Liquid composition containing a VEGF antagonist

Abstract

Provided are compositions for stabilizing proteins, buffers comprising phosphate, histidine, or a combination thereof, and stabilizers that are sodium chloride-free and / or comprise one or more selected from the group consisting of trehalose, sucrose, and mannitol; stabilized liquid compositions of proteins comprising the stabilizing composition and the protein; and methods for preparing stabilized liquid compositions using the stabilizing composition.

Description

Technical Field

[0001] There is provided a stabilized liquid formulation of a protein, which is a composition for stabilizing a protein, comprising a buffer containing phosphoric acid, histidine, or a combination thereof, and a stabilizer, wherein the stabilizer comprises at least one selected from the group consisting of trehalose, sucrose, and mannitol, and / or does not contain sodium chloride; a stabilized liquid composition comprising the composition for stabilization and a protein; and a method for preparing a stabilized liquid composition using the composition for stabilization.

Background Art

[0002] Fusion protein drugs have a larger molecular weight and a more complex structure than general protein drugs, so they are more likely to cause physicochemical instability, and therefore, the development of appropriate formulations is required. The chemical and physical stability of protein drugs can be optimized by various conditions such as pH conditions, selection of buffer, protein concentration, excipients, and temperature. In order to develop a formulation that can ensure the stability of the fusion protein and maintain the pharmacological activity of the fusion protein, various means such as modification of buffer, pH optimization, and addition of stabilizers have been used.

[0003] EYLEA (registered trademark) (aflibercept), a therapeutic agent for macular degeneration, is a liquid formulation drug having a composition of 40 mg / mL aflibercept, pH 6.2, 10 mM sodium phosphate, 40 mM NaCl, 5% (w / v) sucrose, and 0.03% (w / v) polysorbate 20. In the case of intraocular drugs, the regulatory standards for sub-visible particles are more stringent than those for other general biopharmaceuticals (see Table 1), and therefore, it is necessary to develop a formulation considering the particulate aspect..

[0004]

Table 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Accordingly, the present disclosure provides a stabilizing composition for enhancing the stability of protein drugs, and a stable liquid composition of a protein drug containing the same.

[0006] An embodiment provides a composition for stabilizing a protein drug, comprising: a buffer solution containing sodium phosphate, histidine, or a combination thereof, and a stabilizer that does not contain sodium chloride and / or contains at least one selected from the group consisting of trehalose, sucrose, mannitol, and combinations thereof.

[0007] Another embodiment provides a liquid composition comprising a protein; a buffer solution containing sodium phosphate, histidine, or a combination thereof; and a stabilizer that does not contain sodium chloride and / or contains at least one selected from the group consisting of trehalose, sucrose, mannitol, and combinations thereof.

[0008] For example, the liquid composition may contain: (1) a protein at 5 mg / ml to 100 mg / ml; (2) a buffer solution at pH 4 to 8; and (3) a stabilizer, wherein the buffer solution contains sodium phosphate, histidine, or a combination thereof, and the stabilizer does not contain sodium chloride and / or contains at least one selected from the group consisting of trehalose, sucrose, and mannitol. The liquid composition may further contain a surfactant, for example, in an amount of 0.01 to 3% (w / v) based on the total composition.

[0009] Another embodiment provides a pharmaceutical composition comprising the liquid composition.

[0010] For example, for a liquid composition or a pharmaceutical composition, the protein may have a molecular weight of 10 to 500 kDa, 10 to 400 kDa, 10 to 300 kDa, 10 to 200 kDa, or 10 to 150 kDa. In one embodiment, the protein may be a VEGF antagonist, for example, at least one selected from the group consisting of aflibercept (having a molecular weight of about 97 to 115 kDa), bevacizumab (having a molecular weight of about 149 kDa), ranibizumab (having a molecular weight of about 48 kDa), and the like.

[0011] When the protein is one or more selected from the group consisting of VEGF antagonists such as aflibercept, bevacizumab, ranibizumab, etc., the pharmaceutical composition may be an ophthalmic composition, and in particular, a parenteral preparation for intravitreal administration.

[0012] The liquid composition or the pharmaceutical composition may be for intravitreal administration.

[0013] Yet another embodiment provides a method for stabilizing a protein or a method for preparing a stabilized aqueous liquid composition, the method including a step of mixing the protein with a stabilizing composition.

