Patent Description:
A type of cell-inducing dimer mitogen having selectivity to vascular endothelial cells has been identified, and is designated as vascular endothelial growth factor (VEGF). The VEGF is an important factor which increases angiogenesis and vascular permeability.

It has been known that the VEGF activates VEGF receptors (i.e., VEGFR-<NUM>, VEGFR-<NUM>, and VEGFR-<NUM>) which are membrane-spanning tyrosine kinase receptors. Among the VEGF receptors, VEGFR-<NUM> and VEGFR-<NUM> have <NUM> Ig-like sequences in the extracellular domain in order to bind to the VEGF, a single transmembrane region, and a consensus tyrosine kinase region. These features are applied as a sequence for an anti-VEGF agent. Aflibercept, which is an ophthalmic therapeutic agent, is a soluble decoy receptor of about <NUM> kDa (including glycosylation) having a structure in which a second binding domain of VEGFR-<NUM> and a third binding domain of VEGFR-<NUM> are fused with an Fc region of a human IgG1 (see [<NPL>]).

In mechanisms via the VEGF, abnormal angiogenesis is associated with ophthalmic diseases such as wet age-related macular degeneration, diabetic macular edema, and macular edema in retinal vein occlusion, etc. (see [<NPL>]).

Examples of therapeutic agents for these ophthalmic diseases include pegaptanib (trade name: Macugen), ranibizumab (trade name: Lucentis), bevacizumab (trade name: Avastin), and aflibercept (trade name: Eylea). Aflibercept is approved for the treatment of wet age-related macular degeneration in <NUM> (see [<NPL>], and [<NPL>], etc.). Among the therapeutic agents above, aflibercept has been reported to have the best therapeutic effect on patients with diabetic macular edema having advanced amblyopia (see [<NPL>]. Aflibercept has been commercialized as a therapeutic agent for metastatic colorectal cancers (trade name: Zaltrap) and a therapeutic agent for retinal vein occlusion, diabetic macular edema, choroidal neovascularization, and wet age-related macular degeneration (trade name: Eylea).

Physicochemical modifications occur in protein drugs including antibody drugs under the non-optimal condition. In particular, factors such as temperature, pH, concentration of a salt, contact with air, concentration of a protein, and types of buffers significantly affect oxidation, deamidation, isomerization, and polymerization, of a protein. These modifications cause aggregation, and generate fragments, isomers of the protein, so that bioactivity may be reduced. These properties differ among proteins. Particularly, for an Fc fusion protein, due to the problem in folding, separate <NUM> peaks appear in hydrophobic interaction chromatography (see [<NPL>]). The following prior Patent document <NUM> (International Publication <CIT>) discloses "an ophthalmic formulation including <NUM>-<NUM>/ml of aflibercept, <NUM>-<NUM>% of an organic cosolvent (e.g., polysorbate, polyethylene glycol, propylene glycol, etc.), <NUM>-<NUM> of an isotonic agent (e.g., NaCl, KCI etc.), <NUM>-<NUM> of sodium phosphate buffer and <NUM>-<NUM>% of stabilizer (e.g., sucrose, sorbitol, glycerol, trehalose, and mannitol, etc.)" and "a lyophilizable formulation including <NUM>-<NUM>/ml of aflibercept, <NUM>-<NUM> of sodium phosphate buffer, <NUM>-<NUM>% of an organic cosolvent, <NUM>-<NUM>% of a stabilizer, and <NUM>-<NUM> of an isotonic agent. " The formulation disclosed in the following prior Patent document <NUM> may be applied to a prefilled syringe suitable for intravitreal administration. <CIT> and <CIT> disclose formulations of VEGF-specific fusion protein antagonists.

For the ophthalmic formulation and lyophilizable formulation disclosed in the following prior Patent document <NUM>, an effect of inhibiting production of impurities and byproducts due to aggregation, fragmentation and isomerization of aflibercept, was reported. However, the formulation in prior Patent document <NUM> was problematic in that the effect of stabilizing aflibercept was markedly reduced under harsh conditions such as high temperature condition of <NUM> or more, or shaking condition.

