AU2020364480A1 - Stable liquid composition, method for preparing same, and formulation comprising same - Google Patents

Abstract

The present invention relates to a liquid composition comprising an anti-VEGF antibody, and may provide a liquid composition having remarkably improved stability of an unused fraction after opening and/or a diluted solution for drip infusion by using a buffer containing acetate and adjusting pH and/or osmotic pressure. The present invention provides a liquid composition comprising an anti-VEGF antibody and a method for preparing same. The liquid composition has improved stability of an unused fraction after opening and/or a drip diluted solution by using a buffer containing acetate and adjusting pH and/or osmotic pressure.

Description

STABLE LIQUID COMPOSITION, METHOD FOR PREPARING SAME, AND FORMULATION COMPRISING SAME TECHNICAL FIELD

[0001] The present disclosure relates to a stable liquid composition, a preparation method therefor, and a formulation containing the stable liquid composition.

BACKGROUND ART

[0002] In 1986, the mouse anti-CD3 antibodywas approved by the FDAas the first monoclonal antibody treatment for the prevention of transplant rejection (Yednock et al., Nature 356, 63-66 (1992)). Since then, a number of therapeutic antibodies have been developed and marketed and the market competition for therapeutic antibodies is also increasing with the appearance of Biosimilar products of some original drugs that have expired their market exclusivity. Therefore, maximizing the stability of each therapeutic antibody using the optimization and development of formulation is considered one of the ways to develop their competitiveness. Due to the large molecular weight and complex structure of an antibody, it is important to develop a formulation considering the characteristics of each antibody drug and to minimize the destabilization of the antibody due to the storage conditions and external environmental conditions of each drug. This will maximize the efficacy of the drug and minimize the unwanted drug-related adverse events or toxicity.

[0003] Types of destabilization of antibodies include chemical types, including changes in covalent bonds, and physical types, including modifications of three-dimensional spatial structures. The chemical types of destabilization include, for example, antibody hydrolysis, oxidation, deamidation, disulfide modification, and racemization, and the physical types of destabilization include antibody aggregation and adsorption of the antibody, and unfolding that leads to a decrease in antibody activity.

[0004] During pre-use storage or distribution or when opening a container for use or preparing an instillation solution, the antibody may be modified by physical factors, or the activity of the antibody may be reduced by modification, oxidation or aggregation of the antibody, or structural modifications of the antibody may occur. Therefore, there is a need to stabilize the structure of the antibody from various factors and environments generated in the process of processing and handling the antibody.

[0005] For the above reasons, in the case of Avastin@ which is a currently commercially available product, when the container containing the antibody is opened (when the drug is taken out of the container by a syringe) to prepare an instillation solution for administration, an unused portion remaining in the container should be discarded due to stability problems. In addition, it is known that a solution obtained by diluting the drug with 0.9% physiological saline for injection can be stored at 2 to 300 C for 48 hours.

[0006] If the unused portion after container opening or the diluted solution can maintain acceptable stability and quality at a level acceptable for a pharmaceutical product, not only the opened/diluted pharmaceutical product can be efficiently utilized, but also a more flexible working environment can be provided for healthcare workers who are responsible for the waiting time and storage conditions before use. Therefore, an extended "in-use" stability of the drug would allow a more streamlined approach to the handling of opened container and diluted solution, improving both the efficiency of practitioners and the cost-effectively of the drug, e.g. by providing a multi-dose product.

[0007] However, since factors that affect the stability of formulation compositions vary depending on products and situations, there is a difficulty in developing a stable formulation that is able to display long term in-use stability. In particular, in the case of a liquid formulation, the pharmaceutical drug can easily react with the constituent components and is highly sensitive to oxygen. In addition, in the pharmaceutical field, particularly in the protein formulation field, it is difficult to predict the interaction between a specific pharmaceutical drug and constituent components, and there has been a need to develop a stable formulation capable of maintaining its stability in various environments.

DESCRIPTION OF EMBODIMENTS TECHNICAL PROBLEM

[0008] Therefore, the present disclosure provides a formulation containing an anti-VEGF antibody having enhanced storage stability and "in-use" stability. Specifically, the present disclosure provides a formulation containing an anti-VEGF antibody which can (i) ensure the long-term storage stability of a pharmaceutical product prepared using the formulation and (ii) maintain its stability even after the packaging of the formulation is opened for use and/or after dilution for instillation.

SOLUTION TO PROBLEM

[0009] In an embodiment, the present disclosure provides a formulation containing a stable liquid composition containing an anti-VEGF antibody, having a specific range of osmolality and/or pH, which is capable of maintaining long-term storage stability and stability in an actual use environment.

[00010] Afirst aspect of the present disclosure provides a formulation containing a stable liquid composition, the stable liquid composition containing an anti-VEGF antibody and an acetate buffer and having a pH of 5.2 or less.

[00011] In one embodiment, the liquid composition may have an osmolality of 270 mOsm/kg or less. Optionally, the osmolality may be 270 mOsm/kg or < 270 mOsm/kg.

[00012] The formulation or the liquid composition may be free of phosphoric acid, amino acids, or salts thereof. In addition, the acetate buffer may be prepared by mixing an acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1.

[00013] The liquid composition may further contain sugar or polyol. Specifically, the liquid composition may further contain trehalose.

[00014] In another embodiment, the formulation may be an injectable formulation, and the liquid composition may be stable in a vial or an infusion bag. Specifically, the liquid composition contained in the formulation may maintain stability at a temperature of 10°C or lower for at least 72 hours after vial opening. In addition, the liquid composition may maintain stability at a temperature of 10°C or lower for at least 7 weeks after vial opening. The vial may be a glass vial.

[00015] In still another embodiment, the formulation may be a parenteral formulation that is suitable for injection and may maintain stability at 10°C or lower for at least 45 days after diluting the liquid composition and placing it an infusion bag. The infusion bag may be a polyolefin bag.

[00016] In yet another embodiment, the formulation may have

[00017] (i) a percent change in high-molecular-weight species (A%HMW) of 1.0 or less and/or a percent change in acidic variant (A%acidic) of 5.1 or less, as measured after storing the liquid composition at 30 0C or lower for at least 5 weeks,

[00018] (ii) a percent change in high-molecular-weight species (A%HMW) of 1.8 or less and/or a percent change in acidic variant (A%acidic) of 15.5 or less, as measured after storing the liquid composition at 30 0C or lower for at least 6 months; or

[00019] (iii) a percent change in high-molecular-weight species (A%HMW) of 1.6 or less, as measured after storing the liquid composition at 30 0C or higher for at least 4 weeks.

