DESC:
FIELD OF THE INVENTION
The present invention relates to stable ophthalmic pharmaceutical formulations of Aflibercept and a method of making and using such formulations for the treatment of angiogenic eye disorders.

The present invention relates to stable ophthalmic pharmaceutical formulations of Aflibercept in aqueous form as well as formulations that can be lyophilized and reconstituted further for intra-vitreal administration.

More specifically, the present invention relates to stable ophthalmic pharmaceutical formulations comprising Aflibercept in a concentration ranging from 10mg/ml to 400mg/ml and one or more pharmaceutically acceptable excipients selected from buffer, stabilizing agents, tonicity modifying agent, surfactant and the like.

Stable ophthalmic pharmaceutical formulations of the present invention can be filled in suitable pharmaceutical packages such as vial and pre-filled syringe.

Stable ophthalmic pharmaceutical formulations of the present inventions are suitable for the treatment of neovascular disease conditions associated with the eye.

BACKGROUND OF THE INVENTION
Proteins are complex molecules and possess a delicate tertiary structure. Therefore, they are inherently unstable and vulnerable to a variety of degradation. Their stability can be described and measured as conformational and colloidal stabilities, which can easily be influenced by a variety of factors, such as temperature, light, matrix conditions, manufacturing processes, shipping, storage, and administration processes. Therefore, a protein drug candidate needs to be stabilized appropriately.

Aflibercept is a vascular endothelial growth factor (VEGF) Inhibitor. It is a recombinant homodimeric glycoprotein that includes two main components: the vascular endothelial growth factor (VEGF) binding portion from the extracellular domains of human VEGF receptor 1 and 2, fused to the Fc portion of human IgGl. It is a recombinant fusion protein capable of inhibiting type A VEGF, type B VEGF, and placental growth factor (PIGF), developed by Regeneron as intravitreal injection and marketed as Eylea® 2mg (0.05 mL of 40 mg/mL) indicated for the treatment of neovascular (wet) age-related macular degeneration (AMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), retinopathy of prematurity (ROP) and EyleaHD® 8mg (0.07 mL of 114.3 mg/mL solution) for the treatment of patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy (DR).

U.S. Patent No. 8,092,803 and U.S. Patent No. 10,464,992 disclose a stable liquid and lyophilized composition of Aflibercept in a concentration from 1-100 mg/ml, about 0.01-5% of one or more organic co-solvent, about 1-7.5% of stabilizing agent, one or more tonicity modifying agent and about 5-40 mM of buffer having a pH of about 5.8 to about 7.0.

U.S. Patent No. 10,576,128 discloses a liquid pharmaceutical composition consisting of Aflibercept in a concentration range 6 mg/ml to 45 mg/ml, 1 mM to 40 mM histidine hydrochloride/L-histidine, 0.01 to 0.08% (w/v) polysorbate 20, 20 mM to 100 mM NaCl, 3-20% (w/v) sucrose and water and having a pH of 6.2 - 6.5.

U.S. Patent No. 11,103,552 discloses Aflibercept composition having Aflibercept concentration at least about 100 mg/ml; about 5% sucrose; L-arginine; a histidine buffer; and about 0.03% surfactant; wherein the formulation has a pH of about 5.0 to about 6.8. The invention of 11,103,552 discloses high dose Aflibercept formulation specifically comprising histidine and arginine; that is more superior to high dose formulation comprising phosphate buffer in terms of stability.

There are several challenges to develop a stable ophthalmic pharmaceutical formulation of protein drugs such as VEGF inhibitor particularly Aflibercept. The major challenge to develop an ophthalmic formulation with high concentration biological drugs like Aflibercept is to control the formation of insoluble particles and to control aggregate formation or flocculation in the formulation throughout the shelf life of the formulation. Various pharmaceutical formulations for ocular administration, specifically intra-vitreal, are disclosed in prior art; however, there is still a need to prepare a stable ophthalmic pharmaceutical formulation of Aflibercept. The inventors have developed a stable ophthalmic pharmaceutical formulation comprising Aflibercept, buffer, stabilizers, surfactant, and/or tonicity-modifying agents. The invention discloses one or more stable ophthalmic pharmaceutical formulations which are suitable for intra-vitreal administration for the treatment of angiogenic eye disorders.

SUMMARY OF THE INVENTION
The present invention is directed to a stable ophthalmic pharmaceutical formulation of Aflibercept and its manufacturing process. The objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising Aflibercept and one or more pharmaceutically acceptable excipients. The objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising Aflibercept and one or more pharmaceutically acceptable excipients such as buffer, stabilizing agent, surfactant, and tonicity modifying agent.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 1-10% stabilizer, 0.01-0.03% surfactant and optionally, a suitable amount of tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5-10% stabilizer, 0.01-0.03% non-ionic surfactant and optionally, 1mM to 50mM tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% stabilizer, 0.01-0.03% non-ionic surfactant and optionally, 1mM to 50mM tonicity modifying agent, wherein formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose, 0.01-0.03% polysorbate and 1mM to 50mM sodium chloride, wherein formulation has a pH of about 5.5 to 6.5.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose, 0.01-0.03% polysorbate and 1mM to 50mM sodium chloride, wherein formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1mM to 10mM sodium chloride, wherein formulation has a pH of about 5.5 to 6.5.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate, and 1mM to 10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate, and 1mM to 10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% stabilizer, 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% stabilizer, 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer, 0.01-0.03% non-ionic surfactant and 1-50mM tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer, 0.01-0.03% non-ionic surfactant and 1-10mM tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer and 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and is free of tonicity modifying agent.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer and 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and is free of tonicity modifying agent and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 40mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer, 0.01-0.03% non-ionic surfactant and 1-50mM tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer, 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer, 0.01-0.03% non-ionic surfactant and 1-50mM tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 160mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM Sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% stabilizer, 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

Another objective of the present invention is to provide a stable ophthalmic pharmaceutical formulation comprising 160mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

The present invention provides formulations with the pH and viscosity that is suitable for ophthalmic administration. Accordingly, the present disclosure provides a stable ophthalmic formulation of Aflibercept, with low aggregation propensity over longer period.

