC as described in Example 1. A higher main peak value (e.g., percentage) of the formulation measured by SEC-UHPLC indicates a lower level of aggregation, thus indicating greater stability of the formulation. Another indicator of improved stability is the absence of change in the main peak value between the initial and subsequent time points compared to the other formulation.

[0071] The percentage of the main peak for each formulation in Table 4 was measured at 0, 1 week, and 2 weeks (T=0, T=1w, and T=2w, respectively) for formulations stored at 40°C, and is shown in Table 6. The difference or delta value between the main peak percentages at T=0 and T=1 week, and the difference or delta value between the main peak percentages at T=0 and T=2 weeks for each formulation are also shown in Table 6.

[0072] [Table 6]

[0073] Table 6 shows a correlation between increased pH and decreased percentage main peak, except for the lowest pH (Formulation I). As pH decreases, the stability of aflibercept increases, as judged by the reduced level of aggregation.

[0074] Example 3: Stability of aflibercept formulations that do not contain buffering agents and may or may not contain isotonic agents and / or surfactants. Example 2 demonstrated that aflibercept is stable in buffer-free formulations, as determined by the level of aggregation. The effect of salts and surfactants on the stability of aflibercept in buffer-free formulations at pH 6.2 was tested and compared with a buffer-containing formulation at pH 6.2.

[0075] To test the stability of aflibercept and its ability to reach and maintain the target pH in formulations without buffering agents, aflibercept was purified from cell cultures using a CEX column at 6.5 mg / mL in 100 mM Na acetate, 300 mM NaCl, and pH 5.0. The pH of the composition was adjusted to pH 6.4 to achieve the final target pH 6.2, and the pH drift between the pH before and after buffer exchange, as observed in Example 2, was calculated. The protein was then concentrated to 40 mg / mL and buffer-exchanged with the formulations shown in Table 7. After buffer exchange, surfactants were added to the different formulations. The pH and protein concentration after buffer exchange were tested, and the samples were stored under stress conditions at 40°C for up to 2 weeks. Eylea® is formulated at pH 6.2 as 40 mg / mL aflibercept in 10 mM sodium phosphate, 40 mM sodium chloride, 5% sucrose, and 0.03% polysorbate 20. This formulation, containing a buffer, was included for comparison (Formulation A).

[0076] [Table 7]

[0077] Table 8 shows the pH values ​​for each formulation in Table 7 at T=0, 1 week, and 2 weeks.

[0078] [Table 8]

[0079] The starting materials for the formulations in Table 7 were adjusted to pH 6.4 to accommodate the pH shift of the formulations after buffer exchange. The pH of formulation A at T=0 was 6.4 (Table 8), which was higher than the target pH of 6.2 (Table 7), but within the range of ±0.3 units of manufacturing variability. The difference between T=0 and the subsequent two weeks of storage at 40°C was a maximum of 0.2 units of pH, which is within the range of typical manufacturing variability (usually ±0.3 units of pH) and is a pH shift not expected to affect the quality attributes of aflibercept. Furthermore, the presence of buffer resulted in a difference of 0.1 units of pH, similar to when using formulations without buffer. Therefore, for all formulations tested, the pH remained stable over storage under stress conditions.

[0080] To determine the stability of the formulations listed in Table 7, the formulations were tested by SEC-UHPLC as described in Example 1. A higher main peak value (e.g., percentage) of the formulation measured by SEC-UHPLC indicates a lower level of aggregation, thus indicating greater stability of the formulation. Another indicator of improved stability is the absence of change in the main peak value between the initial and subsequent time points compared to the other formulation.

[0081] The percentage of the main peak for each formulation in Table 7 was measured at 0, 1 week, and 2 weeks (T=0, T=1w, and T=2w, respectively) for formulations stored at 40°C, and is shown in Table 9. Table 9 also shows the difference or delta value between the main peak percentages at T=0 and T=1 week, and the difference or delta value between the main peak percentages at T=0 and T=2 weeks for each formulation.

[0082] [Table 9]

[0083] Table 9 shows that all buffer-free formulations, regardless of the presence of salts or surfactants, exhibited higher main peak percentages compared to the buffer-containing formulation (Formulation A), indicating reduced aggregation or improved stability. When comparing the aggregation levels between formulations B and F, which contain and do not contain surfactants, comparable percentage main peaks were observed, indicating that the buffer-free aflibercept formulation is stable regardless of the presence of surfactants, in terms of its aggregation as measured by SE-HPLC.

[0084] A direct correlation was observed between salt concentration and the decrease in the percentage main peak, as shown in Figure 3. However, all formulations without a buffer showed lower levels of aggregation compared to the buffered formulation (Formulation A).