[00020] A second aspect of the present disclosure provides a method for preparing a stable liquid composition containing an anti-VEGF antibody and an acetate buffer.

[00021] In one embodiment, the acetate buffer is prepared by mixing an acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1. The molar ratio between the acetate and the antibody may be to 360: 1.

[00022] In another embodiment, the method may further comprise a step of adjusting the pH of the liquid composition to 5.2 or less. The method may further comprise a step of adjusting the osmolality of the liquid composition to 270 mOsm/kg or less. The liquid composition may be stable in a vial or an infusion bag.

[00023] A third aspect of the present disclosure provides a liquid composition prepared by the method described herein.

ADVANTAGEOUS EFFECTS OF DISCLOSURE

[00024] Without being bound by a theory, it has been found that the pH and/or osmolality of the liquid composition containing an anti-VEGF antibody can be a factor that affects the stability of the formulation of the present disclosure. It is also surprising found that a specific range of osmolality and a specific range of pH is able to enhance the stability of liquid composition comprising an anti-VEGF antibody in a synergistic manner. Thus, adjusting pH and/or osmolality to a specific value(s) makes it possible to provide a formulation which does not only demonstrate an improved storage stability against various factors and environments occurring in an antibody handling process, e.g. at room temperature for over 5 weeks, but also exhibits superior "in-use" stability, such that any unused portion after opening and/or a dilution prepared for instillation remains stable and usable.

BEST MODE

[00025] Unless otherwise defined, all technical and scientific terms used in the present specification have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent thereto are also included in the scope of the present invention. In general, the nomenclature and measurement methods used in the present specification are well known and commonly used in the art. In addition, the numerical values described in the present invention are considered as including the meaning of "about" even though not specified. A number indicated with the term "about" may be a value within a range of ±10% from the indicated value.

[00026] One aspect of the present disclosure is directed to a formulation containing a stable liquid composition, the stable liquid composition containing a VEGF-binding antibody (anti VEGF antibody) and an acetate buffer and having a pH of 5.2 or less.

[00027] Acetate Buffer

[00028] In the present specification, the term "buffer" refers to a solution prepared by mixing a weak acid and a conjugate base or mixing a weak base and a conjugate acid. In one embodiment, the "acetate buffer" may be prepared by mixing effective amounts of an acetate and acetic acid in order to maintain the desired pH of the liquid composition for stabilization of the VEGF-binding antibody, minimize changes in the pH, and ensure the stability of the composition.

[00029] The acetate may be sodium acetate, potassium acetate, calcium acetate, magnesium acetate, or an anhydride or hydrate thereof, but is not limited thereto

[00030] The acetate may include sodium acetate, an anhydride thereof, or a hydrate thereof. In a specific embodiment, the sodium acetate hydrate may be sodium acetate trihydrate.

[00031] The acetic acid could be mixed with the acetate at a specific ratio in order to adjust the pH of the liquid composition and ensure the stability of the formulation. In one embodiment, the acetate buffer may be prepared by mixing the acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1. Specifically, the molar ratio may be 0.1, 0.2, 0.3, 0.4, 0.5, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 (values in the range between the stated values may also be included). In one embodiment, the molar ratio may be 0.3 to 5.4: 1.

[00032] The molar ratio may be determined by calculating the molar concentration of the acetate based on the known molecular weight of the acetate (for example, 82.0 g/mol for sodium acetate or 136.08 g/mol for sodium acetate trihydrate), calculating the molar concentration of acetic acid based on the known molecular weight of acetic acid (60.05 g/mol), and then calculating the ratio of the molar concentration of the acetate to the molar concentration of acetic acid.

[00033] In one embodiment, the molar concentration of the acetate buffer in the buffer may be the concentration of acetate ions. Specifically, the molar concentration of the acetate buffer may be 10 to 100 mM, for example, 20, 30, 40, 60, 70, 80, or 90 mM (values in the range between the stated values may also be included). In one embodiment, it may be 40 to 60 mM.

[00034] The acetate buffer prepared to have the above-described molar ratio between the acetate and acetic acid may have the above-described molar concentration, and furthermore, the molar ratio contributes to the pH of the liquid composition described below.

[00035] pH and Osmolality

[00036] In one embodiment, the liquid composition contains an acetate buffer obtained by mixing the acetate and acetic acid at the above-described molar ratio, and the pH of the liquid composition may be 5.2 or less.

[00037] In one embodiment, the pH of the liquid composition may be 5.2 or less, for example, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 or 4.0 (values in the range between the stated values may also be included). In one embodiment, the pH of the liquid composition may be 4.0 to 5.2, or 4.5 to 5.2.

[00038] In one embodiment, the osmolality of the liquid composition may be 270 mOsm/kg or less. Specifically, the osmolality may be 210, 220, 230, 240, 250, 260 or 270 mOsm/kg (values in the range between the stated values may also be included). In one embodiment, the osmolality may be 270 mOsm/kg or < 270 mOsm/kg., and in another embodiment, the osmolality may be 230 to 270 mOsm/kg.

[00039] The liquid composition having a pH and/or osmolality within the above-described range have improved storage stability, and may maintain stability in a formulation containing the liquid composition even after opening the container or after dilution of the formulation for dispensing.

[00040] Even if the safety and effectiveness of a pharmaceutical drug have been first confirmed, it is difficult to predict the stability and effectiveness of the opened/dispensed pharmaceutical drug in an actual use environment. This is because factors that can affect the use environment and stability after opening significantly vary depending on products or situations. In particular, a liquid formulation is highly sensitive to oxygen, and an injectable formulation is one of formulations having the highest risk. The liquid composition of the present disclosure can not only exhibit long-term storage stability under room temperature and thermal stress conditions by adjusting the pH and/or osmolality thereof, but also maintain stability without affecting quality or efficacy in an actual use environment.

[00041] The term "adjusting pH and/or Osmolality"as used herein in the present specification means not only controlling the pH and/or Osmolality, but the composition/formulation has initially the desired pH/Osmolality so as to exhibit a desired pharmacological activity, long-term storage stability and in-use stability.

[00042] Liquid Composition

[00043] The "liquid composition" according to the present disclosure means a mixture or combination comprising a VEGF-binding antibody, which is an active ingredient capable of exhibiting a biological effect, and one or more inactive ingredients (additives) suitable for therapeutic administration.

[00044] The liquid composition may further contain one or more pharmaceutically acceptable components so that the VEGF-binding antibody can stably exhibit its activity. In some embodiments, the liquid composition refers to a formulation suitable for administration of the antibody so as to exhibit a desired pharmacological activity without side effects within the scope of medical judgment.