The invention further provides ophthalmic formulations in a pre-filled syringe or vial, particularly suitable for intravitreal administration.

Another objective of the present invention is to provide a prefilled syringe containing a stable ophthalmic pharmaceutical formulation of Aflibercept as disclosed herein. The present invention provides a prefilled syringe having non-retractable plunger stopper arrangement and tamper evident closure system, filled with stable ophthalmic pharmaceutical formulation of Aflibercept with dose marking for delivery of 20 µl or 30µl or 50µl or 60 µl or 70µl dose of Aflibercept.

Another objective of the present invention is to provide a vial containing a stable ophthalmic pharmaceutical formulation of Aflibercept as disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail by way of reference using the following definitions and examples. The present invention is not limited to particular methods, and experimental conditions described herein, as such methods and conditions may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting unless indicated. All technical and scientific terms and phrases used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

As used herein, the term “about” means an acceptable change for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” means within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

The present invention provides a stable ophthalmic pharmaceutical formulation comprising Aflibercept and one or more pharmaceutically acceptable excipients. The present invention provides a stable ophthalmic pharmaceutical formulation comprising Aflibercept and one or more pharmaceutically acceptable excipients such as buffer, stabilizing agent, surfactant, tonicity modifying agent, viscosity modifying agent and the like.

In one of the embodiments, the invention provides a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM buffer, 1- 10% a stabilizing agent,0.01-0.03% non-ionic surfactant, and optionally, a suitable amount of tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5.

In one of the embodiments, the invention provides a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose as a stabilizing agent, 0.01-0.03% polysorbate as a non-ionic surfactant and suitable amount sodium chloride as tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5 and the osmolality in a range 250-350mOsmol.

In one of the embodiments, a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose, 0.01-0.03% polysorbate and 1mM to 50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In one of the embodiments, a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose, 0.01-0.03% polysorbate and 1mM to 50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In one of the embodiments, the invention provides a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1mM to 10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In one of the embodiments, a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% stabilizer and 0.01-0.03% non-ionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent and amino acid.

In one of the embodiments, a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In one of the embodiments, the invention provides a stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5- 10% sucrose and 0.01-0.03% polysorbate, wherein formulation is free of sodium chloride, histidine and arginine.

In one of the embodiments, the invention provides a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

In one of the embodiments, the invention provides a stable ophthalmic pharmaceutical formulation comprising 10- 400mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

The term "pharmaceutical formulation" or "composition" as used herein refers to a mixture of a protein, such as a fusion protein, e.g., Aflibercept, together with one or more additional components. In some embodiments, the additional components may include, e.g., one or more excipients, such as a buffer, stabilizer, surfactant, tonicity modifying agent, water and the like, e.g., a pharmaceutically acceptable carrier or excipient that is conventional in the art. In one of the embodiments, the additional component is suitable for ophthalmic administration. In one embodiment, pharmaceutical formulations may be aqueous formulation for intravitreal administration. In another embodiment pharmaceutical formulations may be lyophilized powder for reconstitution suitable for intravitreal administration.

The term "intravitreal injection" refers to the administration of a pharmaceutical formulation in which the substance is injected directly into the eye. More specifically, the substance is injected into the vitreous humor (also called vitreous body or simply vitreous) which is the clear gel that fills the space between the lens and the retina of the eyeball of humans and other vertebrates.

The term "Aflibercept" is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF) -binding portions from the extracellular domains of human VEGF Receptors 1 and 2 fused to the Fc portion of the human IgGl. Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilo Daltons (kDa) and contains glycosylation, constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa. Amino acid and nucleic acid sequences of Aflibercept are known in art.

In all embodiments of the invention, Aflibercept can be present at a concentration to produce therapeutic effect. It can be present at concentration of about 1mg/ml to 400mg/ml, about 20mg/ml to 300 mg/ml, about 20mg/ml to 200mg/ml, about 20 mg/ml to 150 mg/ml, about 40 mg/ml to 400 mg/ml, about 40 mg/ml to 300 mg/ml, about 40 mg/ml to 200 mg/ml, about 40 mg/ml to 150 mg/ml, about 40 mg/ml to 100mg/ml, about 50mg/ml to 400mg/ml, about 50mg/ml to 300mg/ml, about 50mg/ml to 200mg/ml, about 50mg/ml to 150mg/ml, 100mg/ml to 200 mg/ml, about 100mg/ml to150 mg/ml, about 100mg/ml to 200 mg/ml, about 110mg/ml to160 mg/ml, about 110mg/ml to170 mg/ml, about 110mg/ml to150 mg/ml, about 110mg/ml to180 mg/ml, about 125 mg/ml to 175 mg/ml, about 125 mg/ml to 225 mg/ml, more specifically about 20mg/ml to 200mg/ml.

In an embodiment of the invention, the Aflibercept concentration is about 10 mg/ml, about 30 mg/ml, about 40 mg/ml, about 60 mg/ml, about 114 mg/ml, about 120 mg/ml, about 140 mg/ml, about 160mg/ml, about 170mg/ml, about 180mg/ml, about 190mg/ml about 200 mg/ml, about 300 mg/ml. Preferable concentrations of Aflibercept is about 40 mg/ml or about 114 mg/ml or about 160 mg/ml or about 200mg/ml.