[0085] Example 4: Stability of aflibercept formulation without buffering agent in three larger studies Examples 1-3 were small-scale studies; approximately 2-10 mL underwent buffer exchange, and approximately 80 μL-280 μL were used to ensure stability. Some of the formulations tested in the preceding examples were studied on a larger scale in three separate studies to demonstrate the applicability of the results to larger scales (Table 10). Eylea®, formulated as 40 mg / mL aflibercept in 10 mM sodium phosphate, 40 mM sodium chloride, 5% sucrose, and 0.03% polysorbate 20 at pH 6.2, containing a buffer and with a pH within the buffer's buffering range, was included for comparison in all three studies (formulations a, f, and j). Buffer exchange was performed in 100 mL-1000 mL volumes, and after the buffer exchange, the surfactant was added to the different formulations. pH, osmotic pressure, and protein concentration after the buffer exchange were tested to ensure proper buffer exchange was performed. Different formulations were subjected to various process stresses, including freeze-thaw, mixing, filtration, holding, filling, light exposure, and transport simulations (including vibration, pressure, and drop impact stress). Following process stress, samples from all formulations were stabilized at various storage temperatures: 40°C, 30°C, 25°C, 5°C, and -30°C.

[0086] [Table 10]

[0087] The pH values ​​at T=0 and at various time points and temperatures are shown in Tables 11A and 11B. The maximum and minimum pH values ​​for all conditions tested in Tables 11A and 11B, as well as the maximum difference between T=0 and the various conditions for all formulations, are shown in Table 11C.

[0088] [Table 11]

[0089] [Table 12]

[0090] [Table 13]

[0091] Table 11C shows that, regardless of the presence of buffering agents, the maximum difference between T=0 and various conditions for all formulations is 0.2 pH units over long-term storage of up to 6 months. This indicates that formulations without buffering agents can maintain a stable pH over long-term storage under various temperature conditions.

[0092] To determine the stability of the formulations listed in Table 10, the formulations were tested by SEC-UHPLC as described in Example 1. A higher main peak value (e.g., percentage) of the formulation measured by SEC-UHPLC indicates a lower level of aggregation, thus indicating greater stability of the formulation. Another indicator of improved stability is the absence of change in the main peak value between the initial and subsequent time points compared to other formulations. The results are shown in Tables 12A and 12B and Figure 4.

[0093] [Table 14]

[0094] [Table 15]

[0095] As shown in Tables 12A and 12B, for all three studies (formulations a-e, f-i, and j-k for studies 1, 2, and 3, respectively), formulations without buffers (including formulations containing buffers but with pH levels outside the buffer's buffering capacity) showed reduced or equivalent aggregation compared to the Eylea® formulations (formulations a, f, and j, respectively) under all tested stability conditions.

[0096] In addition, the invisible particle level of the formulations was tested by light shielding (HIAC) to investigate the effect of the formulations on the tendency of the particle-generating proteins. The results for the number of particles of 2 μm, 5 μm, 10 μm, 25 μm, and 50 μm per 1 mL of formulations a to k are shown in Tables 13A to 13E, respectively.

[0097] [Table 16]

[0098] [Table 17]

[0099] [Table 18]

[0100] [Table 19]

[0101] [Table 20]

[0102] Formulations without buffering agents (including formulations containing buffering agents but with a pH outside the buffering capacity range of the buffering agent) showed invisible counts that were not significantly different from formulations containing buffering agents but with a pH within the buffering capacity range of the buffering agent (i.e., formulations a, f, and j).

[0103] While the present invention is described in terms of various embodiments, it will be understood that variations and modifications will be conceivable to those skilled in the art. Accordingly, the appended claims are intended to encompass all such equivalent variations that fall within the scope of the claimed invention. Furthermore, the headings used herein are for constituent purposes only and should not be construed as limiting the subject matter described.

[0104] All references cited in this application are expressly incorporated herein by reference for any purpose.

Claims

1. (a) Aflibercept; (b) 5% (w / v) sucrose; (c) 3.5% (w / v) trehalose; and (d) A surfactant comprising 0.001% (w / v) to 1% (w / v) of polysorbate 20 or polysorbate 80, A liquid formulation for intravitreal administration that does not contain buffering agents.

2. The formulation according to claim 1, wherein the pH is approximately 5.2, approximately 5.5, approximately 5.8, approximately 6.0, approximately 6.2, approximately 6.3, approximately 6.4, or approximately 6.

5.

3. The formulation according to claim 1, wherein the pH is approximately 5.

8.

4. The formulation according to claim 1, wherein the pH is 5.8 ± 0.

3.

5. The formulation according to claim 1, wherein the pH is approximately 6.

2.

6. The formulation according to claim 1, wherein the pH is 6.2 ± 0.

3.

7. The formulation according to claim 1, wherein the surfactant is polysorbate 80.

8. The formulation according to claim 7, wherein the concentration of polysorbate 80 is approximately 0.01% (w / v).

9. The formulation according to claim 1, wherein the concentration of aflibercept is 1 to 200 mg / ml.

10. The formulation according to claim 9, wherein the concentration of aflibercept is approximately 40 mg / ml.

11. The formulation according to claim 9, wherein the concentration of aflibercept is 100 to 200 mg / ml.

12. The formulation according to claim 9, wherein the concentration of aflibercept is approximately 100 mg / ml.

13. The formulation according to claim 1, wherein the pH is 6.2, the surfactant is 0.01% (w / v) polysorbate 80, and the concentration of aflibercept is 40 mg / ml.

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