Patent Description:
The present invention relates to aqueous pharmaceutical formulations of anti-VEGF antibodies, a process for the preparation thereof, and uses of the formulations.

Vascular endothelial growth factor (VEGF) is a known regulator of angiogenesis and neovascularization, and has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders (<NPL>)). The VEGF mRNA is overexpressed in many human tumors, and the concentration of VEGF in eye fluids are highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies (<NPL>); <NPL>); <NPL>); <NPL>); and <NPL>); <NPL>)). In addition, recent studies have shown the presence of localized VEGF in choroidal neovascular membranes in patients affected by AMD (<NPL>)). Anti-VEGF neutralizing antibodies can be used to suppress the growth of a variety of human tumor cell lines in nude mice and also inhibit intraocular angiogenesis in models of ischemic retinal disorders (<NPL>); <NPL>); <NPL>); and <NPL>)) (<NPL>)).

A number of antibodies are approved for therapeutic use in humans and other mammals, including anti-VEGF antibodies. The concentration of therapeutic antibodies in liquid pharmaceutical formulations varies widely depending, for example, on the route of administration. There is often a need for a high concentration formulation of an antibody when small volumes are desired. For example, high concentration formulations may be desirable for intravitreal injection or subcutaneous administration.

However, formulations with high concentration of antibody may have short shelf lives, and the formulated antibodies may lose biological activity caused by chemical and physical instabilities during storage. Aggregation, deamidation and oxidation are known to be the most common causes of antibody degradation. In particular, aggregation can potentially lead to increased immune response in patients, leading to safety concerns. Thus it must be minimized or prevented.

Formation of particulates in biotherapeutic formulations is also a major quality concern, as particulates in the tens of microns to sub-millimeter and millimeter size range can generally be seen by the naked human eye (see <NPL>). Particulates in therapeutic ophthalmic preparations can cause damage to the eye. Therefore, there are regulatory standards to ensure sub-visible particulate matter content in ophthalmic formulations is within certain limits. For example, the U. Pharmacopeial Convention (USP) has set requirements for particulate matter in ophthalmic solutions, such as the maximum number of particles ≥ <NUM> diameter is <NUM> per mL, the maximum number of particles ≥ <NUM> diameter is <NUM> per mL, and the maximum number of particles ≥ <NUM> diameter is <NUM> per mL determined by the microscopic method particle count (see USP General Chapter <<NUM>>).

Methods for producing high concentration antibody formulations are known. However, a universal approach does not exist to overcome the unpredictable impact of an antibody's amino acid sequence on its tendency to form aggregates or degrade in the presence of various pharmaceutical excipients, buffers, etc. Further, preparing an ophthalmic formulation with a high concentration of protein (such as an antibody) that contains an acceptable level of sub-visible particles is challenging and not predictable. Furthermore <CIT> and <CIT> refer to compositions comprising anti-VEGF antibodies.

It is an object of the invention to provide further and improved formulations with high concentration of anti-VEGF antibodies and low levels of antibody aggregation and sub-visible particles, that are suitable for administration to a human, in particular to a human eye.

Accordingly, the present invention is directed to an aqueous pharmaceutical composition comprising a high concentration of anti-VEGF antibody suitable for ophthalmic injection. In certain aspects, the aqueous pharmaceutical compositions of the invention exhibit low to undetectable levels of antibody aggregation or degradation, with very little to no loss of the biological activities during manufacture, preparation, transportation and long periods of storage, the concentration of the anti-VEGF antibody being at least about <NUM>/ml, <NUM>/ml, <NUM>/ml, <NUM>/ml, <NUM>/ml, <NUM>/ml, <NUM>/ml, <NUM>/ml, or <NUM>/ml.

The invention provides aqueous pharmaceutical compositions comprising an anti-VEGF antibody, a stabilizer, a buffer, and a surfactant. In certain aspects, as aqueous pharmaceutical composition comprises: (i) at least <NUM>/ml of an anti-VEGF antibody, (ii) sucrose or trehalose as a stabilizer, (iii) a citrate or histidine buffer, and (iv) polysorbate <NUM> as a surfactant according to claim <NUM>.

In certain aspects, the aqueous pharmaceutical composition comprises about <NUM>% - <NUM>% w/v sucrose or <NUM>% - <NUM>% trehalose, <NUM>% to <NUM>% citric acid (w/v), <NUM>% to <NUM>% trisodium citrate dihydrate (w/v), and about <NUM>% to <NUM>% polysorbate <NUM> (w/v), wherein the pH of the formulation is <NUM> to <NUM>.

Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

The invention provides aqueous pharmaceutical compositions comprising a high concentration of an anti-VEGF antibody. In certain embodiments an aqueous pharmaceutical composition of the invention is stable for at least <NUM> months at <NUM>-<NUM> and is suitable for ocular administration, including injection or infusion. In a particular embodiment, an aqueous pharmaceutical composition of the invention meets the USP <<NUM>> requirements relative to the presence of particulate matter. Thus, in certain embodiments, the maximum number of particles ≥ <NUM> diameter in an aqueous pharmaceutical composition of the invention is <NUM> per mL, the maximum number of particles ≥ <NUM> diameter in an aqueous pharmaceutical composition of the invention is <NUM> per mL, and the maximum number of particles ≥ <NUM> diameter in an aqueous pharmaceutical composition of the invention is <NUM> per mL, said particle numbers being determined by the light obscuration and/or microscopic particle count method as required by the U. Pharmacopeial Convention General Chapter <<NUM>>).

As used herein, an "aqueous" pharmaceutical composition is a composition suitable for pharmaceutical use, wherein the aqueous carrier is distilled water. A composition suitable for pharmaceutical use may be sterile, homogeneous and/or isotonic. Aqueous pharmaceutical compositions may be prepared either directly in an aqueous form, for example in pre-filled syringe ready for use (the "liquid formulations") or as lyophilisate to be reconstituted shortly before use. As used herein, the term "aqueous pharmaceutical composition" refers to the liquid formulation or reconstituted lyophilized formulation. In certain embodiments, the aqueous pharmaceutical compositions of the invention are suitable for ophthalmic administration to a human subject. In a specific embodiment, the aqueous pharmaceutical compositions of the invention are suitable for intravitreal administration. In another embodiment, the aqueous pharmaceutical compositions of the invention are suitable for administration by intravitreal infusion.