The term “excipient” used here in refers to the additional component that may include, one or more excipients, such as water, buffer, surfactant, stabilizing agent, anti-aggregating agent, tonicity modifier and the like; a pharmaceutically acceptable carrier or excipient that is conventional in the art.

The term “buffer” refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base of weak acid or a weak base and its conjugate acid of a weak base that resists changes in its pH by the action of its acid-base conjugate components and therefore keeps the pH at a nearly constant value. The buffer is phosphate buffer; an acetate buffer, such as sodium acetate and acetic acid, a citrate buffer, such as sodium citrate or citric acid, succinic acid buffer, tris-aminomethane (Tris) buffer.

The concentration of buffer is about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 12 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 12 mM, about 8 mM to about 50 mM, about 8 mM to about 40 mM, about 8 mM to about 30 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 12 mM, or 10 mM, based on the total liquid formulation. In preferred embodiment, concentration of the buffer is about10mM, about 20mM, about 30mM, about 40mM, about 50mM, more preferably 10mM.

The buffer used in the formulation of the present invention is a phosphate-containing buffer. The phosphate buffer comprises of disodium hydrogen phosphate, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate or a combination thereof. The buffer used in the formulation of the present invention has a pH in the range from about 5.0 to about 7.0, more preferably from about 5.5 to about 6.5. The pH of formulation is about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 7.0. More preferably the pH of formulation is at about 5.8, or about 6.2. The phosphate buffer is prepared in concentration range between 5 mM to 50mM or 10 mM to 40mM. In preferred embodiment, concentration of phosphate buffer is about10mM, about 20mM, about 30mM, about 40mM, about 50mM, more preferably 10mM.

The term “stabilizer” refers to an excipient or mixture of excipients that has been used to optimize the stability of protein present in pharmaceutical formulation. Stabilizers act by minimizing the extent of aggregation, by optimizing the viscosity of formulation during storage and administration. They can also be referred as anti-aggregating agents, viscosity modifiers or osmotic agent. The stabilizer is selected from the group consisting of polyols, polymers, and combinations thereof. In one embodiment, the stabilizer is the sugar. In another embodiment, the stabilizer is sucrose, sorbitol, trehalose, glycerol, or mannitol. The sugar may be present in a concentration ranging from 5 to 12 w/v %, preferably from 6 to 10 w/v %.

In one of the embodiments, stabilizer used in the formulation of present invention is sucrose. It is also used as a nonionic osmotic agent. The concentration of sucrose used in formulation is in the range of 1 to 20% (w/v), 1 to 15% (w/v), 1 to 10% (w/v), 5 to 20% (w/v), 5 to 15% (w/v), 5 to 10% (w/v), 7.8 to 20% (w/v), 7.8 to 15% (w/v), 7.8 to 10% (w/v), 7.8 to 8.2% (w/v). Preferably, concentration of sucrose in formulation is at about 7% (w/v) or about 6% (w/v), about 8% (w/v) or about 9% (w/v). The concentration of sucrose used in formulation is preferably about 9% (w/v).

In one of the embodiments, stable ophthalmic pharmaceutical formulation of the present invention is free of amino acid. The amino acid is any pharmaceutically acceptable amino acid or its salt such as lysine, histidine, arginine, proline, glycine and combination thereof. In specific embodiment, a stable ophthalmic pharmaceutical formulation of Aflibercept is free of histidine and arginine.

A “tonicity modifier” is an excipient that contributes to the osmolality and tonicity of the solution. In one of the embodiments, tonicity modifier is selected from the group consisting of inorganic salt which has osmo-regulatory properties. Preferred inorganic salts for use in the pharmaceutical formulation of the present invention are potassium chloride, calcium chloride, sodium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate. Preferably the inorganic salt is sodium salt, even more preferably it is sodium chloride (NaCl).

The concentration of the inorganic salt used in the formulation of present invention is in suitable concentration to maintain iso-tonicity and osmolality. The concentration of the inorganic salt is present in the range of 1 to 100 mM. In preferred embodiment, the concentration of sodium chloride is in the range of 1 to 80 mM, preferably in the range of 1 to 50 mM, even more preferably 1mM to 40 mM. In a most preferred embodiment, the inorganic salt is sodium chloride with a concentration of about 1mM, about 2mM, about 4mM, about 5mM, about 6mM, about 8mM, about 10mM, about 12mM, about 15mM, about 16mM, about 18mM, about 20mM, about 22mM, about 25mM, about 30mM about 35mM and about 40 mM, more preferably about 5mM or about 10mM.

The osmolality of a pharmaceutical formulation is adjusted to minimize discomfort to the patient upon administration. It is generally preferred that a pharmaceutical formulation for intravitreal administration to a patient should be isotonic to vitreous fluid and have the same or similar osmolality, which is achieved by addition of a tonicity modifier or stabilizer. However, hypertonic formulations which would then be diluted in an isotonic vehicle are also within the scope of this invention.

In one of the embodiment, the stable ophthalmic formulation provided in the present specification is isotonic with a living tissue. The osmolality of the present formulations is from about 180 to about 500 mOsM. In another embodiment, the osmolality of the present formulations is from about 300 mOsM.