[00045] In one embodiment, the formulation or liquid composition is free of phosphoric acid, amino acids, or salts thereof.

[00046] In another embodiment, the liquid composition may further contain sugar or polyol. Specifically, the sugar can be selected from monosaccharides, disaccharides, oligosaccharides, polysaccharides, and derivatives thereof. The sugar derivatives may mean sugar alcohols or sugar acids. Specifically, the sugars or sugar derivatives may be one or more selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, fructooligosaccharide, galactoligosaccharide, mannan oligosaccharide, starch, glycogen, cellulose, chitin, pectin, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, polyglycitol, aldonic acid, ulosonic acid, uronic acid, aldaric acid, stachyose, sorbose, xylose, ribose, myoinisitose, myoinisitol, and polyethylene glycol. In addition, the polyol may mean an organic compound having two or more hydroxyl (-OH) groups at the molecular terminal. In one embodiment, the polyol may be any one selected from the group consisting of propanediol, glycerin, butylene glycol, propylene glycol, dipropylene glycol, pentylene glycol, hexylene glycol, polyethylene glycol, and sorbitol, but it is not limited thereto.

[00047] In still another embodiment, the liquid composition can further contain a surfactant.

[00048] The surfactant is a component that affects the surface tension of the formulation. The surfactant can reduce the surface tension of the formulation, reduce aggregation of the formulated antibody during storage or handling, reduce particle formation, and prevent particles from being adsorbed onto a container, etc. The surfactant may be, for example, polysorbate, poloxamer, sodium dodecyl sulfate (SDS), sodium laurel sulfate (SLS), or the like. The polysorbate may be polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, or a mixture of two or more thereof. In one embodiment, the surfactant is polysorbate 20.

[00049] For adjustment to the desired pH and osmolality of the present disclosure, the concentrations of the sugar, polyol, and surfactant may be adjusted within the ranges described herein. In order to maintain the chemical/physical stability of the liquid composition, the specific concentrations of these components may be further adjusted depending on the type of excipient.

[00050] Hereinafter, the concentrations of the sugar and the surfactant will be described in detail by way of example.

[00051] In one embodiment, the sugar may be trehalose, specifically trehalose dihydrate. The concentration of the trehalose may be 30 to 200 mM, for example, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 or 190 mM (values in the range between the stated values may also be included). In one embodiment, the liquid composition may contain trehalose dihydrate at a concentration of 50 to 200 mM, or 100 to 190 mM.

[00052] In one embodiment, the surfactant may be polysorbate 20, and the concentration of the polysorbate may be 0.001 to 10 g/ml, for example, 0.01, 0.05, 0.1, 0.15,0.2, 0.25, 0.3, 0.35, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0 g/ml (values in the range between the stated values may also be included). In one embodiment, the liquid composition may contain the surfactant at a concentration of 0.1 to 5 mg/ml.

[00053] In addition, the buffer or liquid composition may further contain a bulking agent, a viscosity adjusting agent, an antioxidant, and/or a tonicity agent.

[00054] In an embodiment, the liquid composition comprises an anti-VEGF antibody, an acetate buffer, and one or more pharmaceutically acceptable additives, wherein the composition has a pH of 5.2 or less and an osmolality of 270 mOsm/kg or less. In a particular embodiment, the liquid composition has a pH of 4.5 to 5.2 and an osmolality of 230 to 270 mOsm/kg. In an embodiment, the liquid composition is free of phosphoric acid, amino acids, or salts thereof. In another embodiment, the acetate buffer of the liquid composition is prepared by mixing an acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1. In a particular embodiment, the molar ratio is 0.3 to 5.4: 1. The anti-VEGF antibody is bevacizumab, ranibizumab, or a biosimilar antibody thereof and the acetate is sodium acetate, an anhydride thereof, or a hydrate thereof such as sodium acetate trihydrate.

[00055] In an embodiment, the liquid composition comprises an anti-VEGF antibody, an acetate buffer, and one or more pharmaceutically acceptable additives selected from the group consisting of polyol, sugar, a surfactant, a bulking agent, a viscosity adjusting agent, an antioxidant, and a tonicity agent. In various embodiments, the liquid composition comprises 10 to 100 mg/ml anti-VEGF antibody, 10 to 100 mM acetate buffer, 30 to 200 mM sugar, and 0.001 to 10 g/ml surfactant, wherein the sugar and the surfactant are selected from the groups described above. The liquid composition may further comprise polyol. In a particular embodiment, the liquid composition comprises an anti-VEGF antibody, an acetate buffer, trehalose, and polysorbate 20. In an embodiment, the liquid composition comprises an anti VEGF antibody, an acetate buffer, 100 to 190 mM trehalose dihydrate, and 0.1 to 5 mg/ml polysorbate 20. In an embodiment, the anti-VEGF antibody is bevacizumab, ranibizumab, or a biosimilar antibody thereof and the acetate is sodium acetate, an anhydride thereof, or a hydrate thereof such as sodium acetate trihydrate. The liquid composition may have (i) a percent change in high-molecular-weight species (A%HMW) of 1.0 or less and/or a percent change in acidic variant (A% acidic) of 5.1 or less, as measured after storing the liquid composition at 30 0C or lowerfor at least 5 weeks; (ii) a percent change in high-molecular-weight species (A%HMW) of 1.0 or less and/or a percent change in acidic variant (A%acidic) of 15.5 or less, as measured after storing the liquid composition at 300 C or lower for at least 6 months; or (iii) a percent change in high-molecular-weight species (A%HMW) of 1.6 or less, as measured after storing the liquid composition at 30 0C or higher for at least 4 weeks.

[00056] Stable Liquid Composition

[00057] The term "stable" as used herein in reference to the formulation means that the formulation substantially retains its physical stability, chemical stability and/or biological activity before and after administration of the formulation, additional preparation processes, or storage.

[00058] The term means that the biological activity of the antibody in the composition is about % to about 150%, about 70% to about 140%, or about 80% to about 120%, or 90% to 110%, as measured after exposure of the formulation to a specific stress condition (e.g. storing under thermal stress condition, long-term storage or storing with opened vial, diluted infusion bag), it can be determined that the antibody retains biological activity.

[00059] In addition, the term means that the physical stability, chemical stability or biological activity of the antibody is improved or maintained at an acceptable level even after the liquid composition is stored under certain conditions. For protein formulations sensitive to storage conditions, it is important to develop a formulation that can retain stability in various environments.

