WO2025219484A1

WO2025219484A1 - High concentration aflibercept formulations

Info

Publication number: WO2025219484A1
Application number: PCT/EP2025/060573
Authority: WO
Inventors: Hordur Kari HARDARSON; Hildur LUDVIKSDOTTIR; Maria Lind SIGURDARDOTTIR
Original assignee: Alvotech hf
Current assignee: Alvotech hf
Priority date: 2024-04-16
Filing date: 2025-04-16
Publication date: 2025-10-23
Anticipated expiration: 2026-10-16

Abstract

An aqueous pharmaceutical formulation suitable for intravitreal injection contains: (i) about 100 mg/mL to about 125 mg/mL of aflibercept; (ii) trehalose; (iii) methionine; (iv) optionally a poloxamer; (v) optionally a histidine buffer; and (vi) water. The formulations have an osmolality of about 250 to about 310 mOsm/kg, and a pH of about 5.8 to about 6.5, and a viscosity in the range of 6 mPa*s to 10 mPa*s at 20°C. The use of methionine as a thermal stabilizer allows the presence of less trehalose, which permits a lower viscosity while achieving suitable stability.

Description

HIGH CONCENTRATION AFLIBERCEPT FORMULATIONS

FIELD OF THE INVENTION

[001] The present invention is directed to high concentration aflibercept pharmaceutical formulations suitable for intravitreal administration and to methods of treating angiogenic eye disorders. The invention is also directed to a prefilled syringe that contains the high concentration aflibercept pharmaceutical formulation.

BACKGROUND OF THE INVENTION

[002] Aflibercept is used in the treatment of angiogenic ocular diseases, which are among the leading causes of irreversible vision loss in developed countries. Aflibercept is a vascular endothelial growth factor-A (VEGF-A or VEGFA) antagonist comprising VEGFR-1 and VEGFR-2 domains expressed in fusion with a modified IgG Fc and is typically administered as an aqueous formulation via intraocular (e.g. intravitreal) injection. Aflibercept binds VEGF ligands with a greater affinity than the natural receptors, thereby inhibiting VEGF activity and inhibiting the growth of new blood vessels.

[003] Aflibercept was initially commercialized as an intravitreal injectable formulation under the brand name EYLEA® (Regeneron Pharmaceuticals, Inc.) with an aflibercept concentration of 40 mg/ml, i.e., 0.05 ml contain 2 mg of Aflibercept. This aqueous sterile formulation additionally contained 10 mM sodium phosphate (buffer), 40 mM sodium chloride, 5% sucrose, and 0.03% polysorbate 20 with a pH of 6.2. EYLEA® was approved to treat a variety of conditions including wet age-related macular degeneration (wet AMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), and diabetic retinopathy (DR).

[004] For various reasons, a higher dose and hence higher concentration version of Aflibercept was desired. As described in US 11,103,552, formulations with high concentrations of an antibody or therapeutic protein, however, are often difficult to produce. The higher concentration of protein can increase protein aggregation and viscosity. Such formulations can have lower storage stability and/or more difficult handling and manufacturing characteristics. Thus, merely increasing the concentration of the antibody/protein in a known formulation will often result in unsatisfactory performance, especially in terms of storage stability. Instead, different formulations are needed to provide pharmaceutically acceptable/stable high concentration formulations. The US 11,103,552 purports to provide a variety suitable high concentration aflibercept formulations. Some of these formulations (and all of the claimed formulations) require L-arginine as a thermal stabilizer and/or viscosity reducing agent. Correspondingly, the commercial high concentration aflibercept, sold as EYLEA® HD, comprises 114.3 mg/ml of aflibercept (i.e., 8 mg in 0.07 ml) in a buffer solution containing a histidine buffer, arginine hydrochloride, sucrose, polysorbate 20 and water for injection with a pH of 5.8

[005] WO 2023/031478 by LEK Pharmaceuticals D.D. also proposes formulations for providing high concentrations of VEGF receptor Fc fusion protein, e.g. aflibercept. The formulations contain at least 100 mg/ml of the protein and (i) no buffering agent, (ii) a citrate buffer, or (iii) a histidine buffer preferably without sucrose and preferably with methionine. The citrate buffer is purported in the specification to provide low aggregation. The methionine is taught to be useful as an antioxidant.