In one of the embodiments, stable ophthalmic pharmaceutical formulation may further comprise a surfactant. The surfactant may be selected from any pharmaceutically acceptable surfactants which can disperse the protein evenly in the liquid composition medium. The surfactant may be a non-ionic surfactant; selected from the group consisting of polysorbate for example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), wherein the numerical value denominates the total number of oxyethylene groups (—(CH2CH2O)—); poloxamer (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly(propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compounds (for example, polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C1-C30), polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1-C30), etc.), sodium dodecyl sulphate (SDS), and the like. Preferably the non-ionic surfactant is the polysorbate class of surfactant comprising of polysorbate 20, polysorbate 60, polysorbate 40, and polysorbate 80. The preferred non-ionic surfactant is polysorbate 20.

In one of embodiment, non-ionic surfactant present in concentration range 0.001 to 3% (w/v), 0.001 to 2% (w/v), 0.001 to 1% (w/v), 0.001 to 0.5% (w/v), 0.001 to 0.1% (w/v), 0.001 to 0.05% (w/v), 0.01 to 3% (w/v), 0.01 to 2% (w/v), 0.01 to 1% (w/v), 0.01 to 0.5% (w/v), 0.01 to 0.1% (w/v), 0.01 to 0.05% (w/v), 0.01 to 0.03% (w/v), based on the total composition. The concentration of the polysorbate 20 used in the formulation of the present invention is in the range of 0.001 to 1% (w/v) or 0.001 to 0.5 % (w/v) or 0.001 to 0.05 % (w/v) or 0.001 to 0.005 % (w/v) or 0.001 to 0.03 % (w/v). Preferably in the range 0.001 to 0.03 % (w/v). The concentration of polysorbate 20 is about 0.03 % (w/v).

The stable ophthalmic pharmaceutical formulation may comprise the aforementioned contents of Aflibercept, a buffer, a stabilizer, tonicity modifying agent and a residual aqueous medium or vehicle for example, water (purified water), saline solution, water for injection.

In an embodiment, the invention provides, a stable ophthalmic pharmaceutical formulation having at least 10 mg/ml or a concentration that contains a single dose of Aflibercept (which is discussed herein), a phosphate buffer, a stabilizer, a surfactant and tonicity modifying agent.

In an embodiment, the invention provides, a stable ophthalmic pharmaceutical formulation having at least 10 mg/ml or a concentration that contains a single dose of Aflibercept (which is discussed herein), a phosphate buffer, a stabilizer, a surfactant and tonicity modifying agent, wherein the formulation is free of amino acid.

In an embodiment the invention provides, a stable ophthalmic pharmaceutical formulation of Aflibercept, having at least 10 mg/ml or a concentration that contains a single dose of Aflibercept (which is discussed herein), a phosphate buffer, a stabilizer and a surfactant, wherein the formulation is free of tonicity modifying agent.

In an embodiment the invention provides, a stable ophthalmic pharmaceutical formulation of Aflibercept, having at least 10 mg/ml or a concentration that contains a single dose of Aflibercept (which is discussed herein), a phosphate buffer, a stabilizer and a surfactant, wherein the formulation is free of tonicity modifying agent and amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10-40mM phosphate-buffer, 7%-9% sucrose as a stabilizer, 0.03% polysorbate as a nonionic surfactant and 1-50mM sodium chloride as tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10-40mM phosphate-buffer, 7%-9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 10mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 10mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 10mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 5mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride, wherein the formulation has a pH of about 6.2 and it is free of histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose as a stabilizer and 0.03% polysorbate as a nonionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent and amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose, and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10-40mM phosphate-based buffer system, 7%-9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate-buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 6.2 and it is free of sodium chloride and amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate-buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 6.2 and it is free of sodium chloride, histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate-buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 6.2 and it is free of sodium chloride and amino acid.

In an embodiment, a stable liquid formulation comprises about 40 mg/ml Aflibercept, 10mM phosphate-buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 6.2 and it is free of sodium chloride, histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 114 mg/ml Aflibercept, 10-40mM phosphate-buffer, 7%-9% sucrose as a stabilizer, 0.03% polysorbate as a nonionic surfactant and 1-50mM sodium chloride as tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose, 0.03% polysorbate and 1-50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 10mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 10mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of histidine and arginine.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of histidine and arginine.

In an embodiment, a stable liquid formulation comprises about 114 mg/ml Aflibercept, 10-40mM phosphate-buffer, 7%-9% sucrose as a stabilizer and 0.03% polysorbate as a nonionic surfactant, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of tonicity modifying agent and amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 5.8 and it is free of sodium chloride and amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 114 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 5.8 and it is free of sodium chloride, histidine and arginine.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 160 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 160 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 180 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 180 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 200 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a stable ophthalmic pharmaceutical formulation comprises about 200 mg/ml Aflibercept, 10-40mM phosphate buffer, 7%-9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, the invention provides a pharmaceutically acceptable packaging comprising a stable ophthalmic pharmaceutical formulation of Aflibercept. In one of the embodiments, a pharmaceutically acceptable packaging includes vial or prefilled syringe. In one of the embodiments, a pharmaceutically acceptable packaging includes prefilled syringe in combination with pediatric dosing device to avoid administration of dose higher than recommended volume.

In one embodiment, the stable ophthalmic pharmaceutical formulation of Aflibercept is filled in vial. Vial can be made up of glass or polymer such as Cyclic Olefin Polymer (COP) with a screw-on cap with a tamper-evident seal. The vial’s capacity depends on the intended use (e.g., single dose or multi-dose).

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% non-ionic surfactant and optionally suitable amount of tonicity modifying agent, wherein the formulation has a pH of about 5.5 to 6.5.