[00060] The physical stability, chemical stability and/or biological activity may be evaluated by commonly known methods. Specifically, the stability may be evaluated in a variety of different ways, including evaluation of aggregate formation (for example, using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, imaged capillary isoelectric focusing (iclEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequencing; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example, tryptic or LYS-C) analysis; or evaluating biological activity or antigen binding function of the antibody.

[00061] When the antibody is not aggregated, precipitated, and/or denatured when assessed by visual inspection of color and/or transparency, UV light scattering or size exclusion high performance liquid chromatography (SE-HPLC), the antibody retains physical stability.

[00062] In one embodiment, the stability of the antibody in the liquid composition may be determined by measuring the aggregation percentage (%) and monomer percentage (%) of the liquid composition, the % acidic, % main and % basic profiles of the protein, and the total purity. The total purity may be determined by measuring whether 2H1L(two heavy chain and one light chain), high molecular weight species (HMWS) and/or low molecular weight species (LMWS) are formed in the liquid composition.

[00063] In addition, the stability of the antibody in the formulation may be determined by measuring whether variants of the antibody are formed. The variants may include variants whose charge varies due to modification of the antibody, and may be, for example, charge variants. The charge variants may be more acidic and may have lower pi values compared to a non-modified antibody.Acidic variants may be generated when they acquire a negative charge or lose their positive charge compared to the main antibody type. In addition, the charge variants may be more basic and may have higher pl values compared to a non-modified antibody. Basic variants may be generated when they acquire a positive charge or lose their negative charge compared to the main antibody type. These charge variants may be generated by antibody oxidation, deamidation, lysine residue C-terminus processing, N-terminal pyroglutamate formation, or non-enzymatic glycosylation. Methods for analysis of the charge variants include chromatograph methods and electrophoresis methods, in which the former includes ion exchange chromatography (IEX), and the latter generally includes capillary zone electrophoresis (CZE), imaged capillary isoelectric focusing (icIEF), and the like.

[00064] Since the aggregated or degraded antibodies or variants in the liquid composition are impurities, the concentration of the antibody excluding these impurities is measured when measuring the purity. In this case, stability is inversely proportional to the percentage of the impurities formed. For example, "stable liquid composition" may mean that the purity of the antibody excluding the aggregated or degraded antibodies in the composition is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, as measured after storage under specific conditions.

[00065] The biological activity of the antibody in the formulation may be measured by antigen binding assay or neutralization assay. Specifically, when the biological activity of the antibody in the composition is 60% to 150%, 70% to 140%, or 80% to 120%, or 90% to 110%, as measured after exposure of the formulation to a specific stress condition, it can be determined that the antibody retains biological activity.

[00066] The formulation or the liquid composition has improved long-term storage stability under room temperature or thermal stress conditions exceeding the room temperature (e.g. °C or higher). In the present specification, the temperature condition may be a temperature within a range of± 2 C from the stated temperature, even though not specially mentioned.

[00067] In the present specification, the term "improved" stability means that the experimental results on stability measured after storage under the same conditions are equal or superior to the physical/chemical stabilities of commercially available Avastin and/or the experimental results on stability measured after storage under the same conditions are superior to the liquid compositions having outside of the claimed ranges of pH and/or osmolality in the present disclosure. Specifically, in some embodiments, the stable liquid composition of the present disclosure has an equal or smaller percent change in high-molecular-weight species percent change (A%HMW) and/or an equal or smaller percent change in acidic variant (A%acidic)than Avastin@. This indicates that similar results are also obtained compared to formulations having outside of the claimed ranges of pH and/or osmolality in the present disclosure. That is, the formulation of the present disclosure can exhibit improved stability.

[00068] In the present specification, the "%HMW'or "%acidic" may be calculated as the area under the curve of the HMW species/acidic variants divided by the area under the curve of the entire chromatogram (relative contents). The term of "A%HMW' or "A%acidic" means the difference (or changes) between %HMW or %acidic before and after storage.

[00069] In one embodiment, the liquid composition has improved stability at room temperature. In one embodiment, the liquid composition may have a percent change in acidic variant (A%acidic) of 5.1 or less, as measured after at least 5 weeks of storage at a temperature of 0C or lower. For example, the temperature may be 250C, and theA%acidic measured under the above-described conditions may be 5.0, 4.9, 4.8, 4.7, 4.6, 4.5 or 4.4 (values in the range between the stated values may also be included).

[00070] In addition, the liquid composition may have a percent change in acidic variant (A%acidic) of 15.5 or less, as measured after at least 6 months of storage at a temperature of 0C or lower. For example, the temperature may be 250C, and theA%acidic measured under the above-described conditions may be 15.4, 15.3, 15.2, 15.1, 15.0, 14.0, 13.0, 12.0, 11.0, 10.0 (values in the range between the stated values may also be included). TheA%acidic may be 10.0 to 15.0.

[00071] In another embodiment, the liquid composition may have a percent change in high molecular-weight species (%HMW) of 1.0 or less, as measured after at least 5 weeks of storage at a temperature of 300 C or lower. The temperature may be 25C, and theA%HMW may be 0.9 or less.

[00072] In another embodiment, the liquid composition may have a percent change in high molecular-weight species (%HMW) of 1.8 or less, as measured after at least 6 months of storage at a temperature of 300 C or lower. The temperature may be 25C, and theA%HMW may be 1.5 or less.

[00073] In still another embodiment, the formulation or liquid composition has improved storage stability under thermal stress conditions.

[00074] In one embodiment, the liquid composition may have a percent change in high molecular-weight species (%HMW) of 1.6 or less, as measured after at least 4 weeks of storage at a temperature of 30°C or higher. In an embodiment, the temperature is 40°C, and the A%HMW is 1.5 or lower or 1.4 or lower (values in the range between the stated values may also be included).

[00075] In another embodiment, the formulation or liquid composition maintains stability under thermal stress conditions or exposure stress conditions (opening or dilution for dispensing) in an actual use environment. Specifically, the formulation may be an injectable formulation, and the liquid composition may be stable in a vial or an infusion bag.

[00076] In this specification, the term "maintain" or "maintenance" of stability means not significant change of experimental values measured in physical/chemical/biological activities (or stabilities) before and after storage of the formulation under conditions. In various embodiment, "maintain" or "maintenance" refers to maintenance of more than 90% biological activities after 6-month storage of the formulation when compared to the ones of the same active ingredient before storage. In some other embodiment, the term means maintenance of more than 95%, 96%, 97%, 98%, or 99% of the original biological activities after 6-month storage of the formulation.