[006] Parenteral formulations have a variety of requirements. Product stability is an essential quality attribute for pharmaceutical formulations that is important for both efficacy and safety and is a regulatory requirement for approval by health authorities. It refers to the retention of chemical, physical and biological properties and characteristics after manufacture, formulation, transportation, storage, and administration. Stability can be particularly challenging for biologies, which are prone to aggregation, degradation, oxidation, isomerization, adsorption, and denaturation, which can be accelerated by environmental stresses such as agitation, and variations in pH and temperature. As mentioned above, high concentration biologies present heightened challenges. Beyond stability, the formulation must have a physiologically acceptable osmolality and pH. Furthermore, the viscosity should not be excessive or else administration by injection may be difficult and manufacturing may be problematic.

[007] Intravitreal injection formulations have additional limitations. The volume of the injection is necessarily small, i.e., fractions of a milliliter. The persistence of the formulation due to low clearing rate heightens exposure concerns beyond that of a typical systemic administration. [008] While some proposals for high concentration aflibercept formulation have been made, it would be desirable to find alternative, pharmaceutically useful formulations of high concentration aflibercept. It would also be beneficial to develop a formulation that required few excipients and/or in lower amounts to achieve stability as well as suitable osmolality and viscosity.

SUMMARY OF THE INVENTION

[009] The present invention relates to the discovery that methionine provides thermal stability to high concentration aflibercept intravitreal formulations and can enable the formation of a low viscosity, physiologically acceptable osmolality and pH when combined with trehalose and optionally a histidine buffer. Accordingly, a first aspect of the invention relates to an intravitreal injection formulation comprising:

(i) 100 mg/mL to 125 mg/mL of aflibercept;

(ii) optionally a histidine buffer

(iii) a trehalose;

(iv) a methionine; and

(vi) water, wherein the formulation has: an osmolality in the range of 250 to 310 mOsm/kg, a pH in the range of 5.8 to 6.5, and a viscosity in the range of 6 mPa*s to 10 mPa*s at 20°C.

[010] While trehalose can provide thermal stability, applicant discovered that the amounts required to stabilize high concentration aflibercept would increase viscosity and/or osmolality to unacceptable levels. The addition of methionine, even in small amounts, allowed sufficient thermal stability with reduced amounts of trehalose that resulted in a low viscosity formulation and within the desired physiologically acceptable range for an intravitreal injection.

[Oil] The histidine buffer, when present, is typically in a concentration in the range of 3 mM to 40 mM, preferably 5 mM to 20 mM, and more preferably 5 mM to 15 mM, and in some embodiments 14.3 mM. The trehalose can be present in a concentration in the range of about 140 mM to 200 mM, preferably 145 mM to 180. The methionine can be present in a concentration in the range of 3 mM to 50 mM, preferably 4 mM to 10 mM. [012] In one embodiment, the intravitreal injection formulation comprises (i) about 114.3 mg/ml aflibercept; (ii) about 14.3 mM histidine buffer; (iii) about 170 mM trehalose; (iv) about 5 mM L-methionine; (v) about 0.03% poloxamer 188; and (vi) water. In another embodiment, the intravitreal injection formulation comprises (i) about 114.3 mg/ml aflibercept; (ii) about 170 mM trehalose; (iii) about 5 mM L-methionine; (iv) about 0.03% poloxamer 188; and (v) water. These embodiments often have a pH of about 5.9 to about 6.1 and typically have a viscosity of 7.5 mPa*s to 8.5 mPa*s. Further preferred in these embodiments is that the aqueous formulation does not comprise arginine or sucrose, and typically does not comprise both arginine and sucrose.