In an embodiment, the present invention provides a multi-dose vial containing stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and optionally suitable amount of sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and optionally suitable amount of sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the present invention provides a multi-dose vial containing stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and optionally suitable amount of sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 10mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 10mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of histidine and arginine.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 5mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 5mM sodium chloride, wherein the formulation has a pH of about 5.8 and it is free of histidine and arginine.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.8 and it is free of sodium chloride and amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 5.8 and it is free of sodium chloride and amino acid.

In an embodiment, the present invention provides a vial containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 5.8 and it is free of sodium chloride, histidine and arginine.

In one of the embodiments, the stable ophthalmic pharmaceutical formulation of Aflibercept as disclosed herein is provided in pre-filled pharmaceutical packages. A prefilled syringe or pre-filled pharmaceutical package consists of a plunger, stopper, barrel, and a needle. The barrel is the part of the syringe that holds the medication and is made of either glass or plastic. The plunger and stopper arrangement fit tightly inside the barrel such that it glides unidirectionally through it when pressure is applied. The needle shield or tip cap is used to protect the needle from damage and contamination before use.

In one of the embodiments, a prefilled syringe comprising stable ophthalmic pharmaceutical formulation of Aflibercept. The prefilled syringe may contain multi-compartment barrel with, one of compartment may contain formulation either in lyophilized form or contains powder for reconstitution and another compartment contains the vehicle for reconstitution which may be aqueous or non-aqueous.

In an embodiment, the pre-filled pharmaceutical package provide formulation of a 1-400mg/ml Aflibercept, wherein, the prefilled syringe has tamper evident closure system and the stopper with plunger arrangement such as it glides along the wall of barrel unidirectionally to deliver the formulation at target site.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 10-400mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-50mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 5.5 to 6.5 is free of sodium chloride and amino acid.

In an embodiment, a prefilled syringe containing stable ophthalmic pharmaceutical formulation comprising 114mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose, 0.01-0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of histidine and arginine.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 5% - 10% sucrose and 0.01-0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride, histidine and arginine.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of 160mg/ml Aflibercept, 10-40mM phosphate buffer, 9% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride histidine and arginine.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of about 180 mg/ml Aflibercept, 10-40mM phosphate buffer, 5%-10% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of about 180 mg/ml Aflibercept, 10-40mM phosphate buffer, 5%-10% sucrose, 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of about 200 mg/ml Aflibercept, 10-40mM phosphate buffer, 5%-10% sucrose, 0.03% polysorbate and 1-10mM sodium chloride, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of amino acid.

In an embodiment, the prefilled syringe contains a stable ophthalmic pharmaceutical formulation comprising of about 200 mg/ml Aflibercept, 10-40mM phosphate buffer, 5%-10% sucrose and 0.03% polysorbate, wherein the formulation has a pH of about 5.5 to 6.5 and it is free of sodium chloride and amino acid.

In an another embodiment, the present invention provides, prefilled syringe, with dose marking for delivery of 20 µL or 30µL or 40µL or 50µl, 60 µl or 70µl or 80 µl dose of Aflibercept formulation as disclosed herein.

The Aflibercept formulation is prepared by buffer exchange techniques like dialysis or diafiltration, wherein the excipient such as surfactant, tonicity modifier and stabilizer can be added during the buffer exchange process to avoid any aggregation due to shear involved in processing.

The bulk processed Aflibercept material was formulated using the following procedures. The Aflibercept material is buffer exchanged by the dialysis procedure using tangential flow filtration. After dialysis the osmotic pressure and the pH is tested, if required the dialysis is repeated by addition of HCl or NaOH, to attain the required pH. The sample is then diluted with the buffer to attain protein concentration followed by addition of surfactant, tonicity modifier agent and the stabilizer (diluted in water).

The present invention also provides a method for making any formulation set forth herein comprising the step of combining each component of the formulation into a single composition. Such methods may include the step of adding the resulting formulation into a vial or injection device or syringe. Any composition that is the product of such a method also forms part of the present invention.

Pharmaceutical formulations comprising proteins have multiple stability challenges in view of the unique chemical and physical properties of proteins. Physical instability involves aggregates/ impurities/ flocculation. Inventors of the present invention had found that in Aflibercept formulations disclosed herein prevents aggregates/ impurities/ flocculation resulting in stable formulation.

A stable ophthalmic pharmaceutical formulation is one in which the Aflibercept essentially retains its physical and chemical stability as well as its biological activity upon storage. Aflibercept contained in the formulation or pharmaceutical compositions does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the formulation or composition at the beginning of storage.

The term “high molecular weight” (HMW) species, refer to higher order oligomeric species like dimers, trimers, tetramers, etc., formed by addition of monomers that can either be covalently or non-covalently bonded and which may also contain the misfolded monomers. The term “low molecular weight” (LMW) species, refer to clipped species of intact antibodies composed of heavy and light chains.

As used herein, “stable” formulations of Aflibercept are formulations that exhibit less amount of HMW formation when compared to the marketed formulation of Aflibercept such as Eylea® and Eyela HD ®. Stable formulations of Aflibercept are formulations that exhibit about 30% less HMW formation when compared to the marketed formulation.

The term “long-term storage” in connection with formulation is understood to mean that the formulation or pharmaceutical composition can be stored for three months or more, for six months or more, or for one year or more. The terms ‘long term storage’ and ‘long term stability’ further include storage conditions that are at least comparable to or better than the stability of currently available commercial formulations of Aflibercept, without losses in stability that would render the formulation unsuitable for its intended pharmaceutical application. It is also contemplated that the composition can be frozen and thawed more than once.