Means for Solving the Problems

[0014] This specification provides a stabilizing composition for enhancing the stability of a protein, a stable liquid composition of the protein containing the same, a method for preparing a stable aqueous liquid composition containing the protein, and a method for stabilizing the protein.

[0015] One embodiment provides a stabilizing composition for a protein, which includes a buffer solution containing sodium phosphate, histidine, or a combination thereof, and a stabilizer containing no sodium chloride and / or at least one selected from the group consisting of trehalose, sucrose, and mannitol.

[0016] Another embodiment provides a liquid composition including the following (1) Protein; (2) A buffer solution containing sodium phosphate, histidine, or a combination thereof; and (3) A stabilizer that does not contain sodium chloride and / or contains one or more selected from the group consisting of trehalose, sucrose, and mannitol.

[0017] The protein content in the liquid composition may be 5 mg / ml to 100 mg / ml, 5 mg / ml to 80 mg / ml, 5 mg / ml to 60 mg / ml, 5 mg / ml to 50 mg / ml, 10 mg / ml to 100 mg / ml, 10 mg / ml to 80 mg / ml, 10 mg / ml to 60 mg / ml, 10 mg / ml to 50 mg / ml, 20 mg / ml to 100 mg / ml, 20 mg / ml to 80 mg / ml, 20 mg / ml to 60 mg / ml, 20 mg / ml to 50 mg / ml, 30 mg / ml to 100 mg / ml, 30 mg / ml to 80 mg / ml, 30 mg / ml to 60 mg / ml, or 30 mg / ml to 50 mg / ml, for example, 5 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0018] The buffer solution may be one of pH 4 to 8, for example, pH 5.2 to 7.5, pH 5.2 to 7.2, pH 5.2 to 7, pH 5.2 to 6.8, pH 5.2 to 6.6, pH 5.2 to 6.4, pH 5.2 to 6.2, pH 5.5 to 7.5, pH 5.5 to 7.2, pH 5.5 to 7, pH 5.5 to 6.8, pH 5.5 to 6.6, pH 5.5 to 6.4, pH 5.5 to 6.2, pH 5.8 to 7.5, pH 5.8 to 7.2, pH 5.8 to 7, pH 5.8 to 6.8, pH 5.8 to 6.6, pH 5.8 to 6.4, pH 5.8 to 6.2, or pH 6.2.

[0019] The buffer may contain one or more selected from the group consisting of phosphoric acid, acetic acid, citric acid, succinic acid, pharmaceutically acceptable salts of the acids (e.g., sodium salts, potassium salts, etc.), and histidine. In one embodiment, the buffer may contain sodium phosphate, histidine, or a combination thereof. The buffer may be included at a concentration of about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, 1 mM to about 12 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 12 mM, about 8 mM to about 50 mM, about 8 mM to about 40 mM, about 8 mM to about 30 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 12 mM, or 10 mM based on the total liquid composition.

[0020] In one embodiment, the stabilizer may not contain sodium chloride.

[0021] In other embodiments, the stabilizer may be one or more selected from the group consisting of trehalose, sucrose, mannitol, and combinations thereof. For example, the stabilizer may be trehalose at 1 - 20% (w / v), 1 - 15% (w / v), 1 - 10% (w / v), 5 - 20% (w / v), 5 - 15% (w / v), 5 - 10% (w / v), 7.8 - 20% (w / v), 7.8 - 15% (w / v), 7.8 - 10% (w / v), 7.8 - 8.2% (w / v), or 8% (w / v); sucrose at 1 - 20% (w / v), 1 - 15% (w / v), 1 - 10% (w / v), 5 - 20% (w / v), 5 - 15% (w / v), 5 - 10% (w / v), 7.8 - 20% (w / v), 7.8 - 15% (w / v), 7.8 - 10% (w / v), 7.8 - 8.2% (w / v), or 8% (w / v); and mannitol at 0.5 - 10% (w / v), 0.5 - 7.5% (w / v), 0.5 - 5% (w / v), 1 - 10% (w / v), 1 - 7.5% (w / v), 1 - 5% (w / v), 3 - 10% (w / v), 3 - 7.5% (w / v), 3 - 5% (w / v), 4 - 5% (w / v), or 4.5% (w / v), and may contain one or more selected from the group.

[0022] In certain embodiments, when the liquid composition contains sodium phosphate as a buffer, or contains a phosphate (e.g., sodium phosphate) as a buffer and sucrose as a stabilizer, the liquid composition may not contain sodium chloride.