[00077] Also, "maintenance" of the activities may mean that the rate of change of biological activities value (e.g. value measured by VEGF binding assay or VEGF neutralization assay) is under 1.0 or less. Specifically, the rate of change may be 1.0, 0.8, 0.6, or less than 0.3 in VEGF binding assay (%relative binding activity), and the rate of change may be under 0.5, 0.4, 0.3 or 0.2 in VEGF neutralization assay (% relative potency).

[00078] In one embodiment, the liquid composition may maintain stability at a temperature of °C or lower for at least 72 hours after vial opening. At this time, the temperature may be one between 2 0C and 80 C.

[00079] In another embodiment, the formulation may maintain stability at a temperature of 10°C or lower for at least 7 weeks (35 days) after diluting the liquid composition and placing it in an infusion bag. At this time, the temperature may be one between 2C and 8C. Specifically, the formulation may maintain stability after i) 35 days of storage at 2 to 80 C, ii) 48 hours of storage at 300C after storage under conditions of i), or iii) 72 hours of storage at 300C after 45 days of storage at 2 to 8C.

[00080] The infusion bag may be a polyolefin bag. The formulation is passed through containers or devices made of various materials before finally being administered to a patient.

Since the stability and efficacy of the formulation may change depending on the material of the container and the diluent, the stability of the formulation under actual use conditions (e.g., after opening/dilution) should be considered during development of the formulation. In the Examples described herein, incompatibilities between the anti-VEGF antibody (e.g. bevacizumab or ranibizumab) and polyolefin were not observed.

[00081] In another embodiment, the liquid composition may maintain stability even after a vial containing the liquid composition is stored for at least 36 months at a temperature of 2 to 8C, and then continuously stored for 7 weeks at a temperature of 300 C and at a relative humidity of 65 ±5% in an unopened state.

[00082] Antibody

[00083] The "anti-VEGF antibody"in the liquid composition of the present disclosure refers to an antibody that may bind to VEGF, or reduce the expression level of VEGF, or neutralize, block, inhibit, eliminate, reduce or interfere with the biological activity associated with binding of VEGF to the VEGF receptor. The term "VEGF-binding antibody" is used interchangeably with the term "anti-VEGF antibody". The VEGF-binding antibody includes not only a complete antibody form that binds to VEGF, but also an antigen-binding fragment of the antibody.

[00084] In one embodiment, the antibody may be a recombinant humanized anti-VEGF monoclonal antibody which is a VEGF-binding antibody. Specifically, the antibody is "bevacizumab" known as "Avastin@", "ranibizumab" known as "Lucentis" or a biosimilar antibody thereof.

[00085] The antibody may be an anti-VEGF antibody comprising a sequence having a sequence identity of at least 85%, specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% to the known sequence of the antibody.

[00086] Another aspect of the present disclosure provides a method for preparing a stable liquid composition, the stable liquid composition containing a bevacizumab and an acetate buffer.

[00087] In one embodiment, the acetate buffer may be prepared by mixing an acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1, and the molar ratio between the acetate and the antibody may be 50 to 360: 1.

[00088] In another embodiment, the method may comprise a step of adjusting the pH of the liquid composition to 5.2 or less, or a step of adjusting the osmolality of the liquid composition to 270 mOsm/kg or less. The liquid composition may be stable in a vial or an infusion bag.

[00089] It is to be understood that, among contents not mentioned separately for the above preparation method, contents which are common with those described for the liquid composition or formulation are as mentioned above with respect to the liquid formulation.

[00090] In still another aspect, the present disclosure provides a stable liquid composition prepared by the method according to the other aspect of the present disclosure.

[00091] Hereinafter, the present invention will be described in detail with reference to examples and experimental examples. However, the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

[00092] Measurement Methods

[00093] The physicochemical and biological analysis methods used in the following experimental examples are as follows.

[00094] Biological and Physicochemical Analyses

[00095] The protein concentration was calculated by measuring the absorbance (protein concentration measurement) at a wavelength of 280 nm (A28o) using a UV-VIS spectrophotometer, and pH was measured according to European Pharmacopoeia (Ph. Eur.) 2.2.3. In addition, biological analyses were performed using VEGF neutralization assay and VEGF binding assay according to the same guidelines. Purity and impurity tests were performed using SE-HPLC (%HWM measurement) and icIEF (%acidic measurement). The detailed description of the analysis methods is as follows.

[00096] Size Exclusion High-Performance Liquid Chromatography (SE-HPLC)

[00097] SE-HPLC Method is performed to quantitate the total aggregate level of the liquid composition by neat injection using a non-diluted sample and the monomer levels by dilute injection. The dilute injection is run after dilution with dilution buffer (50 mM sodium acetate, pH 5.0) to 2 mg/mL. All samples are incubated at 300 C for 24 hours and injected onto the TSKgel G3000SWXL (TOSOHAAS, P/N 08541, 5pm/.8mm x 300 mm) column using an appropriate HPLC system. The autosampler is kept at 300C while the column is kept at ambient temperature. Flow rate was 0.5 mLmin and total run time per sample is 30 minutes. Detection occurs at 280 nm and data are acquired and processed using software with integration capabilities. Results for each peak are calculated as % of the total protein area.

[00098] Imaged Capillary Isoelectric Focusing (icIEF)

[00099] Reference Standard and samples are diluted with appropriate buffer and injected onto the capillary using an appropriate ICE3 system. Detection occurs at 214 nm and data is acquired and processed using software with integration capabilities. Results for each peak are calculated as % of the total protein area.

[000100] VEGF Binding Assay

[000101] The surface of an ELISA plate was blocked with bovine serum albumin-containing buffer, and then VEGF-A165was adsorbed onto the blocked plate surface by nonspecific binding. Serially diluted sample was added to bind to VEGF-A165, followed by washing to remove nonspecific binding. Then, an HRP-conjugated anti-human IgG (Fc specific) antibody was added and reacted with the sample for a predetermined time, and then an appropriate chromogenic substrate was added. Thereafter, the test plate was then read at a wavelength of 450 nm using a spectrophotometer. The optical density, which was directly proportional to the sample concentration, was obtained, and the relative VEGF-A165 binding activity of the sample was determined by analyzing a 4-parameter curve plotted from the data in parallel line analysis software.

[000102] VEGF Neutralization Assay

[000103]A VEGFR2-dependent reporter gene system was used to determine the neutralization potency of VEGF-A16s by the sample. Specifically, the HEK293-NFAT-VEGFR2 Luc cell line contained a nucleic acid encoding a reporter operably linked to activated T-cell specific element in the promoter region of the luciferase reporter gene. Binding of VEGF to VEGFR2 induces activation of VEGFR2-dependent signal pathway and the expression of the luciferase reporter gene. Thus, the neutralization potency of VEGF-A165 of each sample was calculated based on dose-dependent changes in luminescence levels that corresponded to the amount of luciferase in the cell line.