[013] Another aspect of the invention relates to a pre-filled syringe for intravitreal injection, which comprises a syringe containing less than 0.1 ml of the intravitreal formulation described above such that the pre-filled syringe exhibits a break free and glide force that does not exceed 10 N. Typically, the intravitreal injection formulation contains about 114.3 mg/ml and the volume of the formulation is about 0.07 ml, for a total dose of about 8 mg. The syringe can be made of glass or plastic, but is preferably glass. The needle is suitable for intravitreal injection and is typically 30 gauge.

DETAILED DESCRIPTION

[014] Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[015] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[016] The use of the term "or" in the claims is used to mean "and/or," unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

[017] As used herein, the terms "comprising" (and any variant or form of comprising, such as "comprise" and "comprises"), "having" (and any variant or form of having, such as "have" and "has"), "including" (and any variant or form of including, such as "includes" and "include") or "containing" (and any variant or form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps.

[018] The use of the term "for example" and its corresponding abbreviation "e.g." means that the specific terms recited are representative examples and embodiments of the disclosure that are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise.

[019] As used herein, "about" can mean plus or minus 10% of the provided value. Where ranges are provided, they are inclusive of the boundary values. "About" can additionally or alternately mean either within 10% of the stated value, or within 5% of the stated value, or in some cases within 2.5% of the stated value; or "about" can mean rounded to the nearest significant digit.

[020] As used herein, "between" is a range inclusive of the ends of the range. For example, a number between x and y explicitly includes the numbers x and y and any numbers that fall within x and y.

[021] The term “intravitreal injection formulation” refers to an aqueous pharmaceutical composition that is suitable for administration to the eye.

[022] The term “aflibercept” refers to the well-known VEGF receptor fusion protein comprising two polypeptides that each comprises an immunoglobin-like (Ig) domain 2 of VEGFR1, an Ig domain 3 of VEGFR2, and a multimerizing component, as well as to any accepted variations or biosimilars thereof. See, e.g., US 11,103,552, Ba J, et al., Drug Des Devel Ther 2015; 9:5397-405, and Stewart MW, et al., Br J Ophthalmol. 2008 May;

92(5):667-8. Because aflibercept is produced by a biological process, certain minor variations can occur from batch to batch, which variations are considered to be aflibercept. A biosimilar, as is understood by workers skilled in the art, refers to a biologic that is highly similar to the reference product (in this case to the aflibercept produced by Regeneron Pharmaceuticals) and has no clinically meaningful differences therefrom in terms of efficacy and safety. The high degree of similarity between the biosimilar and reference product generally includes the physical, chemical, and biological properties and the minor differences permitted are not clinically meaningful. This understanding is used by the U.S. FDA and the European Medicines Agency (EMA) as well as by pharmaceutical companies seeking permission to market a biosimilar of a reference product.

[023] Also, aflibercept includes glycosylated forms. For example, aflibercept may be glycosylated at one, two, three, four, five, six or seven N-glycosylation sites. In some embodiments, aflibercept is glycosylated at five N-glycosylation sites. In some embodiments, one, two or three of the aflibercept glycosylation sites are on VEGFR-1. In some embodiments, one, two or three of the aflibercept glycosylation sites are on VEGFR-2. In some embodiments, one, two or three of the aflibercept glycosylation sites is on IgG. In some embodiments, two of the aflibercept glycosylation sites are on VEGFR-1, two of the aflibercept glycosylation sites are on VEGFR-2, and one of the aflibercept glycosylation sites is on IgG. In some embodiments, the aflibercept is glycosylated at VEGFR-1 Asn 36, VEGFR-1 Asn 68, VEGFR-2 Asnl23, VEGFR-2 Asnl96, IgG Asn282, or combinations thereof. See e.g. Lee et al., Int J Mol Sci 23(19): 11807, 2022. As is conventional, the aflibercept is usually expressed in Chinese Hamster Ovary (CHO) cells.

[024] The intravitreal injection formulation has an aflibercept concentration of about 100 to about 125 mg/ml. Typically, the aflibercept concentration is in the range of 110 mg/ml to 120 mg/ml, and often about 114.3 mg/ml.