[0023] The liquid composition may contain the above-mentioned contents of protein, buffer and stabilizer, and a residual aqueous medium (e.g., water (purified water), physiological saline, water for injection, etc.).

[0024] In one embodiment, the liquid composition may further contain a surfactant in an amount of, for example, 0.001 to 3% (w / v), 0.001 to 2% (w / v), 0.001 to 1% (w / v), 0.001 to 0.5% (w / v), 0.001 to 0.1% (w / v), 0.001 to 0.05% (w / v), 0.01 to 3% (w / v), 0.01 to 2% (w / v), 0.01 to 1% (w / v), 0.01 to 0.5% (w / v), 0.01 to 0.1% (w / v), 0.01 to 0.05% (w / v), or 0.03% (w / v) based on the total liquid composition. The surfactant may be any pharmaceutically acceptable surfactant capable of uniformly dispersing the protein in the liquid composition medium. The surfactant may be a non-ionic surfactant; for example, at least one selected from the group consisting of polysorbates (e.g., polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate)), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate)), where the numerical value after polyoxyethylene, i.e., (20), represents the oxyethylene group (-(CH2CH2O)-); poloxamer (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly(propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compound (e.g., polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C1-C 30)), polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1 - C 30 ) etc.), and means the total number of sodium dodecyl sulfate (SDS) etc. For example, the surfactant may be polysorbate (for example, polysorbate 20).

[0025] The liquid composition provided herein may be isotonic with living tissue. For example, the osmotic pressure of the liquid composition may be from about 200 mOsm / kg to about 400 mOsm / kg, for example, from about 250 mOsm / kg to about 300 mOsm / kg. Such osmotic pressure may be adjusted by a stabilizer.

[0026] The conductivity of the liquid composition provided herein is about 0.1 mS / cm or more, for example, from about 0.1 mS / cm to about 10 mS / cm, from about 0.1 mS / cm to about 7 mS / cm, from about 1 mS / cm to about 10 mS / cm, from about 1 mS / cm to about 7 mS / cm, from about 2.5 mS / cm to about 10 mS / cm, from about 2.5 mS / cm to about 7 mS / cm, from about 5 mS / cm to about 10 mS / cm, or from about 5 mS / cm to about 7 mS / cm.

[0027] In the liquid composition provided herein, the protein may be a protein drug, for example, a protein (such as a fusion protein) having a molecular weight of 10 to 500 kDa, 10 to 400 kDa, 10 to 300 kDa, 10 to 200 kDa, or 10 to 150 kDa. In one embodiment, the protein may be a VEGF (vascular endothelial growth factor) antagonist, for example, a VEGF-specific fusion protein in which the VEGF binding site derived from the extracellular domains of human VEGF receptor 1 and VEGF receptor 2 is fused to the Fc region of human IgG1. In a particular embodiment, the VEGF-specific fusion protein may be a protein in which the region containing immunoglobulin-like (Ig) domain 2 of human VEGF receptor 1 (Flt1) and Ig domain 3 of human VEGF receptor 2 (Flt1 or Flt4) is fused to the Fc region of human IgG1; for example, the VEGF-specific fusion protein may be aflibercept having the following amino acid sequence of SEQ ID NO: 1.

[0028] Aflibercept Amino Acid Sequence (SEQ ID NO: 1) SDTGRPFVEM YSEIPEIIHM TEGRELVIPC RVTSPNITVT LKKFPLDTLI PDGKRIIWDS RKGFIISNAT YKEIGLLTCE ATVNGHLYKT NYLTHRQTNT IIDVVLSPSH GIELSVGEKL VLNCTARTEL NVGIDFNWEY PSSKHQHKKL VNRDLKTQSG SEMKKFLSTL TIDGVTRSDQ GLYTCAASSG LMTKKNSTFV RVHEKDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G (Disulfide cross-linking: 30-79; 124-185; 246-306; 352-410, dimer: 211; 214)

[0029] The fusion protein may be produced recombinantly or synthetically.

[0030] The liquid composition provided herein may be stably maintained at a high temperature of about 40 °C for 4 weeks or more.