[000104] I. Storage Stability

[000105] The composition and its concentration of Avastin@ is known: pH 6.2, 25 mg/ml bevacizumab, 51 mM sodium phosphate, 60 mg/ml a,a-trehalose dihydrate and 0.4 mg/ml polysorbate 20.

[000106] Preparation Example 1: Preparation of Liquid Compositions

[000107] As shown in Table 1 below, liquid composition samples were prepared as Sample 1 1, 1-2 and 1-3, which each contained 25.0 mg/ml of bevacizumab, 60 mg/ml of trehalose dihydrate, and 0.4 mg/ml of polysorbate 20. Sample 1-4 is a comparison example based on Avastin@. The buffer information, pH and osmolality of each composition sample are shown in Table 1 below.

[000108] [Table 1] pH Osmolality Sample Buffer (measured, table 16) (mOsm/L) 1-1 51 mM sodium acetate 4.5 248.3 1-2 51 mM sodium acetate 4.9 264.0 1-3 51 mM sodium acetate 5.4 277.3 51 mM sodium 282.7 1-4 6.1 phosphate

[000109] The amount of sodium acetate trihydrate and acetic acid, molar ratio of acetate to acetic acid of the samples are shown in Table 2 below.

[000110] [Table 2] Sodium Acetate Molar Ratio of Acetate to Sample pH Trihydrate (g/L) Acetic Acid (g/L) AceticAcid 1-1 4.5 2.43 1.98 0.54:1 1-2 5.0 4.40 1.14 1.70:1 1-3 5.5 5.89 0.47 5.53:1

[000111] Example 1: Evaluation of Storage Stability

[000112] The samples of Preparation Example 1 were stored under the following conditions, and then the percent changes in high-molecular-weight species (%HMW) and percent changes in acidic variant (A%acidic) of each sample were measured. The storage conditions: i) 25 2 0C, 60± 5 % RH, 5 weeks, ii) 40± 20C, 75 5 % RH, 5 weeks

[000113] The results of the measurement are shown in Table 3 and 4 below.

[000114] [Table 3]

High-molecular-weight species %

Storage Sample. % changes Condition 0 wk 5 wk (A%HMWS) 1-1 2.0 2.9 0.9 i 1-2 2.4 3.9 1.5 1-3 3.4 5.5 2.1

1-4 3.2 4.2 1.0 1-1 2.0 5.4 3.4 1-2 2.4 7.1 4.7 ii 1-3 3.4 9.3 5.9 1-4 3.2 9.5 6.3

[000115] [Table 4] Acidic variants

% Storage Sample No. %changes Condition 0 wk 5 wk (A %acidic) 1-1 42.9 43.5 0.6 1-2 42.9 44.4 1.5 I 1-3 42.7 44.9 2.2 1-4 30.4 33.8 3.4 1-1 42.9 64.2 21.3 1-2 42.9 60.8 17.9 II 1-3 42.7 61.7 19.0 1-4 30.4 62.4 32.0

[000116] From the results in Tables 3 and 4 above, it can be confirmed that, the composition samples have equal or superior to the physical/chemical stabilities of commercially available Avastin@. Also, the samples can maintain the stabilities even under the thermal stress conditions.

[000117] Preparation Example 2: Preparation of Liquid Compositions

[000118] As shown in Table 5 below, liquid composition samples were prepared, which each contained bevacizumab, acetate buffer, 60 mg/ml of trehalose dihydrate, and 0.4 mg/ml of polysorbate 20. The pH and osmolality of each composition sample are shown in Table 5 below.

[000119] [Table 5] Sample p Osmolality Bevacizumab Acetate buffer mOsm/kg mg/ml mM 2-1 4.52 252 45 51

2-2 4.53 251 55 51 2-3 4.97 267 50 51 2-4 4.95 264 50 51 2-5 5.42 277 45 51 2-6 5.45 282 55 51

[000120] Example 2: Evaluation of Storage Stability

[000121] The samples of Preparation Example 2 were stored under the following condition, and then the percent changes in high-molecular-weight species (%HMW) and percent changes in acidic variant (A%acidic) of each sample were measured. The results of the measurement are shown in Table 6 below.

[000122] Storage condition: 25± 20C and 5 weeks.

[000123] [Table 6] Sample %High-molecular-weight %Acidic variant species 0 weeks 5 weeks A% 0 weeks 5 weeks Aacidic HMW 2-1 2.02 2.56 0.54 32.0 35.9 3.9 2-2 2.11 2.77 0.66 31.9 35.3 3.4 2-3 2.94 3.72 0.78 31.8 36.1 4.3 2-4 2.97 3.85 0.88 31.9 36.1 4.2 2-5 4.51 5.60 1.09 31.8 37.0 5.2 2-6 5.04 6.37 1.33 31.8 37.0 5.2

[000124] Table 6 shows that the composition samples of the present disclosure exhibited improved stability.

[000125] Preparation Example 3: Preparation of Liquid Compositions

[000126] As shown in Table 7 below, liquid composition samples were prepared, which each contained bevacizumab, acetate buffer, 60 mg/ml of trehalose dihydrate, and 0.4 mg/ml of polysorbate 20, and which had various pHs and osmolalities.

[000127] [Table 7]

Osmolality Bevacizumab Acetate Sample pH mOsm/kg mg/ml mM

3-1 5.0 268 25.0 51.0 3-2 4.5 243 22.5 45.9 3-3 4.5 243 27.5 45.9 3-4 4.5 260 22.5 56.1 3-5 5.0 249 25.0 42.4 3-6 5.7 280 25.0 51.0 3-7 5.4 281 27.5 56.1

[000128] Example 3: Evaluation of Storage Stability at Room Temperature (6 Months)

[000129] The samples of Preparation Example 3 were stored under the following condition, and then the percent changes in high-molecular-weight species (%HMW) and percent changes in acidic variant (A%acidic) of each sample were measured. The results of the measurement are shown in Table 4 below.

[000130] Storage condition: 25± 2C and 6 months.

[000131] [Table 8] Sample %High-molecular-weight species %Acidicvariant

Initial 6 months AHMW Initial 6 months Aacidic 3-1 2.5 3.8 1.3 35.9 50.9 15.0 3-2 1.9 2.6 0.7 37.2 49.4 12.2 3-3 2.0 2.7 0.8 35.8 49.4 13.6 3-4 2.0 2.7 0.7 36.0 49.6 13.6 3-5 2.4 3.6 1.2 835.8 50.2 14.4 3-6 5.4 7.5 2.2 36.2 51.8 15.6 3-7 3.6 5.4 1.9 35.5 51.4 15.9

[000132] Table 8 shows that the composition samples of the present disclosure exhibited improved stability.