The present invention provides novel pharmaceutical formulations, in particular novel pharmaceutical formulations in which the active ingredient comprises antibodies to human VEGF. In one aspect, the invention relates to an aqueous pharmaceutical composition with high concentration of anti-VEGF antibodies. Preferred anti-VEGF antibodies in formulations of the invention are described in <CIT>,.

The term "antibody" as used herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion," "antigen binding polypeptide," or "immunobinder") or single chain thereof. An "antibody" includes a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., VEGF). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')<NUM> fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a single domain or dAb fragment (<NPL>), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., <NPL>; and <NPL>). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of different isotype, for example, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

In a preferred embodiment, an aqueous pharmaceutical composition of the invention comprises a variable heavy chain having the sequence as set forth in SEQ ID NO: <NUM> and a variable light chain having the sequence as set forth in SEQ ID NO: <NUM>.

In another preferred embodiment, the anti-VEGF antibody is a single-chain Fv (scFv) antibody fragment comprising the sequence:
<IMG>.

An anti-VEGF antibody in an aqueous pharmaceutical composition of the invention can be produced, for example, as described in <CIT>. An scFv can be produced using an expression vector, as described therein. A methionine derived from the start codon in an expression vector is present in the final protein in cases where it has not been cleaved posttranslationally (see SEQ ID NO: <NUM> in the Examples).

In certain embodiments, the anti-VEGF antibody in an aqueous pharmaceutical composition of the invention comprises heavy chain CDR1, CDR2 and CDR <NUM> as set forth in SEQ ID NO: <NUM>, <NUM>, and <NUM> respectively, and light chain CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: <NUM>, <NUM>, and <NUM>.

In one embodiment, the concentration of an anti-VEGF antibody in the aqueous pharmaceutical composition of the invention is at least <NUM>/ml. Preferably, the aqueous pharmaceutical composition of the invention comprises about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml, about <NUM>/ml or about <NUM>/ml of an anti-VEGF antibody.

In certain embodiments, the aqueous pharmaceutical composition of the invention comprises between <NUM>/ml and <NUM>/ml of an anti-VEGF antibody, for example, an antibody comprising SEQ ID NO: <NUM> and SEQ ID NO: <NUM>.

In one embodiment, the aqueous pharmaceutical composition of the invention comprises <NUM>/ml of an anti-VEGF antibody comprising SEQ ID NO: <NUM>.

In another embodiment, the aqueous pharmaceutical composition of the invention comprises <NUM>/ml of an anti-VEGF antibody comprising SEQ ID NO: <NUM>.

The aqueous pharmaceutical compositions of the invention include, in addition to the anti-VEGF antibody, further components such as one or more of the following: (i) a stabilizer; (ii) a buffering agent; (iii) a surfactant; amino acid according to claim <NUM>. Inclusion of each of such additional components can give compositions with low aggregation of the anti-VEGF antibody. Preferably, the aqueous pharmaceutical compositions of the invention include, in addition to the anti-VEGF antibody: (i) a stabilizer; (ii) a buffering agent; and (iii) a surfactant.

Suitable stabilizer for use with the invention can act, for example, as viscosity enhancing agents, bulking agents, solubilizing agents, and/or the like. The stabilizer can be ionic or non-ionic (e.g. sugars). As sugars they include, but are not limited to, monosaccharides, e.g., fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides, e.g. lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, e.g. raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like. For example, the sugar may be sucrose, trehalose, raffinose, maltose, sorbitol or mannitol. The sugar may be a sugar alcohol or an amino sugar. Sucrose and trehalose are preferred. Most preferred is sucrose. As ionic stabilizer they may include salts such as NaCl or amino acid components such as arginine-HCl.

Suitable buffering agents for use with the invention include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, thomethamine hydrochloride, or phosphate buffer. In addition, amino acid components can also be used as buffering agent. Citrate or histidine buffer are particularly useful, including <NUM>-<NUM> of histidine buffer, for example, <NUM>% to <NUM>% (w/v) histidine and <NUM>% - <NUM>% (w/v) histidine Hydrochloride monohydrate), or <NUM>-<NUM> citrate buffer, for example <NUM>% to <NUM>% citric acid (w/v) and <NUM>% to <NUM>% trisodium citrate dihydrate (w/v). Citric acid used in a formulation of the invention can be any hydration form, for example anhydrous or monohydrate.

The aqueous pharmaceutical compositions include such buffering agent or pH adjusting agent to provide improved pH control. In certain embodiment, an aqueous pharmaceutical composition of the invention has a pH between <NUM> and <NUM>, between <NUM> and <NUM>, between <NUM> and <NUM>, or between <NUM> and <NUM>. In one embodiment, the pH of an aqueous pharmaceutical composition of the invention is about <NUM> to about <NUM>. In a specific embodiment, an aqueous pharmaceutical composition of the invention has a pH of about <NUM>.

As used herein, the term "surfactant" herein refers to organic substances having amphipathic structures; i.e., they are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic and dispersing agents for various pharmaceutical compositions and preparations of biological materials.