[0031] The terms "excellent stability" or "stably maintained" may mean that the physical, chemical and / or biological properties and / or the structure of the protein in the composition can be maintained during storage (e.g., during storage, low protein polymer formation rate, low protein aggregation rate, low protein degradation rate, and / or low denaturation rate, etc.). Various analytical techniques for measuring protein stability are well known in the relevant technical fields.

[0032] For example, when the protein (antibody) content of the liquid composition provided herein is 40 mg / ml, the change in the protein polymer formation rate or aggregation rate (% high molecular weight (w / v); %HMW) measured by conventional SEC (size exclusion chromatography) during storage at 40 °C for 4 weeks (%HMW at the 4th week of storage - %HMW at the 0th week (start of storage)) is less than about 9, for example, about 8.8 or less or about 8.3 or less, but not limited thereto.

[0033] Another embodiment provides a pharmaceutical composition comprising a liquid composition. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, and / or excipient. Pharmaceutically acceptable carriers are those conventionally used and may include, but are not limited to, one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., purified water), saline, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0034] The liquid composition or the pharmaceutical composition may be administered via an oral or parenteral route. In the case of parenteral administration (e.g., injection), it may be administered by intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intracutaneous administration, topical administration, intranasal administration, intratracheal administration, rectal administration, intratumoral administration, intravitreal administration, etc.

[0035] In a particular embodiment, the liquid composition or the pharmaceutical composition may be an eye drop solution containing the above-mentioned VEGF antagonist, and in this case, it may be a parenteral preparation administered to the vitreous humor of the eye.

[0036] Another embodiment provides a method for stabilizing a protein or a method for preparing a stabilized liquid composition, which comprises the step of mixing the protein with the aforementioned stabilizing composition.

[0037] In a particular embodiment, there is provided a method for stabilizing a protein or a method for preparing a stabilized aqueous liquid composition, which comprises the step of mixing the following: (1) a protein; (2) a buffer containing sodium phosphate, histidine, or a combination thereof; (3) a stabilizer that does not contain sodium chloride and / or contains one or more selected from the group consisting of trehalose, sucrose, and mannitol; and (4) optionally, a surfactant.

[0038] The specific descriptions of the types and contents of each component used in the method for stabilizing proteins or the method for preparing a stabilized aqueous liquid composition are the same as those described above.

Advantages of the Invention

[0039] The present invention provides a stabilizing composition and a liquid preparation for enabling proteins such as fusion proteins to stably maintain their physical, chemical, and / or biological effectiveness for a long period of time, thereby inhibiting the generation of polymers and / or aggregates, the generation of fragments, and / or the denaturation into charged variants that may occur during the storage of proteins, and thus enabling the pharmacological effects of the proteins to be maintained for a long period of time.

Brief Description of the Drawings

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0041] The present invention will be described in more detail through the following examples and test examples. However, these examples and test examples are intended to illustrate the present invention and should not be construed as limiting the present invention.

Examples

[0042] (Example 1. Preparation of Liquid Composition) Using aflibercept (a fusion protein of the VEGF binding site derived from the extracellular domains of human VEGF receptors 1 and 2 and the Fc region of human IgG1; CAS number: 862111-32-8; SEQ ID NO: 1), a fusion protein that functions as a VEGF antagonist and is a protein drug, liquid compositions 1 to 14 of the protein drug were prepared as the compositions in Table 2 below:

[0043]

Table 2

[0044] (Example 2. Measurement of HMW Content (%)) To test the stability of Compositions 1 to 14 prepared in Example 1, %HMW (% high molecular weight; based on weight), which indicates the degree of aggregation of the protein drug in the composition, was measured by SE-HPLC during 4 weeks of storage under the conditions of a protein drug concentration of 40 mg / mL, pH 6.2, and 40°C.

[0045] More specifically, substances detected earlier than when monomers were detected were defined as HMW, and %HMW was measured using HPLC (Waters 2695 separation module alliance) and a column (Tosoh, TSK-gel G3000 SWXL) under the conditions of a flow rate of 1.0 mL / min and an injection time of 17 minutes (%HMW = Area H / Area TOTAL × 100).

[0046] The measured %HMW (initial %HMW, %HMW at week 4, Δ%HMW (%HMW at week 4 - initial %HMW)) are shown in Table 3, and %HMW at week 4 is shown in Figure 1 (P: Na - phosphate, H: histidine, T: trehalose, S: sucrose, M: mannitol):

[0047] [Table 3]

[0048] As shown in Table 3 and Figure 1, in all test compositions containing sucrose, trehalose, or mannitol as stabilizers, both the %HMW at week 4 and the increase rate of %HMW at week 4 (compared to the initial %HMW (week 0)) were lower than those of the control (EYLEA® and EYLEA® composition - applied formulations).