Most common protein-based impurities include aggregates such as high molecular weight (HMW) and low molecular weight (LMW) species, charged variants such as acidic and basic variants, and host-cell proteins. High molecular weight (HMW) and low molecular weight (LMW) species have the potential to affect the safety and efficacy of biopharmaceuticals. As a result, the levels of these protein impurities in biologic drug substances and drug products must be controlled and are typically considered to be critical quality attributes. The Size Exclusion Chromatography (SEC) analyses were conducted to measure the monomer concentration and rate of formation LMW and HMW.

In an embodiment of the invention, when a formulation of the present invention is analyzed by Size-Exclusion HPLC (SE-HPLC), at least 93 to 95% of the total peak area is the main peak and rest area % are the aggregates. The % main peak may also be referred to as % monomer. The monomer content peak was differentiated from aggregated peak using TSK GEL G3000 SWXL column having 7.8mm diameter and 300mm length with 5µ particle size. (Make – Tosoh). The buffer having 100mM disodium hydrogen phosphate, 250mM sodium chloride and 10% ethanol at pH 6.7 is flowed through the column at 0.75mL/min using HPLC Agilent 1200 series. The peak was confirmed at 214nm, and area calculated as a % area.

The Imaged Capillary Isoelectric Focusing (icIEF) assay is used to separate charge isoforms / impurities based on charge and isoelectric point. The increase and decrease in charge variants likely result from chemical modification of the peptide backbone indicating the change in protein charge. The isoelectric point is determined against the pI marker using Maurice systems.

The oxidized impurities are determined using Poros A/20 column having dimensions 4.6cm x 100cm (Applied biosystems). One mobile phase contains PBS pH 7.2 and other mobile phase contains 0.1M acetic acid and 0.15M sodium chloride, pH 3. The buffer system runs through the column at a flow rate of 2.0 mL/min. The oxidized impurity was detected at 280nm against positive control.
Various analytical techniques for measuring protein stability, including measuring the type and degree of particulates that may be present in protein formulations, are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 (Vincent Lee ed., New York, N.Y., 1991) and Jones, 1993 Adv. Drug Delivery Rev. 10: 29-90.

Stability can be measured at a selected temperature for a selected time period, e.g., as described in the examples below.

Unless otherwise indicated, formulations may be maintained at an indicated temperature, e.g. 40° C, 25° C, 2-8° C, or a freezing temperature (e.g. 0 °C and below) for a period of time, e.g. days, weeks, or months, and assessed for stability and/or other parameters. For example, the pH, conductivity and/or viscosity of the samples may be measured at time zero and periodically throughout the experiment. The chemical and physical stability of the Aflibercept formulations and other properties such as viscosity, osmolarity, optical density etc. are analyzed using chromatography or other methods; non-limiting examples include SEC (size exclusion chromatography); HIC (Hydrophobic Interaction Chromatography); CEX (Cation Exchange Chromatography); AF4 (Asymmetrical flow field-flow fractionation); RP (reverse phase chromatography); UV (Ultra-violet spectroscopy); CE-IEF (Capillary Isoelectric Focusing); CE-SDS (Capillary Electrophoresis Sodium Dodecyl Sulfate).

The ophthalmic formulation of the present invention may be administered by various routes to the eye, such as topical, local ocular (subconjunctival, intravitreal, retrobulbar, intracameral), and systemic, depending on purposes. The present ophthalmic formulation is preferably administered through intravitreal injection for the treatment of ophthalmic diseases. The invention further features ophthalmic formulations provided in a pre-filled syringe or vial, particularly suitable for intravitreal administration.

The suitable amount of administration of the 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.

The present invention provides below illustrative formulation of Aflibercept free of amino acid. Formulation disclosed herein can be provided in prefilled syringe or vial.

Formulation 1: 40mg/ml Aflibercept, 10 mM phosphate buffer, 6% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 2: 40mg/ml Aflibercept, 10 mM phosphate buffer, 7% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 3: 40mg/ml Aflibercept, 10 mM phosphate buffer, 8% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 4: 40mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 5: 40mg/ml Aflibercept, 10 mM phosphate buffer, 10% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 6: 114mg/ml Aflibercept, 10 mM phosphate buffer, 6% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 7: 114mg/ml Aflibercept, 10 mM phosphate buffer, 7% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 8: 114mg/ml Aflibercept, 10 mM phosphate buffer, 8% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 9: 114mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 10: 114mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 10mM Sodium chloride with a pH of about 5.8.
Formulation 11: 114mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.8.
Formulation 12: 114mg/ml Aflibercept, 10 mM phosphate buffer, 10% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride with a pH of about 5.5 to 6.5.
Formulation 13: 160mg/ml Aflibercept, 10 mM phosphate buffer, 6% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride, with a pH of about 5.5 to 6.5.
Formulation 14: 160mg/ml Aflibercept, 10 mM phosphate buffer, 7% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride, with a pH of about 5.5 to 6.5.
Formulation 15: 160mg/ml Aflibercept, 10 mM phosphate buffer, 8% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride, with a pH of about 5.5 to 6.5
Formulation 16: 160mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride, with a pH of about 5.5 to 6.5.
Formulation 17: 160mg/ml Aflibercept, 10 mM phosphate buffer, 10% sucrose, 0.03% polysorbate 20 and 5mM Sodium chloride, with a pH of about 5.5 to 6.5.
Formulation 18: 180 mg/ml Aflibercept, 10mM phosphate buffer, 6% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 19: 180 mg/ml Aflibercept, 10mM phosphate buffer, 7% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 20: 180 mg/ml Aflibercept, 10mM phosphate buffer, 8% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 21: 180 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 22: 180 mg/ml Aflibercept, 10mM phosphate buffer, 10% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 23: 200 mg/ml Aflibercept, 10mM phosphate buffer, 6% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 24: 200 mg/ml Aflibercept, 10mM phosphate buffer, 7% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 25: 200 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 26: 200 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.
Formulation 27: 200 mg/ml Aflibercept, 10mM phosphate buffer, 10% sucrose, 0.03% polysorbate 20 and 5mM sodium chloride with a pH of about 5.5 to 6.5.