Suitable surfactants for use with the invention include, but are not limited to, non-ionic surfactants, ionic surfactants and zwitterionic surfactants. Typical surfactants for use with the invention include, but are not limited to, sorbitan fatty acid esters (e.g. sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerine fatty acid esters (e.g. glycerine monocaprylate, glycerine monomyristate, glycerine monostearate), polyglycerine fatty acid esters (e.g. decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters (e.g. polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerine fatty acid esters (e.g. polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g. polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g. polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oils (e.g. polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g. polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g. polyoxyethylene stearic acid amide); C<NUM>-C<NUM> alkyl sulfates (e.g. sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C<NUM>-C<NUM> alkyl ether sulfate with an average of <NUM> to <NUM> moles of ethylene oxide units added (e.g. sodium polyoxyethylene lauryl sulfate), and C<NUM>-C<NUM> alkyl sulfosuccinate ester salts (e.g. sodium lauryl sulfosuccinate ester); and natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (e.g. sphingomyelin), and sucrose esters of C<NUM>-<NUM> fatty acids. A composition may include one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters e.g. polysorbate <NUM>, <NUM>, <NUM> or <NUM>. Polysorbate <NUM> is particularly preferred.

Suitable free amino acids for use with the invention include, but are not limited to, arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine glutamic acid or aspartic acid. The inclusion of a basic amino acid is preferred i.e. arginine, lysine and/or histidine. If a composition includes histidine then this may act both as a buffering agent and a free amino acid, but when a histidine buffer is used it is typical to include a non-histidine free amino acid e.g. to include histidine buffer and lysine. An amino acid may be present in its D- and/or L-form, but the L-form is typical. The amino acid may be present as any suitable salt e.g. a hydrochloride salt, such as arginine-HCl. In one preferred embodiment, an aqueous pharmaceutical composition of the invention does not comprise any such free amino acids.

In a preferred embodiment, a sugar is present in the aqueous pharmaceutical composition of the invention, e.g. after reconstitution of a lyophilisate in water, at a concentration of between <NUM> and <NUM> % (w/v). In certain embodiments, the sugar is sucrose at a concentration of about <NUM>% to about <NUM>%, or trehalose at a concentration of about <NUM>% to about <NUM>%. A concentration of <NUM>% (w/v) sucrose is preferred.

In a preferred embodiment, a buffering agent is present in the aqueous pharmaceutical composition of the invention, e.g. after reconstitution of a lyophilisate in water, at a concentration of between <NUM> and <NUM> e.g. <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>. In certain embodiments, the buffering agent is citrate or histidine. A concentration of <NUM> citrate buffer is preferred.

In a preferred embodiment, a surfactant is present in the aqueous pharmaceutical composition of the invention, e.g. after reconstitution of a lyophilisate in water, at a concentration of up to <NUM>% (by volume) e.g. <NUM>-<NUM>%, <NUM>-<NUM>%, <NUM>-<NUM>%. A concentration of <NUM>% polysorbate <NUM> is preferred.

Other contemplated excipients, which may be utilized in the aqueous pharmaceutical compositions of the invention include, for example, antimicrobial agents, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin, gelatin, casein, salt-forming counterions such sodium and the like. These and additional known pharmaceutical excipients and/or additives suitable for use in the formulations of the invention are known in the art, e.g., as listed in "<NPL>); and <NPL>).

Preferred formulations of the invention are shown in Table <NUM> in the Examples below.

In certain embodiments, lyophilisation of an anti-VEGF antibody is contemplated to provide an aqueous pharmaceutical composition of the invention for treating a patient.

Techniques for lyophilisation of antibodies are well known in the art e.g. see <NPL>); <NPL>).

Before a lyophilisate can be administered to a patient it should be reconstituted with an aqueous reconstituent. This step permits antibody and other components in the lyophilisate to redissolve to give a solution which is suitable for injection to a patient.

The volume of aqueous material used for reconstitution dictates the concentration of the antibody in a resulting pharmaceutical composition. Reconstitution with a smaller volume of reconstituent than the pre-lyophilisation volume provides a composition which is more concentrated than before lyophilisation. The reconstitution factor (volume of formulation after lyophilization:volume of formulation before lyophilization) may be from <NUM>:<NUM> to <NUM>:<NUM>. A reconstitution factor of <NUM>:<NUM> is useful. As mentioned above, lyophilisates of the invention can be reconstituted to give aqueous compositions with an anti-VEGF antibody concentration of at least <NUM>/ml (i.e., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>/ml), and the volume of reconstituent will be selected accordingly. If required, the reconstituted formulation can be diluted prior to administration to a patient as appropriate to deliver the intended dose.

Typical reconstituents for lyophilized antibodies include sterile water or buffer, optionally containing a preservative. If the lyophilisate includes a buffering agent then the reconstituent may include further buffering agent (which may be the same as or different from the lyophilisate's buffering agent) or it may instead include no buffering agent (e.g. WFI (water for injection), or physiological saline).

The aqueous pharmaceutical compositions of the invention comprising anti-VEGF antibodies can be used to treat a variety of diseases or disorders. Pharmaceutical compositions comprising anti-VEGF antibodies are particularly useful to treat neovascular ocular diseases in a subject.

A "neovascular ocular disease" that can be treated using an aqueous pharmaceutical composition of the invention includes, a condition, disease, or disorder associated with ocular neovascularization, including, but not limited to, abnormal angiogenesis, choroidal neovascularization (CNV), retinal vascular permeability, retinal edema, diabetic retinopathy (particularly proliferative diabetic retinopathy), diabetic macular edema, neovascular (exudative) age-related macular degeneration (AMD), including CNV associated with nAMD (neovascular AMD), sequela associated with retinal ischemia, Central Retinal Vein Occlusion (CRVO), and posterior segment neovascularization.

The aqueous pharmaceutical compositions of the invention may include further active ingredients in addition to the anti-VEGF antibody. Further pharmacological agents may include, for instance, other antibodies useful for treating ocular diseases.

Aqueous pharmaceutical compositions of the invention can be administered to a patient. As used herein, the term "subject" or "patient" refers to human and non-human mammals, including but, not limited to, primates, rabbits, pigs, horses, dogs, cats, sheep, and cows. Preferably, a subject or patient is a human.