[000133] Preparation Example 4: Preparation of Liquid Compositions

[000134] As shown in Table 9 below, liquid composition samples were prepared, which each contained bevacizumab, acetate buffer, trehalose dihydrate, and polysorbate 20, and which had various pHs and osmolalities.

[000135] [Table 9]

Acetate Trehalose Osmolality Bevacizumab PS20 Sample pH buffer dihydrate mOsm/kg mg/ml mM mg/ml mg/ml

4-1 4.93 268 25.0 51.0 60 0.4 4-2 4.52 221 22.5 45.9 54 0.6 4-3 4.48 223 27.5 45.9 54 0.2 4-4 4.49 239 27.5 56.1 54 0.6 4-5 4.49 241 22.5 56.1 54 0.2 4-6 4.47 265 22.5 45.9 66 0.2 4-7 4.46 263 27.5 45.9 66 0.6 4-8 5.41 245 22.5 45.9 54 0.2 4-9 5.41 243 27.5 45.9 54 0.6 4-10 5.42 268 22.5 56.1 54 0.6 4-11 5.41 267 27.5 56.1 54 0.2 4-12 5.4 283 22.5 45.9 66 0.6 4-13 5.41 284 27.5 45.9 66 0.2 4-14 5.40 307 22.5 56.1 66 0.2 4-15 5.40 310 27.5 56.1 66 0.6

[000136] Example 4: Evaluation of Storage Stability under Thermal Stress Conditions

[000137] The samples of Preparation Example 4 were stored under the following condition, and then the percent changes in high-molecular-weight species (%HMW) and percent changes in acidic variant (A%acidic) of each sample were measured. The results of the measurement are shown in Table 10 below. Storage condition: 400C and 4 weeks.

[000138] [Table 10]

% High-molecular-weight species Sample 0 weeks 4 weeks AHMW

4-1 2.53 3.90 1.4 4-2 2.09 2.79 0.7 4-3 2.10 2.81 0.7 4-4 2.10 3.32 1.2 4-5 2.08 2.83 0.8 4-6 2.05 2.85 0.8 4-7 2.07 2.88 0.8 4-8 3.56 5.59 2.0 4-9 3.77 5.63 1.9 4-10 3.48 5.47 2.0 4-11 3.78 5.69 1.9 4-12 3.53 5.26 1.7 4-13 3.59 5.37 1.8 4-14 3.52 5.37 1.9 4-15 3.67 5.76 2.1

[000139] Table 10 shows that the compositions of the present disclosure exhibited improved stability.

[000140] Preparation Example 5: Preparation of Liquid Compositions

[000141] As shown in Table 11 below, liquid composition samples were prepared, which each contained bevacizumab, buffer, trehalose dihydrate, and polysorbate 20, and which had various pHs and osmolalities. Sample 5-2 is based on the formulation of Avastin@.

[000142] [Table 11] Trehalose PS20

Sample pH Osmolality Bevacizumab Buffer dihydrate

mOsm/kg mg/ml mM mg/ml mg/ml

Sodium 60 0.4 5-1 5.0 267 25 acetate 51mM Sodium 60 0.4 5-2 6.2 279 25 phosphate 51mM

[000143] Example 5: Evaluation of Storage Stability and Biological Activity under Thermal Stress Conditions

[000144] The samples of Preparation Example 5 were stored under the following condition, and then compared with commercially available Avastin@. Storage condition: 400C and 5 weeks. The percent changes in high-molecular-weight species (A%HMW), percent changes in acidic variant (A%acidic) and biological activity of each sample were measured. The results of the measurement are shown in Table 12 below.

[000145] [Table 12] %HMW %acidic Sample 0 weeks 5 weeks AHMW 0 weeks 5 weeks Aacidic 5-1 1.6 4.6 3.0 32.2 54.2 22.0 5-2 2.8 8.0 5.2 27.3 57.6 30.3 VEGF binding, %relative binding Reporter gene assay, % relative activity potency Sample Decrease Decrease 0 weeks 5 weeks rate of 0 weeks 5 weeks rate of change change 5-1 99 91 0.08 89 80 0.10 5-2 103 94 0.09 91 76 0.16

[000146] The decrease rate of change was calculated by dividing the amount of change compared to the initial measured value by the initial measured value. From the results in Table 12 above, it can be confirmed that the composition of the present disclosure has equivalent or higher chemical/physical/biological stabilities than commercially available Avastin@, and can maintain the stabilities even under the thermal stress conditions.

[000147]1II. Evaluation of Long-Term Storage Stability and Stability After Opening/Dilution

[000148] Preparation Example 6. Preparation of Liquid Composition

[000149] A liquid composition was prepared, which contained 25 mg/ml of bevacizumab, 4.8 mg/ml of sodium acetate trihydrate, 0.95 mg/ml of acetic acid, 60 mg/ml of trehalose dihydrate, and 0.4 mg/ml of polysorbate 20.

[000150] Example 6. Evaluation of Long-Term Storage Stability

[000151] The liquid composition prepared in Preparation Example 6 had a pH of 5.0 and an osmolality of 267 mOsm/kg. The liquid composition was stored at a temperature of 2 to 80C for at least 36 months, and then stored under the following condition. Storage condition: temperature of 30 20C, relative humidity of 65 ±5%, and 5 weeks. The percent changes in high-molecular-weight species (A%HMW), percent changes in acidic variant (A%acidic) and biological activity of the liquid composition were measured. The results of the measurement are shown in Table 13 below.

[000152] [Table 13] %HMW %acidic 0 weeks 5 weeks AHMW 0 weeks 5 weeks Aacidic 3.3 3.6 0.3 41.0 48.0 4.0 VEGF binding, % relative binding VEGF neutralization assay, % relative activity potency Decrease Decrease 0 weeks 5 weeks rate of 0 weeks 5 weeks rate of change change 100 102 -0.02 98 95 0.03

[000153] Example 7. Evaluation of Stability of Opened/Pierced Vials

[000154] Vials were prepared, which each contained 400 mg (16 mL) of a liquid composition containing the same components as those of Preparation Example 6. The liquid composition had a pH of 5.1 and an osmolality of 263 mOsm/kg.

[000155] 15 mL of the liquid composition was aseptically taken from each of the vials by a syringe having a 21G needle. The opened/pierced vial samples prepared as described above were stored at a temperature of 5±3C for 35 days under alight-protected condition, and then the percent changes in high-molecular-weight species (A%HMW), percent changes in acidic variant (A%acidic) and biological activity of each sample were measured. The results of the measurement are shown in Table 14 below.