Therefore, the present inventors have completed the present invention by developing a liquid formulation having enhanced stability under the harsh conditions as well as stably maintaining a fusion protein having an IgG Fc domain such as aflibercept under the storing condition for a long period of time.

The present invention is derived to solve the problems described above, and provides a formulation having stabilized protein in which an Fc domain of a human immunoglobulin G (IgG) and a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor are fused.

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

Still another object of the present invention is to provide a liquid formulation including the composition for stabilizing a protein in which an Fc domain of an IgG and a soluble extracellular domain of a VEGF receptor are fused; and aflibercept.

The present invention provides a liquid formulation, including:.

According to another preferred embodiment of the present invention, the fusion protein aflibercept may be present in an amount of <NUM> to <NUM>/ml.

According to the present invention, the histidine salt is histidine-HCI or histidine-acetate.

According to the present invention, wherein the concentration of the histidine salt is <NUM> to <NUM>.

According to the present invention, the sugar is at least one selected from the group consisting of <NUM>% (w/v) to <NUM>% (w/v) of sucrose trehalose, mannitol, and glucose.

According to still another preferred embodiment of the present invention, the sugar may be <NUM>% (w/v) to <NUM>% (w/v) of sucrose.

According to the present invention, the surfactant is <NUM>% (v/v) to <NUM>% (v/v) of polysorbate <NUM> or polysorbate <NUM>, for example, <NUM>% (v/v) or <NUM>% (v/v) to <NUM>% (v/v).

The claims of present invention regard a liquid formulation including a buffer including a histidine salt and having pH ranging from <NUM> to <NUM>; one or more stabilizers selected from the group consisting of sugars and surfactants; and aflibercept; wherein the histidine salt is <NUM> to <NUM> of histidine-HCI or histidine-acetate, and the sugar is at least one selected from the group consisting of <NUM>% (w/v) to <NUM>% (w/v) of sucrose, trehalose, mannitol and glucose, and the surfactant is <NUM>% (v/v) to <NUM>% (v/v) of polysorbate <NUM> or polysorbate <NUM>, for example, <NUM>% (v/v) or <NUM>% (v/v) to <NUM>% (v/v).

According to a preferred embodiment of the present invention, the liquid formulation may be suitable for intravitreal injection.

In the liquid formulation provided by the present invention, production of impurities and byproducts due to aggregation, fragmentation and isomerization of the fusion protein having an Fc domain of a human immunoglobulin G (IgG) (in particular, the protein in which an Fc domain of a human immunoglobulin G (IgG) and a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor are fused (i.e. aflibercept)) under harsh conditions such as a high temperature condition of <NUM> or more or shaking condition, as well as general storing condition, is significantly reduced, and thus stability for long-term storage may be enhanced.

Also, the present invention improves therapeutic effects on various ophthalmic diseases (e.g., retinal vein occlusion, diabetic macular edema, choroidal neovascularization and wet age-related macular degeneration, etc.) caused by abnormal angiogenesis, while pursuing stabilization of bioactivity through a stable liquid formulation suitable for intravitreal injection of an anti-VEGF-Fc fusion protein including aflibercept.

As described above, the previously provided formulation in which a fusion protein such as aflibercept having an IgG Fc domain is stabilized has a problem in that stability is significantly reduced under the harsh conditions, so that it has been required to develop a formulation capable of maintaining the fusion protein under the harsh conditions in addition to the general storing conditions.

Therefore, the present invention has found a solution for above-described problems by providing a liquid formulation as of claim <NUM> including: aflibercept, which is a protein in which an Fc domain of a human immunoglobulin G (IgG) and a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor are fused; and a buffer including a histidine salt and pH ranging from <NUM> to <NUM>. Since the liquid formulation of the present invention stably maintains the fusion protein under general storing conditions for a long period of time and also significantly reduces production of impurities and byproducts under harsh conditions, it is possible to provide a formulation having significantly improved stability relative to the known fusion protein formulation under the inevitable condition where the optimal storing condition would not be provided.

The present invention provides a liquid formulation as of claim <NUM> including aflibercept, which is a protein in which an Fc domain of a human immunoglobulin G (IgG) and a soluble extracellular domain of a vascular endothelial growth factor (VEGF) are fused; and a buffer including a histidine salt and having a pH ranging from <NUM> to <NUM>.