Administration will typically be via a syringe. Thus the invention provides a delivery device (e.g. a syringe) including a pharmaceutical composition of the invention (e.g., pre-filled syringe). Patients will receive an effective amount of the anti-VEGF antibody as the principal active ingredient (i.e., an amount that is sufficient to achieve or at least partially achieve the desired effect). A therapeutically effective dose is sufficient if it can produce even an incremental change in the symptoms or conditions associated with the disease. The therapeutically effective dose does not have to completely cure the disease or completely eliminate symptoms. Preferably, the therapeutically effective dose can at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system.

The dose amount can be readily determined using known dosage adjustment techniques by a physician having ordinary skill in treatment of the disease or condition. The therapeutically effective amount of an anti-VEGF antibody used in an aqueous pharmaceutical composition of the invention is determined by taking into account the desired dose volumes and mode(s) of administration, for example. Typically, therapeutically effective compositions are administered in a dosage ranging from <NUM>/ml to about <NUM>/ml per dose. Preferably, a dosage used in a method of the invention is about <NUM>/ml to about <NUM>/ml (i.e., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>/ml). In a preferred embodiment, the dosage of an anti-VEGF antibody used in a method of the invention is <NUM>/ml or <NUM>/ml.

In certain embodiments, a dose is administered directly to an eye of a patient. In one embodiment, a dose per eye is at least about <NUM> up to about <NUM>. Preferred doses per eye include about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>. Doses can be administered in various volumes suitable for ophthalmic administration, such as <NUM>µl or <NUM>µl, for example, including <NUM>/<NUM>µl or <NUM>/<NUM>µl. Smaller volumes can also be used, including <NUM>µl or less, for example about <NUM>µl or about <NUM>µl. In certain embodiments, a dose of <NUM>/<NUM>µl or <NUM>/<NUM>µl (e.g., <NUM>/<NUM>µl) is delivered to an eye of a patient for treating or ameliorating one or more of the diseases and disorders described above. Delivery can be, for example, by intravitreal injection or infusion.

The invention also provides formulations (i.e., aqueous pharmaceutical compositions) of the invention for use as medicaments, e.g. for use in delivering an antibody to a patient, or for use in treating or ameliorating one or more of the diseases and disorders described above.

The invention further provides a method for delivering an anti-VEGF antibody to a patient, comprising a step of administering to the patient an aqueous pharmaceutical composition of the invention.

In certain embodiments, a method for delivering an anti-VEGF antibody to a patient invention comprises the steps of: (i) reconstituting a lyophilisate of the invention to give an aqueous formulation, and (ii) administering the aqueous formulation to the patient. Step (ii) ideally takes place within <NUM> hours of step (i) (e.g., within <NUM> hours, within <NUM> hours, within <NUM> hours, or within <NUM> hour).

Certain specific embodiments of the invention are described as numbered hereafter:.

As used herein, all percentages are percentages by weight, unless stated otherwise.

As used herein and unless otherwise indicated, the terms "a" and "an" are taken to mean "one", "at least one" or "one or more". Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

It is intended that the present specification and examples be considered as exemplary only with a true scope and spirit of the invention being indicated by the following claims and equivalents thereof.

The following examples describe formulation development efforts designed to identify suitable stabilization approaches and compositions to provide stable, highly concentrated solutions comprising the antibody <NUM>, enabling an IVT formulation with at least an <NUM>-month shelf-life at refrigerated storage conditions that meets the regulatory requirements for ophthalmic products.

The <NUM> antibody is a single-chain antibody that binds to and inhibits the biologic activity of human vascular endothelial growth factor A (VEGF-A). The amino acid sequence of expressed <NUM> is:
<IMG>.

Significant amounts of sub-visible particulates were observed at a concentration of <NUM>/ml <NUM> when <NUM> was formulated as an isotonic solution in <NUM> citrate buffer with <NUM>% polysorbate <NUM> at pH <NUM>. The major issue with this initial formulation was the particulate matter exceeding regulatory limits for ophthalmic solutions for injection (USP<<NUM>>), even when stored at -<NUM>.

The following examples summarize the formulation development of <NUM> and <NUM>/ml <NUM> intravitreal (IVT) solutions stable at <NUM>-<NUM> storage for at least <NUM> months. The formulation development effort focused on inhibition of the formation of sub-visible particles and meeting the USP requirement for content, purity and potency.

The following methods were used throughout the Examples as indicated.

MFI method used for analysis of excipient screening, Study <NUM> and Study <NUM> for <NUM>/ml optimization studies was as follows:.

The optimize illumination step was performed with purified filtered particle free water.

MFI method used for analysis of <NUM>/ml <NUM> Study <NUM> and Study <NUM>:.

SE-HPLC (Size exclusion chromatography) separates proteins according to their size. Separation was achieved by the differential exclusion, or inclusion, of the sample molecules as they passed through the porous-particle stationary phase. High performance liquid chromatography system capable of maintaining a flow rate of <NUM>/minute and a sample temperature of <NUM>, equipped with a TOSOH SuperSW3000 column (Tosoh Bioscience LLC, King of Prussia, PA), and a detector capable of operating at <NUM> and <NUM> simultaneously. This method was used for purity testing.

AIEX-HPLC (Anion exchange high performance liquid chromatography) separates proteins according to their net charge. This procedure was performed using high-performance liquid chromatography (HPLC), capable of maintaining a flow rate of <NUM>/minute, with a temperature controlled column compartment (set at <NUM>) containing a strong anion exchange column, an auto-sampler (set at <NUM>), and a variable wavelength UV detector, capable of operating at <NUM>.

The capillary gel electrophoresis method was performed for the determination of the identity and purity of proteins between the molecular weights of <NUM> kDa and <NUM> kDa by SDS gel Capillary Electrophoresis. The capillary was dynamically filled with a Beckman Coulter <NUM>% SDS Gel Buffer, pH <NUM>, proprietary formulation. The separation of the protein was performed by molecular sieving electrophoresis. The logarithm of protein molecular weight was linear with its reciprocal electrophoretic mobility. The identity of a protein was determined by comparing its migration with a molecular weight standard. The purity was determined by area percent analysis of the parent peak and impurities. A photodiode array detector (PDA) was used to analyze the sample at <NUM>.