[000156] [Table 14] %HMW %acidic 0 weeks 5 weeks AHMW 0 weeks 5 weeks Aacidic 3.3 3.4 0.1 38.3 39.0 0.7 VEGF neutralization assay, %relative VEGF binding, % relative binding activity poncy potency Decrease Decrease 0 weeks 5 weeks rate of 0 weeks 5 weeks rate of change change 94 96 -0.02 93 92 0.01

[000157] As can be seen from the results in Table 14 above, no significant changes in the physiochemical/biological stabilities after opening were observed.

[000158] Example 8. Evaluation of Stability of Dilutions for Instillation

[000159] Vials were prepared, which each contained 100 mg (4 mL) of a liquid composition containing the same components as those of Preparation Example 6. The liquid composition had a pH of 5.1 and an osmolality of 263 mOsm/kg.

[000160] The vials were stored at a temperature of 30± 20C and a relative humidity of 65 ±5% for 24 hours in an unopened state, and then the content of each vial was diluted with 0.9% physiological saline (sodium chloride), thus preparing dilution samples at a low concentration (1.4 mg/mL). Each dilution sample was stored in a polyolefin bag under the following conditions.

[000161] <Storage conditions>

[000162] Cl: stored at 5 ±30C for 35 days

[000163] C2: stored at 5 ±30C for 35 days, and then stored at 30± 20C for 48 hours

[000164] C3: stored at 5 ±30C for 45 days, and then stored at 30± 20C for 72 hours

[000165] After storage under each condition, the protein concentrations before and after dilution were measured. The results of the measurement are shown in Table 15 below.

[000166] [Table 15] Storage Protein concentration (A2o) (mg/mi) condition Initial Concentration after A% concentration dilution C1 1.4 0 C2 1.4 1.4 0 C3 1.4 0

[000167] Although it has been reported that immunoglobulins at low concentrations can be considerably absorbed more in plastic containers, it was confirmed that the protein concentration in the formulation of the present disclosure was maintained after dilution.

[000168] In addition, after the dilution samples were stored under the above-described conditions, the percent changes in high-molecular-weight species (%HMW), percent changes in acidic variant (A%acidic) and biological activity of each dilution sample were measured. The results of the measurement are shown in Table 16 below.

[000169] [Table 16] Storage %HMW %acidic condition Initial End point AHMW Initial End point Aacidic

C1 2.9 0 39.2 1.1 C2 2.9 3.1 0.2 38.1 39.3 1.2 C3 3.1 0.2 38.5 0.4 Storage VEGF binding, % relative binding VEGF neutralization assay, %

condition activity relative potency Decrease Decrease Initial End point rate of Initial End point rate of change change C1 96 0.02 87 0.18 C2 98 96 0.02 106 91 0.14 C3 97 0.01 102 0.04

[000170] As shown in Table 16 above, the formulation of the present disclosure maintained biological activity in terms of binding activity and potency, in addition to maintain the physicochemical stability thereof.

INDUSTRIALAPPLICABILITY

[000171] The liquid composition containing an anti-VEGF antibody according to the present disclosure is effective to prevent or treat an angiogenesis-related disease, such as colorectal cancer, lung cancer, breast cancer, renal cell carcinoma, glioblastoma, ovarian cancer, fallopian tube carcinoma, peritoneal cancer, cervical cancer, and the like, and thus may be used as a pharmaceutical product in the pharmaceutical industry and medical fields.

Claims (19)

1. [CLAIMS] 1. A liquid composition comprising an anti-VEGF antibody and an acetate buffer, wherein the liquid composition has a pH of 5.2 or less and is a stable liquid composition.
2. 2. The liquid composition of claim 1, wherein the liquid formulation has an osmolality of 270 mOsm/kg or less.
3. 3. The liquid composition of claim 1, wherein the liquid composition is free of phosphoric acid, amino acid, or a salt thereof.
4. 4. The liquid composition of claim 1, wherein the acetate buffer is prepared by mixing an acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1.
5. 5. The liquid composition of claim 1, wherein the liquid composition further comprises sugar or polyol.
6. 6. The liquid composition of claim 1, wherein the liquid composition further comprises trehalose.
7. 7. The liquid composition of claim 1, wherein the liquid composition is an injectable formulation, and the liquid composition is stable in a vial or an infusion bag.
8. 8. The liquid composition of claim 1, wherein the liquid composition maintains stability at a temperature of 10°C or lower for at least 72 hours after vial opening.
9. 9. The liquid composition of claim 1, wherein the liquid composition maintains stability at a temperature of 10°C or lower for at least 7 weeks after vial opening.
10. 10. The formulation of claim 7, wherein the vial is a glass vial.
11. 11. The liquid composition of claim 1, wherein the liquid composition maintains stability at a temperature of 10°C or lowerfor at least 45 days after diluting the liquid composition and placing it in an infusion bag.
12. 12. The liquid composition of claim 7, wherein the infusion bag is a polyolefin bag.
13. 13. The liquid composition of claim 1, which has i) a percent change in high-molecular-weight species (A%HMW) of 1.0 or less and/or a percent change in acidic variant (A% acidic) of 5.1 or less, as measured after storing the liquid composition at 30 0C or lower for at least 5 weeks; ii) a percent change in high-molecular-weight species (A%HMW) of 1.8 or less and/or a percent change in acidic variant (A%acidic) of 15.5 or less, as measured after storing the liquid composition at 30 0C or lower for at least 6 months; or iii) a percent change in high-molecular-weight species (A%HMW) of 1.6 or less, as measured after storing the liquid composition at 30 0C or higher for at least 4 weeks.
14. 14. The liquid composition of claim 1, wherein the anti-VEGF antibody is bevacizumab, ranibizumab, or a biosimilar antibody thereof.
15. 15. A method for preparing a liquid composition comprising an anti-VEGF antibody and an acetate buffer, comprising: preparing the acetate buffer by mixing an acetate and acetic acid at a molar ratio of 0.1 to 5.5: 1.
16. 16. The method of claim 15, further comprising i) adjusting the pH of the liquid composition to 5.2 or less, or ii) adjusting the osmolality of the liquid composition to 270 mOsm/kg or less.
17. 17. The method of claim 15, wherein the anti-VEGF antibody is bevacizumab, ranibizumab, or a biosimilar antibody thereof.
18. 18. The method of claim 15, wherein the liquid composition is stable in a vial or an infusion bag.
19. 19. A liquid composition prepared by the method of claim 15.