[0049] The results of analyzing the statistically significant factors based on the Δ%HMW results shown in Table 3 are shown in Figure 2. In Figure 2, it was confirmed that the factors significantly affecting △%HMW were mannitol, sucrose, and trehalose as stabilizers. As shown in Figure 2, there was no significant difference due to the type of buffer. In the case of stabilizers, it was shown that the %HMW was lower when using mannitol, sucrose, or trehalose compared to the composition using dextrose.

[0050] For the formulations containing mannitol, sucrose, or trehalose as stabilizers, which were confirmed to reduce Δ%HMW as described above, the same test was repeated (n = 3), and the obtained results are shown in Table 4:

[0051] [Table 4]

[0052] As shown in Table 4, formulations containing mannitol, sucrose, or trehalose as stabilizers showed a low Δ%HMW compared to the control, had excellent stability, and indicated that such excellent stability was repeatedly achieved.

[0053] (Example 3. Measurement of Acidic Component (%) and Major Component (%)) As shown in Table 5, Compositions 1 - 10 (protein drug concentration: 40 mg / mL; pH 6.2; surfactant: 0.03% (w / v) PS20) were prepared using mannitol, sucrose, or trehalose as stabilizers whose excellent stabilizing effects were confirmed in Example 1 and Table 3, and using dextrose as a control stabilizer. To test the stability of these compositions, the % acidic (% acidic variant content; based on weight) and % main (content of protein maintaining the surface charge of the initial state of the charge variant aspect (based on weight)) indicating the degree of denaturation of the protein drug in the composition were measured using imaging capillary isoelectric focusing (icIEF) during storage at 40°C for 4 weeks.

[0054] More specifically, % acidic, % basic, and % main were measured by analyzing the acidic, main, and basic isoforms of the sample incubated under the conditions of a sample injection time of 110 seconds and a sample injection pressure of 2000 psi using an icIEF apparatus (Protein simple, iCE3) after treating with an enzyme (neuraminidase A, Sigma - Aldrich).

[0055] The measured % acidic and % main (initial, 4th week, results at the 4th week - initial results (Δ% acidic and Δ% main)) are shown in Table 5:

[0056]

Table 5

[0057] As shown in Table 5, all of the test compositions showed a lower degree of change in both % acidity and % main compared to the comparative composition containing dextrose as a stabilizer, indicating that the test compositions had increased stability.

[0058] Statistically significant factors determined based on the results of Table 5 were analyzed, and the results are shown in Figure 3. As shown in Figure 3, it was confirmed that there was no significant difference in Δ% acidity depending on the type of buffer, but all stabilizers except dextrose significantly affected Δ% acidity.

[0059] (Example 4. Measurement of the VEGF Binding Potency of Aflibercept) To test the maintenance of the activity of aflibercept, the pharmacological active ingredient in Compositions 1 to 14 of Table 2 prepared in Example 1, the %RBA (Relative Binding Activity) and %RPA (Relative Potency Activity) of aflibercept in the composition against VEFG were measured during storage for 4 weeks under the conditions of a protein drug concentration of 40 mg / mL, pH 6.2, and 40 °C.

[0060] More specifically, %RBA was measured by the following method: After VEGF165 coating (including washing and blocking; R&D system) on a MaxiSorp 96-well plate (Nunc), aflibercept and a secondary antibody (Sigma) were loaded thereon in sequence, and then a TMB ELISA substrate solution (Thermo Fisher Scientific) was treated; thereafter, %RBA (relative potency fraction) was measured using an ELISA reader (SpectraMax).

[0061] The %RBA measured at 0 week and 4 weeks at 40 °C and the %RBA change rate (Δ%RBA) for 4 weeks are shown in Table 6.

[0062]

Table 6

[0063] As shown in Table 6, the %RBA reduction rate was shown to be lower than or equivalent to that of the control in all tested compositions except for the composition containing dextrose as a stabilizer, and such results indicated that aflibercept maintained sufficient binding activity to VEGF in all tested compositions.