The competition ELISA was used for potency testing. Competitive ELISA, the ability of <NUM> to compete with VEGFR2/Fc for biotinylated VEGF was measured. The signal observed was inversely related to the concentration of <NUM>, as increasing amounts of <NUM> effectively blocked the binding of biotinylated VEGF with its receptor VEGFR2/Fc. Each sample was analyzed in a <NUM>-well microtiter plate against a <NUM> reference standard, and the relative potency of the sample to that of the reference standard was reported.

<NUM> was formulated in a citrate buffer at pH <NUM>. The formulation composition is shown in Table <NUM>.

Significant numbers of sub-visible particulates were observed in the above formulation. The particulate matter exceeded regulatory limits for ophthalmic solutions for injection USP<<NUM>> even in the storage of -<NUM>.

The effect of various excipients on formation of sub-visible particulates was investigated to develop a more stable liquid solution of <NUM>. The protein solutions at <NUM>/ml containing different excipients were stored at <NUM> and analyzed for particulates in the size of <NUM> - <NUM> by MFI. The experimental data demonstrated that most excipients tested, including arginine, dextran, ascorbic acid, methionine, and ammonium acetate, did not reduce the formation of the particulates. Only in presence of non-reducing sugars, such as sucrose and trehalose, was particulate formation significantly reduced.

Further excipient screening was conducted. Effect of excipients on <NUM> stability was evaluated in ~<NUM>/ml <NUM> solution. Protein solutions containing <NUM>% human serum albumin (HSA), <NUM>% poloxamer <NUM>, <NUM>% Brij <NUM>, <NUM>% glycerin, <NUM> glycine were stored at <NUM>, and the samples were assayed by MFI, SEC and IEX after <NUM>, <NUM>, and <NUM> days of storage. The results are shown in Table <NUM>. The experimental data demonstrated the instability of the protein in the presence of the tested excipients.

Stability of <NUM>/ml <NUM> was investigated in <NUM> in phosphate buffer at pH6. The formulation containing <NUM>% sucrose and <NUM>% PS80 was assayed at <NUM>, <NUM> and <NUM> days after storage at <NUM>. The results are listed in Table <NUM>.

Protein solution of <NUM>/ml <NUM> in <NUM> citrate buffer containing <NUM>% polysorbate <NUM> and <NUM>% NaCl at pH <NUM> was used for sample preparation. In order to remove the surfactant in the original protein solution, buffer exchange using NAP-<NUM> column was first performed using the vehicle without polysorbate <NUM>. The exchanged protein solution was then concentrated to about <NUM>/ml using Vivaspin filter with 10kDa MWCO. The excipients of HSA, glycine, glycerin, poloxamer <NUM>, and Brij <NUM> were spiked individually. The prepared samples were then syringe-filtered through <NUM> PVDF membrane to a <NUM> clear glass vial. About <NUM>µL sample was used for initial assay, while the rest of the samples were stored at <NUM>. One milliliter sample of each formulation was pulled at <NUM>, <NUM>, and <NUM> days following storage at <NUM> for analysis by MFI, SEC and IEX.

Based on excipient screening studies performed internally, two buffers (citrate and histidine), sugars (sucrose and trehalose), and surfactants (Polysorbate <NUM> and Polysorbate <NUM>) were selected for formulation component selection. Buffer strength of <NUM>, sugar concentration of <NUM>, and surfactant concentration of <NUM>% were chosen for the study. A full factorial experimental study was designed as shown in the table below.

The full design used is shown in Table <NUM> below:.

Protein solution of <NUM>/ml <NUM> in the original buffer containing <NUM>% polysorbate <NUM> was used for the sample preparation. Four new vehicle buffers with different buffer/sugar combinations (<NUM> Citrate/<NUM> Trehalose, <NUM> Citrate/<NUM> Sucrose, <NUM> Histidine/<NUM> Trehalose, <NUM> Histidine/<NUM> Sucrose) were prepared. <NUM> drug substance (DS) was first exchanged into the vehicle buffers using Illustra NAP-<NUM> column (GE Healthcare). The column was equilibrated with <NUM> of citrate buffer. Then <NUM> of <NUM> DS was loaded onto the column. After the DS solution completed moved into the column, the column was washed with <NUM> of the same buffer used to equilibrate the column. The <NUM> solution eluted from the column was collected in a Vivaspin <NUM> concentrator (10KD MWCO, GE Healthcare). The <NUM> eluted solution was then concentrated to about <NUM> using a Beckman GS-15R centrifuge at <NUM> ×g and <NUM>. The concentration of protein was then measured with a NanoDrop <NUM> spectrophotometer using OD280. Polysorbate <NUM> (PS20) or Polysorbate <NUM> (PS80) was spiked into the formulations to a final concentration of <NUM> %. The prepared formulations were then filtered through <NUM> PVDF syringe filter and filled into a <NUM> clear glass vial. About <NUM> uL sample was used for initial assay, while rest of the samples was stored at <NUM>. One milliliter sample was pulled following storage of <NUM>, <NUM> and <NUM> weeks at <NUM> for analysis by MFI, SEC and IEX.

The qualitative study was conducted in order to select the optimum sugar/surfactant/buffer components for the formulation. Two buffers (citrate and histidine), sugars (sucrose and trehalose), surfactants (Polysorbate <NUM> and Polysorbate <NUM>) were used in the study design as discussed above. Four weeks stability study was conducted under the accelerated conditions (<NUM>). The stability samples were assayed by SEC, IEX and MFI. Table <NUM> shows the stability results by SEC, IEX and melting point (Tm) by µDSC.

The stability results were analyzed using Minitab (Minitab Inc. , State College PA). Since SEC and IEX showed the same trend, only SEC data were analyzed and reported by Minitab. The SEC results demonstrated surfactant was the only significant factor; the other factors that appeared to impact the response included buffer selection and interaction between buffer and sugar (to a less extent). SEC data suggested the optimum formulation component being citrate as buffer, polysorbate <NUM> as surfactant and sucrose as sugar.