[025] The intravitreal injection formulation of the present invention optionally comprises a histidine buffer. A "histidine buffer" is a buffer comprising histidine and/or histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, and histidine sulfate solutions, with histidine chloride (histidine/histidine HC1) being particularly preferred. The histidine buffer generally uses L-histidine. The concentration of the histidine buffer is in the range of 3 mM to 40 mM, preferably 5 mM to 20 mM, and more preferably 5 mM to 15 mM. In a particularly preferred embodiment, the concentration of the histidine buffer is about 14.3 mM.

[026] The histidine buffer is optional in the intravitreal injection formulation of the present invention. The formulation can be bufferless and still achieve sufficient stability. Without wishing to be bound by theory, it is supposed that aflibercept in the high concentrations of the invention is self-buffering. Thus, the histidine buffer may be present or absent. When absent, the formulation of the invention preferably does not contain any other buffering agent and is thus “bufferless”. For example, the formulation preferably does not contain phosphate buffer, citrate buffer, acetate buffer, or tris buffer.

[027] The intravitreal injection formulation of the present invention also comprises the sugar trehalose. The trehalose is present in a concentration in the range of 140 mM to 200 mM, preferably 145 mM to 180. This generally provides sufficient stabilizing effects without unduly increasing the viscosity. In some embodiments, the formulations do not comprise sucrose. The applicant has found that the use of trehalose in the formulations described herein generally provides for superior stability for aflibercept as compared to formulations comprising other sugars. Thus, in some embodiments, the intravitreal injection formulations do not comprise sucrose and more generally do not comprise sucrose, glucose or fructose.

[028] The intravitreal injection formulations of the present invention contain methionine. Typically, L-methionine is used. The methionine is present at a concentration of about 3 mM to about 50 mM, and typically 4 mM to 20 mM. In some embodiments, the concentration of methionine is 4 mM to 10 mM, such as 5 mM or 7 mM (each ±0.5 mM). Methionine can surprisingly provide a superior stabilizing effect compared to arginine in the context of a high concentration aflibercept formulation of the present invention. Accordingly, the intravitreal injection formulations of the present invention preferably do not comprise arginine.

[029] The intravitreal injection formulations may also comprise a surfactant, in particular a poloxamer. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). The poloxamer used herein is not intended to be particularly limited and typically the poloxamer is poloxamer 188, otherwise known as Pl 88, where the first two digits (18) multiplied by 100 give the approximate molecular mass of the polyoxypropylene core (i.e., 1800), and the last digit (8) multiplied by 10 gives the percentage of polyoxyethylene content (i.e., 80%). The concentration of the poloxamer (such as P188), if present, is in an amount of from about 0.02% (w/v) to about 0.04% (w/v) based on the total formulation. Often the concentration of poloxamer is about 0.03% (w/v).

[030] The pH of the intravitreal injection formulations is about 5.8 to about 6.5, typically about 5.8 to about 6.2, more typically about 5.9 to about 6.1, and even more preferably about [031] The intravitreal injection formulations have an osmolality of about 250 to about 310 mOsm/kg, typically about 260 to about 300 mOsm/kg, in some embodiments about 270 to about 290 mOsm/kg, and even about 270 to about 285 mOsm/kg. In other embodiments the formulation has an osmolality of about 250 to about 290 mOsm/kg, typically about 260 to about 285 mOsm/kg. The osmolality of the vitreous humor of the eye is generally considered to be 287-293 mOsm/kg, so having an osmolality within or near this range is often useful for an intravitreal injection formulation.

[032] The intravitreal injection formulations have a viscosity of about 6.0 mPa*s to about 10 mPa*s at 20°C. This is considered within the low viscosity range and facilitates easier handling and eventually administration of the intravitreal injection formulation. Typically, the viscosity is in the range of 7.5 mPa*s to about 8.5 mPa*s, though such is not required.

[033] In a particularly preferred embodiment, the intravitreal injection formulation comprises (i) about 114.3 mg/mL aflibercept; (ii) about 170 mM trehalose; (iii) about 5 mM L-methionine; (iv) about 0.03% poloxamer 188; (v) about 14.3 mM histidine buffer; and (vi) water. The formulation has a pH of about 5.9 to about 6.1, and a viscosity of about 7.5 mPa*s to about 8.5 mPa*s. The formulation of this embodiment generally has an osmolality of about 270 to about 300 mOsm/kg. Further preferred in this embodiment is that the aqueous formulation does not comprise arginine.