In the liquid formulation of the present invention, the fusion protein aflibercept is included in an amount of about <NUM>-<NUM>/ml, preferably about <NUM>-<NUM>/ml, and more preferably about <NUM>-<NUM>/ml.

In the liquid formulation according to the present invention, the "buffer" refers to a buffered solution enduring changes in pH due to the action of an acid-base conjugate ingredient of the buffer. In the present invention, a buffer including a histidine salt and having pH ranging from <NUM> to <NUM> was used, wherein the histidine salt is histidine-HCI or histidine-acetate, and the histidine salt concentration may is <NUM> to <NUM>.

As shown in <FIG>, for the formulation using the histidine salt buffer having pH of <NUM> to <NUM>, the effect of inhibiting production of fragments and aggregates was significantly increased more than that of control formulation (<NUM>). In particular, stability of the formulation at pH <NUM> is most excellent.

When pH of the buffer was decreased to less than <NUM>, as shown in <FIG>, stability of the formulation was significantly reduced. For pH of above <NUM>, a problem of increasing impurities caused by disruption or aggregation of a target protein may occur.

In addition, as shown in <FIG>, it has been found that the effect of inhibiting production of impurities and aggregates is most excellent, when the histidine-HCI or histidine-acetate buffer was used among various buffer conditions.

Further, the formulation (<NUM>) of <FIG> was to measure stability of aflibercept in the <NUM> histidine salt buffer having pH <NUM>, and it has been found that aflibercept stability similar to that of control formulation (<NUM>) was maintained under the buffer condition.

The liquid formulation of the present invention as claimed further includes one or more stabilizer selected from the group consisting of sugars and surfactants in addition to the fusion protein, which is an active ingredient of the liquid formulation, and the histidine salt buffer.

In the liquid formulation of the present invention, the sugar is any one selected from the group consisting of trehalose, sucrose, mannitol and glucose. The sugar concentration is <NUM>-<NUM>% (w/v).

As shown in <FIG>, all of formulations (<NUM>) to (<NUM>) which respectively include trehalose, sucrose, mannitol, and glucose in the histidine salt buffer showed enhanced aflibercept stability more than the control formulation (<NUM>). In particular, in the case where the sugar was sucrose, aflibercept stability was most excellent.

Further, as shown in <FIG>, formulation (<NUM>) without sucrose showed similar formulation stability as that of control formulation (<NUM>). As sucrose concentration increased, the effect of inhibiting production of aggregates and impurities increased with respect to control (<NUM>).

In the liquid formulation of the present invention, the surfactant may be used to ophthalmic drug delivery. The surfactant concentration as claimed is <NUM>-<NUM>% (v/v), for example, <NUM>% (v/v) or <NUM>% (v/v) to <NUM>% (v/v).

To evaluate stability of formulations depending whether an additional salt ingredient besides components included in the liquid formulation as claimed is added or not, formulations respectively including various salts and a formulation without salt were prepared. Consequently, as shown in <FIG>, it has been observed that the effect of inhibiting production of impurities and aggregates was most excellent in the formulation without additional salt ingredients.

The liquid formulation according to the present invention maintains stability of the fusion protein under general storing conditions for a long period of time and has an excellent effect of inhibiting production of impurities and byproducts of the fusion protein under harsh conditions, and thus stability of the fusion protein is enhanced more than that of the typical liquid formulation. Herein, the term "harsh conditions" refers to an environment chemically and physically unfavorable to the fusion protein, and leads to unacceptable protein stability. The harsh conditions include, for example, high temperature or shaking condition.

Herein, the term "stable" indicates that a protein essentially maintains physical and/or chemical stability and/or biological activity thereof during storing. Various analytical techniques for measuring protein stability are available in the art. Stability may be measured at selected temperature and selected period.

The protein may be considered to "maintain physical stability thereof" in a formulation, when the protein shows less or no change in aggregation, precipitation, and/or modification during observation of color and/or transparency with naked eyes, UV light-scattering measurement (i.e., notable aggregates are measured), or size exclusion chromatograph (SEC) measurement.