Experimental results on particulates by MFI were also analyzed by Minitab. All the three factors evaluated (sugar, surfactant, and buffer) appeared to be insignificant (α=<NUM>). Comparing the main effects on particulates, citrate was better than histidine; PS80 was better than PS20; Sucrose was similar or slightly better than trehalose. Therefore, citrate as buffer, polysorbate <NUM> as surfactant and sucrose as sugar were chosen for further studies.

A full factorial study containing three factors at two levels with two center points were performed in citrate and histidine buffers respectively, in order to determine the optimum concentration for each selected formulation component. The three factors are buffer (citrate or histidine), polysorbate <NUM> and sucrose. Factors and design space are tabulated in Table <NUM> and the detailed experimental designs are shown in Tables <NUM> and <NUM>.

To prepare individual formulation, protein solution of <NUM>/ml <NUM> in citrate buffer containing <NUM>% polysorbate <NUM>/<NUM>% NaCl at pH <NUM> was used. The protein solution was first buffer exchanged to a vehicle using NAP-<NUM> column. This vehicle contained buffer and sucrose at their target concentrations. The exchanged protein solution was concentrated to a concentration of about <NUM>/ml using Vivaspin <NUM> kDa MWCO.

Batch amounts of polysorbate <NUM> and sodium chloride were then added to make the final formulation compositions. The protein concentration in each sample was verified with theoretical extinction coefficient using absorbance measured by UV at <NUM>.

The prepared formulations were then filtered through <NUM> PVDF syringe filter to a <NUM> clean clear glass vial. About <NUM>µL sample was used for initial assay by SEC and IEX, while about <NUM>-<NUM> samples were stored at <NUM>. About <NUM>µL sample was pulled after storage for <NUM>, <NUM>, and <NUM> days at <NUM> for analysis by MFI, SEC and IEX.

Based on Study <NUM> results, a full factorial study containing three factors at two levels with two center points were performed in citrate and histidine buffers respectively, in order to determine the optimum concentration for each selected formulation component. The tested samples were stored at <NUM>, and pulled at <NUM>, <NUM>, <NUM> and <NUM> weeks for MFI, SEC and IEX analysis. The results are tabulated in the Tables <NUM> and <NUM>.

The experimental data were analyzed by Minitab. For SEC results in citrate buffer, surfactant was the major factor influencing the protein aggregation, while sucrose, interaction between sucrose and polysorbate <NUM>, and buffer strength played less significant roles. Higher sucrose concentration and lower polysorbate <NUM> concentration favored the protein stability. The protein stability affected aggregation slightly and higher citrate buffer strength promoted slightly better protein stability.

For MFI results in citrate buffer, factors of sucrose and polysorbate <NUM> as well as interaction of sucrose and polysorbate <NUM> played equally significant roles in particulates formation. High sucrose and high polysorbate <NUM> suppressed particulates formation significantly, while buffer strength in the <NUM>-<NUM> range slightly affect the particulate formation, lower buffer strength was preferred.

The results by Minitab analysis of histidine buffer solutions showed the surfactant, polysorbate <NUM>, was the major factor influencing the protein aggregation, while interaction between buffer strength and polysorbate <NUM>, as well as sucrose, played less significant roles. Less polysorbate <NUM> favored the protein stability. Buffer strength and sucrose concentration did not appear to affect protein aggregation.

For MFI results in histidine buffer, polysorbate <NUM>, sucrose, and interaction of sucrose and polysorbate <NUM> played significant roles in particulates formation. High sucrose and low polysorbate <NUM>, and low buffer strength were preferred to suppress the particulates formation.

Effects of pH on the aggregation behavior of <NUM> were studied under six pH conditions (<NUM>, <NUM>, <NUM>. <NUM>, <NUM> and <NUM>) in <NUM> citrate buffer containing <NUM>% NaCl, <NUM>% sucrose and <NUM>% PS80. <NUM> drug substance was first exchanged into the vehicles (without PS80) of different pH using Illustra NAP-<NUM> column (GE Healthcare). The column was first equilibrated with <NUM> of citrate buffer. Then <NUM> of <NUM> DS was loaded onto the column. After the DS solution completed moved into the column, the column was washed with <NUM> of the same citrate buffer used to equilibrate the column. Collect the <NUM> solution eluted from the column in a Vivaspin <NUM> concentrator (10KD MWCO, GE Healthcare). The <NUM> eluted solution was then concentrated to about <NUM> using a Beckman GS-15R centrifuge at <NUM> ×g and <NUM>. The concentration of protein was then measured with a NanoDrop <NUM> spectrophotometer. The final <NUM> concentration for this study was determined to be about <NUM>/ml. PS80 was spiked into the formulations to a final target concentration of <NUM>%. The kinetic turbidity assay was conducted using a PerkinElmer Lambda <NUM> spectrophotometer with a two-position cell holder and a water batch for temperature regulation. In a Starna submicro volume quartz cell (Starna Scientific Ltd. , England) with <NUM> path-length, <NUM>µL of <NUM> formulation or the corresponding vehicle was added. The cells were placed into the cell holder that was preheated to <NUM>. Change of OD350 in the <NUM> formulation was monitored for <NUM> minutes. The OD350 data obtained for formulations of different pH was then plotted against time for comparison.

Using the turbidity-based kinetic assay, effects of pH on the aggregation behavior of <NUM> were studied with the formulations from pH <NUM> to <NUM> containing about <NUM>/ml <NUM>. For the turbidity assay, a sharp increase in OD<NUM> was associated with the major aggregation events of the protein. Thus, the difference in the incubation time required for OD<NUM> of each formulation to increase could reflect the different physical stability of the protein in the formulations. As shown in <FIG>, <NUM> appeared to be the most stable at pH <NUM> under the tested formulation conditions, as it had the longest incubation time before the sharp increase of OD<NUM>. Repeated experiments with similar results confirmed that the tested formulation was most stable at pH <NUM>.