The present invention also provides below illustrative formulation of Aflibercept, free of sodium chloride and amino acid. Formulation disclosed herein can be provided in prefilled syringe or vial.

Formulation 1: 40mg/ml Aflibercept, 10 mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 2: 40mg/ml Aflibercept, 10 mM phosphate buffer, 7% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 3: 40mg/ml Aflibercept, 10 mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5
Formulation 4: 40mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 5: 40mg/ml Aflibercept, 10 mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 6: 114mg/ml Aflibercept, 10 mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 7: 114mg/ml Aflibercept, 10 mM phosphate buffer, 7% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 8: 114mg/ml Aflibercept, 10 mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 9: 114mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 10: 114mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 with a pH of about 5.8.
Formulation 11: 114mg/ml Aflibercept, 10 mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 12: 160mg/ml Aflibercept, 10 mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 13: 160mg/ml Aflibercept, 10 mM phosphate buffer, 7% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 14: 160mg/ml Aflibercept, 10 mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20, with a pH of about 5.5 to 6.5.
Formulation 15: 160mg/ml Aflibercept, 10 mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20, with a pH of about 5.5 to 6.5.
Formulation 16: 160mg/ml Aflibercept, 10 mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20, with a pH of about 5.5 to 6.5.
Formulation 17: 180 mg/ml Aflibercept, 10mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 18: 180 mg/ml Aflibercept, 10mM phosphate buffer, 7% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 19: 180 mg/ml Aflibercept, 10mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 20: 180 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 21: 180 mg/ml Aflibercept, 10mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 22: 200 mg/ml Aflibercept, 10mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 23: 200 mg/ml Aflibercept, 10mM phosphate buffer, 7% sucrose, 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 24: 200 mg/ml Aflibercept, 10mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 25: 200 mg/ml Aflibercept, 10mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.
Formulation 26: 200 mg/ml Aflibercept, 10mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20 with a pH of about 5.5 to 6.5.

The present invention is further illustrated by the following representative examples and does not limit the scope of the invention.
Example 1: Aflibercept formulations were prepared according to Table1 and stored at 40°C for 28 days for stability studies. Formulation C1 corresponds to the marketed formulation of Aflibercept high dose i.e. (114mg/ml).

Table 1: Aflibercept Formulation
Excipient C1
(mg/ml) L1
(mg/ml) L2
(mg/ml)
Aflibercept 114.3 114.3 114.3
Histidine 0.6 NA NA
L-Histidine hydrochloride monohydrate 1.3 NA NA
Sodium Phosphate Monobasic Monohydrate NA 1.14 1.28
Sodium Phosphate dibasic Heptahydrate NA 0.41 0.19
Sucrose 50.0 90.0 90.0
Arginine hydrochloride 10.5 NA NA
Polysorbate 20 0.3 0.3 0.3
pH 5.8 6.2 5.8

Table 2:
%HMW Sample Details T0 7D 14D 21D 28D
C1 2.78 21.00 37.05 46.56 55.97
L1 3.18 13.91 25.58 34.06 44.43
L2 3.06 13.14 24.60 32.82 43.13

%LMW Sample Details T0 7D 14D 21D 28D
C1 0.27 0.75 2.16 10.90 12.24
L1 0.25 0.57 1.68 2.02 2.22
L2 0.20 0.57 1.57 2.58 2.34

%Monomer Sample Details T0 7D 14D 21D 28D
C1 96.94 78.27 60.81 42.53 31.8
L1 96.57 85.52 72.75 63.93 53.37
L2 96.74 86.28 73.83 64.61 54.54

The stability data as presented in Table 2, indicates that formulation L1 and L2 contain about 30% less formation of HMW impurity as compared to C1. Formulation L1 and L2 retains a higher percentage of monomer which signifies lower propensity of aggregation formation than C1.

Example 2: Aflibercept formulation F1 to F3 were prepared according to Table 3 and stored at 40°C for 28 days (about 4 weeks). Formulation “C1” corresponds to the marketed formulation of Aflibercept high dose i.e. (114mg/ml).

Table 3: Aflibercept Formulation
Excipient C1
(mg/ml) F1
(mg/ml) F2
(mg/ml) F3
(mg/ml)
Aflibercept 114.3 114.3 114.3 114.3
Histidine 0.6 - - -
L-Histidine hydrochloride monohydrate 1.3 - - -
Sodium Phosphate Monobasic Monohydrate -- 1.28 1.28 1.28
Sodium Phosphate dibasic Heptahydrate -- 0.19 0.19 0.19
Sucrose 50.0 90.0 90.0 90.0
Arginine hydrochloride 10.5 - 52.7 -
Proline - - - 28.8
Sodium chloride - 0.29 0.29 0.29
Polysorbate 20 0.3 0.3 0.3 0.3
pH 5.8 5.8 5.8 5.8
Table 4:
Formulation %HMW formation at 40°C % Rate of HMW formation
T0 Day 7 Day 14 Day 21 Day 28
C1 2.78 21.00 37.05 46.56 55.97 53.19
F1 3.94 23.59 33.08 41.84 45.45 41.51
F2 2.89 28.75 42.37 51.98 54.87 51.98
F3 4.04 25.82 Gel formation Gel formation Gel formation --

The data as presented in Table 4, indicates that F1 has about 30% less formation of HMW impurity as compared to formulation C1. It was observed that F1 demonstrated lower propensity of aggregation than commercially available formulation C1 of Aflibercept.