As shown in <FIG> and <FIG>, the various conditions of liquid formulations of the present invention showed significantly inhibited production of impurities and aggregates more than control formulation (<NUM>), thereby being relatively stable for <NUM> to <NUM> days at a temperature ranging from <NUM> to <NUM>.

Various conditions of liquid formulations of the present invention were stored at <NUM> with shaking at <NUM> rpm for <NUM> days, and then each formulation was subjected to SE-HPLC and HI-HPLC. Consequently, irrespective of presence and absence of polysorbate <NUM>, production of aggregates and modifications was inhibited. Likewise, in the SDS-PAGE (non-reducing) (<FIG>) and SE-HPLC results, it has been observed that production of aggregate and protein fragments was inhibited more than that of control (<NUM>) irrespective of presence and absence of polysorbate under the shaking condition at <NUM> for <NUM> days. In particular, the effect of inhibiting production of aggregates and protein fragments was slightly better for the formulation without polysorbate.

The present invention illustrates a method of stabilizing a protein in which an Fc domain of a human immunoglobulin G (IgG) and a soluble extracellular domain of a vascular endothelial growth factor VEGF receptor are fused by using a buffer including a histidine salt and having pH of <NUM> to <NUM>.

Since the description about feature of the buffer and fusion protein is the same as described above, overlapping contents are not described in order to avoid excess complexity of the present specification.

In the method of stabilizing the fusion protein of the present invention, the buffer may further include one or more stabilizers selected from the group consisting of sugars and surfactants.

Since description about additionally added sugar and surfactant are the same as described above, these features are not described.

The present invention provides a composition as claimed for stabilizing aflibercept, which is a fusion protein in which an Fc domain of an IgG and a soluble extracellular domain of a VEGF receptor are fused, the composition including a buffer including a histidine salt and having pH of <NUM> to <NUM>; and one or more stabilizers selected from the group consisting of sugars and surfactants, wherein the histidine salt is <NUM> to <NUM> histidine-HCI, or histidine-acetate; the sugar is any one selected from the group consisting of <NUM>% (w/v) to <NUM>% (w/v) of sucrose, trehalose, mannitol, and glucose; and the surfactant is any one selected from <NUM>% (v/v) to <NUM>% (v/v), of polysorbate <NUM> or polysorbate <NUM>, for example, <NUM>% (v/v) or <NUM>% (v/v) to <NUM>% (v/v).

The description about features of the stabilizing composition is the same as described above, and thus overlapping contents are not described in order to avoid excess complicity of the present specification.

The present invention as claimed, hence, provides a liquid formulation including the composition for stabilizing a protein in which an Fc domain of an IgG and a soluble extracellular domain of a VEGF receptor are fused; and aflibercept.

The liquid formulation may be used for medicinal use for preventing or treating various diseases without limitation as long as the diseases can be treated with aflibercept treatment. Preferably, the formulation may be used to treat or prevent various ophthalmic diseases such as retinal vein occlusion, diabetic macular edema, choroidal neovascularization and wet age-related macular degeneration, etc..

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

The pharmaceutically acceptable carrier is a typically used in a formulation, and may include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but not limited thereto.

In addition to above ingredients, the liquid formulation of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspensions, preservants, etc. The suitable pharmaceutically acceptable carrier and formulation are described in detail in <NPL>).

The liquid formulation of the present invention may be administered in various routes depending on purposes. The formulation is preferably administered through intravitreal injection for the treatment of ophthalmic diseases. Also, the formulation may be applied to a pre-filled syringe suitable for intravitreal injection.

The suitable amount of administration of the liquid formulation of the present invention may vary depending on factors such as formulation methods, administration modes, age, weight, sex, and disease state of patients, foods, administration time, administration routes, excretion rates, and reaction sensitivity. Doctors with ordinary skills may easily determine and prescribe the amount of administration effective for desired therapy or prevention.

It should be understood that the scope of the present invention is not limited to the following examples, and all technical and scientific terms used in the specification have the same meaning as those typically appreciated by a person with ordinary skill in the technical field to which the present invention belongs ("a person skilled in the art").