A half factorial study (Table <NUM>) was performed to investigate the effects of the major formulation conditions including concentrations of protein (<NUM>-<NUM>/ml), sucrose (<NUM> - <NUM>%), citrate buffer (<NUM> to <NUM>) and polysorbate <NUM> (<NUM> - <NUM>%).

To prepare the above formulations, a <NUM>% citrate buffer at pH <NUM> and a <NUM>% sucrose stock solution in <NUM> citrate buffer at pH <NUM> were first prepared. Then the citrate buffer, sucrose stock and water for injection were mixed at appropriate ratios to generate five buffers with PS80, which were <NUM>% sucrose in <NUM> citrate, <NUM>% sucrose in <NUM> citrate, <NUM>% sucrose in <NUM> citrate, <NUM>% sucrose in <NUM> citrate and <NUM>% sucrose in <NUM> citrate. <NUM> DS were subsequently exchanged into these five buffers using Illustra NAP-<NUM> column (GE Healthcare) followed by concentrating with Vivaspin <NUM> concentrator (<NUM> KD MWCO, GE Healthcare) at <NUM> × at <NUM>. The protein concentrations in each sample were determined with a NanoDrop <NUM> spectrophotometer during centrifugation. For the samples in the first four buffers, when <NUM> concentration reached ~<NUM>/ml, part of the sample was removed from the concentrator and used to prepared formulations (#<NUM>, <NUM>, <NUM> and <NUM> in Table <NUM>) with <NUM>/ml API by adding appropriate amount of corresponding buffer and spiking in <NUM>% PS80. The rest of the samples were concentrated further to above <NUM>/ml and then added buffer and <NUM>% PS80 to obtain the final formulations (#<NUM>, <NUM>, <NUM> and <NUM> in Table <NUM>). Formulations #<NUM> and <NUM> in Table <NUM> were prepared by concentrating the <NUM> samples exchanged into buffer #<NUM> to about <NUM>/ml and then mixed with buffer and <NUM>% PS80 to reach the final formulation concentrations. The pH values of all the final formulations were measure and adjusted to <NUM>.

A turbidity assay modified from the one described above was used as the analytical tool for evaluation. In this modified turbidity assay, a <NUM>-cell position Cary <NUM> UV-Vis spectrophotometer was used. The cuvettes used in this assay were <NUM> quartz cuvettes with stoppers. In each cuvette, <NUM>µL of sample was added for the test. The optical density change at <NUM> was monitored for the formulations incubated at <NUM>.

The measured <NUM> concentrations and final pH of all the formulations for Study <NUM> are summarized in Table <NUM>. Formulations with low, medium and high <NUM> concentrations (Table <NUM>. -<NUM>) with different sucrose, PS80 concentrations and buffer strength were incubated at <NUM> and changes in OD<NUM> were monitored. As shown in <FIG>, different aggregation propensity was observed for the formulations. The two formulations almost aggregated immediately when incubation started were the formulations <NUM> and <NUM>, which contained the relatively higher <NUM> concentration (~<NUM>/ml) and lower sucrose concentration (<NUM>%). On the other hand, formulations <NUM> and <NUM>, containing the relatively lower <NUM> concentration (~<NUM>/ml) and higher sucrose concentration (<NUM>%), aggregated slower than other formulations. The aggregation propensity of other combinations of medium <NUM> and sucrose concentrations fell in between the above two groups.

Using the time for OD<NUM> of each formulation to reach <NUM> as the criteria, the effects of different formulation factors were compared. The results suggested that sucrose had the largest impact on the aggregation of <NUM>, followed by API concentration. The effects of sucrose and API concentrations were opposite, indicating higher sucrose and lower API concentrations were preferred for better formulation stability. Other two factors, buffer strength and PS80 showed less effect.

Based on the results from above Study <NUM>, Study <NUM> was conducted to further investigate the effects of protein concentration (<NUM> - <NUM>/ml) and sucrose concentration (with a narrower range of <NUM> to <NUM>%) in <NUM> citrate buffer containing <NUM>% PS <NUM> at pH <NUM>. The study design is shown in the table below.

To prepare the formulations, about <NUM> of <NUM> drug substance was loaded into a TFF system and then buffer exchange was conducted with <NUM> citrate buffer containing <NUM>% sucrose at about <NUM> times the volume of the drug substance. After buffer exchange, the volume of the protein solution was reduced to about half of the initial volume using the TFF system. About <NUM> of concentrated <NUM> solution was recovered and the protein concentrated was about <NUM>/ml measured by the NanoDrop <NUM> spectrophotometer. The concentrated <NUM> solution was then used to prepare formulations #<NUM>, <NUM>, <NUM>, <NUM>-<NUM> by mixing with appropriate amount of <NUM>% PS80 and <NUM> citrate buffers containing <NUM>, <NUM>% and/or <NUM>% sucrose to obtain the corresponding final API, sucrose and PS80 concentrations in each of these formulations. The rest of the <NUM> solution was further concentrated to about <NUM>/ml to prepare formulations #<NUM> and <NUM> in the same fashion. Lastly, formulation #<NUM> was prepared by concentrating <NUM> solution to about <NUM>/ml and the concentrations of protein, sucrose and PS80 were adjusted with <NUM>% PS80 and <NUM> citrate buffers containing <NUM>, <NUM>% and/or <NUM>% sucrose. The final concentration of <NUM> in each formulation was determined with the NanoDrop <NUM> spectrophotometer and the pH was adjusted to <NUM>.

The samples were analyzed using the turbidity assay described above. Eight selected formulations (#<NUM>-<NUM> and #<NUM>-<NUM>) were also stored in a <NUM> exploratory incubator for <NUM> weeks and analyzed with MIF, IEX and SEC methods at different time points.

Twelve formulations, as listed in Table <NUM> were investigated.