Example 3: Aflibercept formulations were prepared according to Table 5, and stability data of the formulations was generated at various temperatures days and presented in Tables 6 to 9. The data indicates that formulation of the current invention B3, shows lower percent of HMW and LMW formation and higher percentage of monomer as compared to alternate formulation B1.

Table 5: Aflibercept Formulation
Excipient (mg/ml) B1 B2 B3
Aflibercept 160 160 160
Histidine 0.6 -- --
L-Histidine hydrochloride monohydrate 1.3 -- --
Sodium Phosphate Monobasic Monohydrate -- 1.28 1.28
Sodium Phosphate dibasic Heptahydrate -- 0.19 0.19
Sucrose 50.0 90.0 90.0
Sodium chloride -- -- 0.29
Arginine hydrochloride 10.5 -- --
Polysorbate 20 0.3 0.3 0.3
pH 5.8 5.8 5.8

Table 6:
Formulation %HMW formation at 40°C % Rate of HMW formation
T0 Day 7 Day 14 Day 21 Day 28
B1 2.39 20.38 32.51 45.57 51.34 48.95
B3 2.44 10.78 18.36 26.99 33.43 30.99

Formulation %LMW formation at 40°C % Rate of LMW formation
T0 Day 7 Day 14 Day 21 Day 28
B1 0.19 1.68 10.85 16.08 18.11 17.92
B3 0.13 0.41 0.7 0.76 0.9 0.77

Table 7:
Formulation % Monomer formation at 40°C
T0 Day 7 Day 14 Day 21 Day 28
B1 96.99 77.93 56.64 38.35 30.56
B3 97.44 88.82 80.95 72.26 65.68

Table 8:
Formulation % HMW formation at 25°C RH 60% % Rate of HMW formation
T0 1M 3M
B1 2.39 3.89 8.33 5.94
B3 2.44 4.74 7.55 5.11

Formulation % LMW formation at 25°C RH 60% % Rate of LMW formation
T0 1M 3M
B1 0.19 2.38 32.33 32.14
B3 0.13 0.32 0.61 0.48

Table 9:
Formulation % Monomer formation at 5°C % Monomer formation at 25°C
T0 1M 3M T0 1M 3M
B1 97.42 97.05 97.05 97.42 93.39 59.13
B3 97.44 96.95 96.29 97.44 94.95 91.84

Example 4: Aflibercept formulations were prepared according to Table 10. Formulation C2 corresponds to the marketed formulation of Aflibercept (40mg/ml).

Table 10: Aflibercept formulations
Excipient C2
(mg/ml) R2
(mg/ml) R3
(mg/ml)
Aflibercept 40 40mg/ml 40mg/ml
Sodium Phosphate Monobasic Monohydrate 1.1 1.17 1.17
Sodium Phosphate dibasic Heptahydrate 0.54 0.41 0.41
Sucrose 50 90.0 90.0
Sodium chloride (NaCl) 2.34 NA 2.9
Polysorbate 20 0.3 0.3 0.3
pH 6.2 6.2 6.2
,CLAIMS:
1. A pharmaceutical formulation, comprising 10-400mg/ml aflibercept, 10-40mM phosphate buffer, 5-10% stabilizer, 0.01-0.03% surfactant and pH of about 5.5 to 6.5.

2. The pharmaceutical formulation of claim 1, wherein the phosphate buffer is selected from disodium hydrogen phosphate, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate or a combination thereof.

3. The pharmaceutical formulation of claim 1, wherein the stabilizer is selected from polyols, polymers or sugar.

4. The pharmaceutical formulation of claim 3, wherein sugar is selected from sucrose, sorbitol, trehalose, glycerol, or mannitol.

5. The pharmaceutical formulation of claim 1, wherein the surfactant is selected from polysorbate 20, polysorbate 60, polysorbate 40, or polysorbate 80.

6. The pharmaceutical formulation of claim 1, comprising 10- 400mg/ml aflibercept, 10-40mM phosphate buffer, 5-10% sucrose, 0.01-0.03% polysorbate and pH of about 5.5 to 6.5.

7. The pharmaceutical formulation of claim 6, comprising 40mg/ml aflibercept, 10 mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

8. The pharmaceutical formulation of claim 6, comprising 40mg/ml aflibercept, 10 mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

9. The pharmaceutical formulation of claim 6, comprising 40mg/ml aflibercept, 10 mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

10. The pharmaceutical formulation of claim 6, comprising 40mg/ml aflibercept, 10 mM phosphate buffer, 7% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

11. The pharmaceutical formulation of claim 6, comprising 40mg/ml aflibercept, 10 mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

12. The pharmaceutical formulation of claim 6, comprising 114mg/ml aflibercept, 10 mM phosphate buffer, 10% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

13. The pharmaceutical formulation of claim 6, comprising 114mg/ml aflibercept, 10 mM phosphate buffer, 9% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

14. The pharmaceutical formulation of claim 6, comprising 114mg/ml aflibercept, 10 mM phosphate buffer, 8% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

15. The pharmaceutical formulation of claim 6, comprising 114mg/ml aflibercept, 10 mM phosphate buffer, 7% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.

16. The pharmaceutical formulation of claim 6, comprising 114mg/ml aflibercept, 10 mM phosphate buffer, 6% sucrose and 0.03% polysorbate 20 and pH of about 5.5 to 6.5.