In Example <NUM> and following Examples <NUM> to <NUM>, the liquid formulation disclosed in prior art document International Publication <CIT> was stored at <NUM>±<NUM>, and used as control (<NUM>). Also, control (<NUM>) was subjected to the harsh condition (a temperature of <NUM> or more) and used as control (<NUM>). Then, stability of the formulation was compared to stability of various forms of liquid formulations of the present invention.

In the present example, to identify the optimal pH of the liquid formulation containing, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein, samples were prepared for each pH based on the histidine salt buffer as follows.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>±<NUM>); (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>) and formulations having <NUM>% trehalose, <NUM>% (w/v) polysorbate <NUM> and <NUM> histidine-HCI buffer respectively having pH of (<NUM>) <NUM>; (<NUM>) <NUM>; (<NUM>) <NUM>; (<NUM>) <NUM>; and (<NUM>) <NUM> were stored at <NUM> for <NUM> days. Then, impurities produced with lapse of time were analyzed through size exclusion high-performance liquid chromatography (SE-HPLC) and HI-HPLC.

In SE-HPLC analysis, TSK-gel G3000SWXL (<NUM> × <NUM>) column (TOSOH Co. , Japan) was used, and <NUM> potassium phosphate (pH <NUM>), <NUM> KCI and <NUM>% (w/v) NaN<NUM> buffer were used at a flow rate of <NUM>/min. The peak of the anti-VEGF-Fc fusion protein was analyzed at an absorbance of <NUM>.

In SE-HPLC analysis for the sample stored at <NUM> for <NUM> days, the monomer content was calculated, and aggregates, which are high molecular weight impurities, and fragments, which are low molecular weight impurities, were combined and calculated. <FIG> showed SE-HPLC result on day <NUM>. It is indicated that, when comparing with control (<NUM>), the formulation of the present invention shows significantly reduced production of aggregates and fragments at pH <NUM>, <NUM>, <NUM> and <NUM>, and thus the formulation is stable. Also, among various pH conditions, it has been identified that stability of the formulation was highest at pH <NUM>.

In HI-HPLC analysis, TSK Phenyl-5PW (<NUM> × <NUM>) column was used. As a mobile phase, <NUM> sodium phosphate (pH <NUM>) was used. <NUM> NaCl, <NUM> sodium phosphate (pH <NUM>) and <NUM>% (v/v) acetonitrile buffer was used and allowed to flow at a concentration gradient of <NUM>%-<NUM>%. Then, the peak of the anti-VEGF-Fc fusion protein was analyzed at an absorbance of <NUM>. During the HI-HPLC analysis, three main peaks, i.e. peak <NUM>, peak <NUM> and peak <NUM> appeared. When changes in the peak were less, it was considered as stable.

<FIG> shows a result of HI-HPLC analysis of the sample stored at <NUM> with lapse of time. <FIG> showed changes in peak <NUM>, peak <NUM>, and peak <NUM> under each condition, and it has been found that, when comparing to control (<NUM>), the formulation of the present invention under the pH <NUM>, <NUM>, <NUM> and <NUM> conditions showed significantly less changes in peaks. Also, among various other pH conditions, the formulation was most stable at pH <NUM> condition, relatively.

In the present example, to identify the optimal buffer condition for the liquid formulation containing, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein, samples were prepared as follows.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>±<NUM>); (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>), and various formulation respectively including <NUM>% polysorbate <NUM> and <NUM>% sucrose in a buffer of (<NUM>) <NUM> sodium phosphate, pH <NUM>; (<NUM>) <NUM> histidine-HCI, pH <NUM>; (<NUM>) <NUM> histidine-acetate, pH <NUM>; (<NUM>) <NUM> succinate, pH <NUM>; and (<NUM>) <NUM> sodium acetate, pH <NUM> were stored at <NUM> for <NUM> days. Then, same as example <NUM>, a monomer, aggregates, and variants were analyzed with lapse of time through SE-HPLC and HI-HPLC.

Thereafter, SDS-PAGE analysis was performed. To <NUM> ul of analytic sample diluted to <NUM>/ml, <NUM> ul of 5X sample buffer (reducing or non-reducing) was added, and the resultant was reacted at <NUM> for <NUM> minutes and used for analysis. <NUM> ul of the final analytic sample was loaded per well. As a marker (M), DokDo-Mark™ protein marker was used, and loaded at <NUM> ul per well. <NUM>% SDS-PAGE (reducing), and <NUM>% SDS-PAGE (non-reducing) gel electrophoresis were performed. Then, staining was performed with coomassie blue. Until clear bands appear, destaining was performed.