[0064] Also, %RPA was measured in the following manner: After loading KDR293 cells (Promega) into a 96-well plate loaded with aflibercept and VEGF in sequence and incubating, %RPA (relative potency analysis) was analyzed using an Envision microplate reader (Perkin Elmer, Envision 2014).

[0065] The %RPA measured at 0 weeks and 4 weeks at 40°C and the %RPA change rate (Δ%RPA) over 4 weeks are shown in Table 7.

[0066]

Table 7

[0067] As shown in Table 7, the reduction rate of %RPA in all tested compositions except for the composition containing dextrose as a stabilizer was lower than or equivalent to that of the control, suggesting that the relative titer of aflibercept against VEGF neutralizing capacity could be sufficiently maintained in all tested compositions.

[0068] Statistically significant factors were analyzed based on the %RPA results in Table 7, and the obtained results are shown in Figure 4. As shown in Figure 4, all tested buffers showed similar or improved %RPA reduction compared to the control, and in the case of stabilizers, all tested stabilizers except dextrose showed similar or less %RPA reduction compared to the control.

[0069] (Example 5. Measurement of the stability of six types of compositions) Among the compositions prepared in Example 1, the % main of six compositions (Compositions 4, 5, 6, 8, 9, and 10 in Table 2) with excellent stability confirmed in Examples 2 to 4 was measured using CE-SDS (capillary electrophoresis sodium dodecyl sulfate) (40 °C, 4 weeks).

[0070] More specifically, the % main was measured by the following method: A sample obtained by mixing SDS sample buffer (AB Sciex) and BME (2-mercaptoethanol, Sigma Aldrich) (mixing ratio: approximately 1:50 (v:v)) was heat-treated at 70 °C, and then the % total protein area detected below 220 nm was analyzed using a CE analyzer (Beckman Coulter, PA800 plus) and 32 karat software (Beckman Coulter).

[0071] The % main measured at 0 week and 4 weeks at 40 °C and the % main change rate (Δ% main) for 4 weeks are shown in Table 8:

[0072]

Table 8

[0073] As shown in Table 8, the % main decrease of the six tested compositions was equal to or lower than that of the control. Such results indicated that all six tested compositions had stability equal to or higher than that of the control.

Claims

1. A liquid composition comprising: (1) Aflibercept at 5 mg / ml to 100 mg / ml; (2) A phosphate buffer having a pH in the range of 4 to 8, at 1 to 50 mM; (3) A stabilizer at 7.8 to 10% (w / v), wherein the stabilizer is sucrose; and (4) A surfactant in an amount of 0.01 to 0.1% (w / v), wherein the surfactant is at least one selected from the group consisting of polysorbate 20, polysorbate 80, and combinations thereof, wherein the liquid composition does not contain sodium chloride, the composition is aqueous and has an osmotic pressure of 200 to 400 mOsm / kg, the 4-week %HMW (high molecular weight %) change rate (Δ%HMW) during storage of the liquid composition at 40 °C for 4 weeks is less than 9, liquid composition.

2. The liquid composition according to claim 1, wherein the amount of aflibercept is 20 mg / ml to 60 mg / ml.

3. The liquid composition according to claim 1 or 2, wherein the pH of the liquid composition is 5.8 to 6.

2.

4. The liquid composition according to any one of claims 1 to 3, wherein the concentration of the buffer in the composition is 1 to 20 mM.

5. The liquid composition according to any one of claims 1 to 4, wherein the buffer contains sodium phosphate.

6. The liquid composition according to any one of claims 1 to 5, wherein the amount of the stabilizer is 7.8 to 8.2% (w / v).

7. The liquid composition according to any one of claims 1 to 6, which is aqueous and isotonic.

8. A liquid composition comprising the following (1) aflibercept at 30 mg / ml to 50 mg / ml; (2) a phosphate buffer of 1 to 20 mM containing sodium phosphate; (3) 7.8 to 10% (w / v) sucrose; and (4) 0.01 to 0.1% (w / v) polysorbate 20; wherein the liquid composition has a pH of 6.2 and does not contain sodium chloride, the composition is aqueous and has an osmotic pressure of 200 to 400 mOsm / kg, the 4-week %HMW (high molecular weight %) change rate (Δ%HMW) during storage of the liquid composition at 40 °C for 4 weeks is less than 9, a liquid composition.

9. A parenteral formulation for intravitreal administration, comprising the liquid composition according to any one of Claims 1 to 8.

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