The turbidity assay at <NUM> was used to monitor the aggregation of the formulations. As shown in <FIG>, different degrees of aggregation were shown by the formulations under the same thermal stress. Formulations with the relatively higher <NUM> and low sucrose concentrations (such as formulations # <NUM> and <NUM> in Table <NUM>) were the ones that aggregated rapidly upon incubation. The formulations with relatively lower <NUM> and high sucrose concentrations (formulations # <NUM> and <NUM> in Table <NUM>) aggregated slower than all the rest of the formulations. The curves obtained from the turbidity assay for the various formulations were fitted to a sigmoidal function. The incubation time corresponds to the middle of a transition was recorded as Tm (transition mid-point time) for each formulation. The results are listed in Table <NUM>. Tm values against sucrose and <NUM> concentrations showed the opposite effects of sucrose and <NUM> concentrations on the protein aggregation.

Besides studies using the turbidity assay at <NUM>, stability of eight selected formulations at <NUM> were also monitored by MFI, SEC and IEX over <NUM> weeks. Formulations and results are listed in Tables <NUM> to <NUM>. In agreement with turbidity assay, MFI results showed that formulations with high <NUM> concentration (~<NUM>/ml) formed more particles especially particles greater than <NUM>, in comparison with formulations with lower API concentrations. The MFI results didn't show clear effects of sucrose concentration for the formulations with similar amount of <NUM> (formulations #<NUM> to <NUM>). As shown in <FIG>, after <NUM>-week storage at <NUM>, the two formulations (#<NUM> and <NUM>) with the high <NUM> concentration degraded the most, which the formulations with ~<NUM>/ml <NUM> degraded less than all other formulations. As for formulations with ~<NUM>/ml <NUM>, slightly less degradation was observed at higher sucrose concentration (formulations #<NUM>) than at the lower sucrose concentration (formulation #<NUM>). IEX results showed the same trend as the SEC.

Exploratory stability was performed for a formulation of <NUM>/ml <NUM> solution in <NUM> citrate, <NUM>% PS20 at pH <NUM> as a control. The control formulation was filtered through <NUM> syringe filter and stored at <NUM>, ambient room temperature, and refrigerator. The samples were pulled at <NUM>, <NUM> and <NUM> weeks for <NUM> samples, and <NUM> weeks for samples stored at ambient room temperature and refrigerator. The selected stability samples were tested for pH, osmolality, content by A280, SEC, IEX CGE and ELISA. The results are shown in Table <NUM>.

Another study on the control formulation was performed for <NUM>/ml <NUM> solution in <NUM> citrate, <NUM>% PS20 and <NUM>% NaCl at pH6. The formulation was filtered through <NUM> syringe filter and stored in <NUM>. The samples were pulled at <NUM>, <NUM>, <NUM>, and <NUM> weeks for MFI analysis. The results are shown in Table <NUM>.

Exploratory stability was performed for <NUM>/ml <NUM> formulation containing <NUM>% sucrose and <NUM>% PS80. The formulation was filtered through <NUM> syringe filter and stored at <NUM>. The samples were pulled at <NUM>, <NUM> and <NUM> weeks and analyzed by MFI, SEC and IEX. The results are tabulated in Table <NUM>.

Real time stability study of <NUM>/ml <NUM> solution was conducted for the formulation containing <NUM>% sucrose, <NUM> citrate, <NUM>% PS80 at pH6. <NUM> as shown in Table <NUM>. The stability samples were stored at <NUM>-<NUM>, <NUM>, <NUM> and light cabinet (LC); they also went through three cycles of freeze-thaw (FT). The samples were pulled according to the schedule shown in Table <NUM>, and analyzed by various analytical methods such as pH, osmolality, MFI, content, SEC, IEX, CGE and potency.

In the early stage, real time stability study of <NUM>/ml <NUM> solution was initiated. The formulation contained <NUM>% sucrose, <NUM> citrate, <NUM>% PS80 at pH6. <NUM> as shown in Table <NUM>. The samples were stored at <NUM>-<NUM>, <NUM>, <NUM> and light cabinet (LC); they also went through three cycles of freeze-thaw (FT). The samples were pulled according to the schedule shown in Table <NUM>, and analyzed by various analytical methods. The stability results are tabulated in Tables <NUM>-<NUM>.

An exploratory stability in citrate buffer was conducted for the formulation of <NUM>/ml <NUM> in <NUM> Citrate/<NUM>% Sucrose / <NUM>% PS80 / pH6. The stability samples were stored at <NUM> and <NUM>, as well as undergone freeze-thaw cycles. The samples were pulled according to the schedule shown in the table below, and analyzed for appearance, pH, osmolality, MFI, content, SEC, and IEX.

Similarly, exploratory stability in histidine buffer was conducted for formulation of <NUM>/ml <NUM> in <NUM> histidine/<NUM>% Sucrose / <NUM>% PS80 / pH6. The stability samples were stored at <NUM> and <NUM>. The samples were also undergone freeze-thaw cycles. The samples were pulled according to the schedule shown in the table below, and analyzed for appearance, pH, osmolality, MFI, content, SEC, and IEX.

The experimental data are tabulated in the tables below.

The experimental data indicated that the stability of the formulations tested were significantly enhanced, especially in suppressing the formation of particulate matters, comparing to that of the control formulation at a protein concentration of <NUM>/ml.

The stable solution formulation identified contained <NUM> Citrate/<NUM>% Sucrose/<NUM>% PS80 as shown in Table <NUM>.

Claim 1:
An aqueous ophthalmic pharmaceutical composition, comprising at least <NUM>/ml of an anti-VEGF antibody that comprises the sequences of SEQ ID NO: <NUM> and SEQ ID NO: <NUM>, and comprising about <NUM>% to <NUM>% (w/v) sucrose or about <NUM>% to <NUM>% (w/v) trehalose, <NUM>% to <NUM>% citric acid (w/v), <NUM>% to <NUM>% trisodium citrate dihydrate (w/v), and <NUM>% to <NUM>% polysorbate <NUM> (w/v), wherein the pH of the composition is about <NUM> to about <NUM>.