As a result of SDS-PAGE (non-reducing) (<FIG>), and SE-HPLC and HI-HPLC analysis, it has been found that, when comparing to control (<NUM>), the formulations of <NUM> histidine-HCI and <NUM> histidine-acetate of the present invention significantly inhibited production of aggregates and impurities.

In the present example, to identify the optimal sugar for the liquid formulation containing, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein, samples were prepared as follows.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>±<NUM>); (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>), and formulations respectively including, in <NUM> histidine-acetate buffer (pH <NUM>) with <NUM>% polysorbate <NUM>, (<NUM>) <NUM>% trehalose; (<NUM>) <NUM>% sucrose; (<NUM>) <NUM>% mannitol; and (<NUM>) <NUM>% glucose were stored at <NUM> for <NUM> days. Then, same as Examples <NUM> and <NUM>, monomers, aggregates, and variants produced with lapse of time were analyzed through SDS-PAGE (reducing, non-reducing), SE-HPLC, and HI-HPLC.

As a result of SDS-PAGE (reducing, non-reducing) analysis (<FIG>) and SE-HPLC and HI-HPLC analysis, when comparing to control (<NUM>), all formulations respectively including trehalose, sucrose, mannitol, and glucose of the present invention significantly inhibited production of aggregates and impurities. In particular, the effect of inhibiting production of impurities and aggregates was most excellent in the formulation including sucrose.

In the present example, to identify the optimal sugar concentration for the liquid formulation containing, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein, samples were prepared as follows.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>±<NUM>); (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>), and formulations respectively having, in <NUM> histidine-acetate buffer (pH <NUM>) with <NUM>% polysorbate <NUM>, sucrose of varying concentrations of (<NUM>) <NUM>%;(<NUM>) <NUM>%; (<NUM>) <NUM>%; and (<NUM>) <NUM>% were stored at <NUM> for <NUM> days. Then, same as Examples <NUM> and <NUM>, monomers, aggregates, and variants produced with lapse of time were analyzed through SDS-PAGE (reducing, non-reducing), SE-HPLC, and HI-HPLC.

In the SE-HPLC analysis results, it has been found that the formulation without sucrose of the present invention (<NUM>) showed formulation stability similar to that of control (<NUM>), and as the sucrose concentration in each formulation of the present invention increased, the effect of inhibiting production of aggregates and impurities was increased with respect to control (<NUM>) (<FIG>).

In the present example, to identify an additional salt ingredient optimal for the liquid formulation containing, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein, samples were prepared as follows.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>±<NUM>); (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>), and formulations respectively having, in <NUM> histidine-acetate buffer (pH <NUM>) with <NUM>% polysorbate <NUM>, (<NUM>) without a salt, (<NUM>) <NUM> sodium chloride; (<NUM>) <NUM> ammonium chloride; (<NUM>) <NUM> ammonium sulfate; and (<NUM>) <NUM> potassium chloride were stored at <NUM> for <NUM> days. Then, same as Examples <NUM> and <NUM>, monomers, aggregates, and variants produced with lapse of time were analyzed through SDS-PAGE (reducing, non-reducing), SE-HPLC, and HI-HPLC analysis.

As a result of SDS-PAGE and SE-HPLC (<FIG>) analysis, it has been observed that, when comparing to control (<NUM>), the formulation without an additional salt ingredient of the present invention (<NUM>) showed the most excellent effect of inhibiting production of impurities and aggregates.

In the present example, samples were prepared as follows in order to evaluate stability for the formulation depending on the presence and absence of polysorbate <NUM> and sucrose concentration during shaking, wherein the liquid formulation is based on the histidine-HCI buffer and contains, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and formulations having (<NUM>) <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM>; (<NUM>) <NUM> histidine-acetate, pH <NUM>, <NUM>% sucrose; (<NUM>) <NUM> histidine-HCI, pH <NUM>, <NUM>% sucrose; (<NUM>) <NUM> histidine-HCI, pH <NUM>, <NUM>% sucrose, <NUM>% polysorbate <NUM>; (<NUM>) <NUM> histidine-HCI, pH <NUM>, <NUM>% sucrose, <NUM>% polysorbate <NUM>; and (<NUM>) <NUM> histidine-HCI, pH <NUM>, <NUM>% sucrose, <NUM>% polysorbate <NUM> were respectively stored at <NUM>±<NUM> or25°C for <NUM> days with shaking at <NUM> rpm through an orbital shaker.

The formulation described in (<NUM>) corresponded to the formulation disclosed in the prior patent document <NUM>, i.e. International Publication <CIT>. The condition described in (<NUM>) was used as a control for changes in the histidine salt buffer.

As shown in SE-HPLC and HI-HPLC analysis results, it has been found that, irrespective of the presence and absence of polysorbate, the formulation stored for <NUM> days at <NUM> with shaking showed inhibited production of aggregates and variants compared to control (<NUM>). In SDS-PAGE (non-reducing) (<FIG>) and SE-HPLC results, it has been observed that, irrespective of the presence and absence of polysorbate, the formulation stored for <NUM> days at <NUM> with shaking showed inhibited production of aggregates and variants compared to control (<NUM>) as well. In particular, the effect of inhibiting production of protein fragments and aggregates of the formulation without polysorbate was slightly better.

In the present example, to identify the optimal polysorbate <NUM> concentration for the liquid formulation containing, as an active ingredient, aflibercept which is an anti-VEGF-Fc fusion protein, samples were prepared as follows.

Specifically, the final aflibercept concentration was adjusted to <NUM>/ml, and (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>±<NUM>); (<NUM>) a formulation having <NUM> sodium phosphate, pH <NUM>, <NUM>% sucrose, <NUM> NaCl, <NUM>% polysorbate <NUM> (formulation stored at <NUM>), and formulations having, in <NUM> histidine-acetate buffer (pH <NUM>), <NUM>% sucrose, and polysorbate <NUM> of varying concentrations of (<NUM>) <NUM>%; (<NUM>) <NUM>%; (<NUM>) <NUM>%; (<NUM>) <NUM>%; and (<NUM>) <NUM>% were stored at <NUM> for <NUM> days. Then, same as Examples <NUM> and <NUM>, monomers, aggregates, and variants produced with lapse of time were analyzed through SDS-PAGE (reducing, non-reducing), SE-HPLC, and HI-HPLC.

In the SE-HPLC analysis results, it has been found that the formulation (<NUM>) without polysorbate <NUM> of the present invention and formulations (<NUM>), (<NUM>), (<NUM>) and (<NUM>) with polysorbate <NUM> had the superior effect of inhibiting production of aggregates and impurities than the effect of control formulation (<NUM>) (<FIG>).

Consequently, through Examples <NUM> to <NUM>, it has been found that, various conditions of liquid formulations of the present invention have greatly inhibited fragmentation and aggregation more than the formulation in prior patent.

Through the formulation stability test, it has been found that the liquid formulation of the present invention has the excellent effect of inhibiting production of impurities and byproducts caused by aggregation, fragmentation and isomerization of the fusion protein having an IgG Fc domain, and thus the fusion protein can be stably stored for a long period of time and also enhanced stability can be provided under harsh conditions.

Claim 1:
A liquid formulation, comprising:
<NUM> to <NUM>/ml of aflibercept;
a buffer including a histidine salt and having pH ranging from <NUM> to <NUM>;
a sugar selected from the group consisting of <NUM>% (w/v) to <NUM>% (w/v) of sucrose, trehalose, mannitol, and glucose; and
<NUM>%(v/v) to <NUM>%(v/v) of surfactant selected from the group consisting of polysorbate <NUM> or polysorbate <NUM>,
wherein the soluble extracellular domain of the VEGF receptor comprises immunoglobulin-like domain <NUM> of a first VEGF receptor and immunoglobulin-like domain <NUM> of a second VEGF receptor, and the histidine salt is <NUM> to <NUM> of histidine-HCI or histidine-acetate,
wherein the formulation does not contain additional salt ingredients.