[034] In another particularly preferred embodiment, the intravitreal injection formulation comprises (i) about 114.3 mg/mL aflibercept; (ii) about 170 mM trehalose; (iii) about 5 mM L-methionine; (iv) about 0.03% poloxamer 188; and (v) water. The formulation has a pH of about 5.9 to about 6.1, and a viscosity of about 7.5 mPa*s to about 8.5 mPa*s. The formulation of this embodiment generally has an osmolality of about 255 to about 285 mOsm/kg. Further preferred in this embodiment is that the aqueous formulation does not comprise arginine and is bufferless.

[035] The formulations described herein are stable during manufacture and storage. The term "stable" refers to formulations that include aflibercept that retain both chemical and physical stability over the period of formulation manufacturing and storage, e.g., that maintain integrity and have minimal degradation, denaturation, or unfolding. Aflibercept stability can be determined using analytical techniques available in the art at different temperatures and over different periods of time. In particular, physical stability can be determined by size exclusion chromatographic (SEC) analysis, high pressure liquid chromatography (HPLC), SEC-HPLC, differential scanning fluorimetry or nano differential scanning fluorimetry (DSF or nanoDSF), dynamic light scattering (DLS), static light scattering (SLS), by counting particle number (e.g. using a HORIZON system), visual appearance, and by any combination thereof; chemical stability can be determined by reducing/denaturing capillary electrophoresis (rCE-SDS), capillary isoelectric focusing (cIEF), mass spectroscopy techniques such as liquid chromatography - mass spectroscopy (LC-MS), and by any combination thereof.

[036] A “high molecular weight species” (HMWS) as used herein, with reference to an aqueous formulation comprising aflibercept, refers to any species of polypeptide or polypeptide complex in the formulation which elutes from a size exclusion column (e.g. SEC-HPLC) ahead of (e.g. with a higher molecular weight than) aflibercept and/or a homodimer thereof. The percentage of HMWS refers to the percentage of such species relative to the overall quantity of polypeptides in the formulation, e.g. by SEC-HPLC analysis. A HMWS can be an indication of denatured aflibercept, or agglomerated aflibercept, as a result of instability with the protein.

[037] In some embodiments, the HMWS is measured at the completion of manufacturing, and then at various periods after manufacturing, i.e., storage. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at To. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at Ti month when stored at 2-8°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at T2 month when stored at 2-8°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at T3 months when stored at 2-8°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at Tt> month when stored at 2-8°C.

[038] In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at Ti month when stored at 25°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at T2 month when stored at 25°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at T3 months when stored at 25°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% HMWS at Tt> month when stored at 25°C.

[039] In some embodiments, the aqueous formulation comprises less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% HMWS at Ti month when stored at 40°C. In some embodiments, the aqueous formulation comprises 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% HMWS at T2 month when stored at 40°C. In some embodiments, the aqueous formulation comprises 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% HMWS at T3 months when stored at 40°C. In some embodiments, the aqueous formulation comprises 50%, less than about 40%, less than about 30%, or less than about 20% at Tt> month when stored at 25°C.

[040] A “low molecular weight species” (LMWS) as used herein, with reference to an aqueous formulation comprising aflibercept, refers to any species of polypeptide or polypeptide complex in the formulation which runs further in rCE-SDS (i.e., with a lower molecular weight than) aflibercept. LMWS can include truncated or degraded forms of aflibercept as a result of instability with the protein in the formulation.

[041] In some embodiments, the LMWS is measured at the completion of manufacturing, and then at various periods after manufacturing, i.e., storage. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% LMWS at To. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2%, LMWS at Ti month when stored at 2-8°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2% LMWS at T2 month when stored at 2-8°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2%, or less than about 1% LMWS at T3 months when stored at 2-8°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2%, LMWS at Tt> month when stored at 2-8°C. [042] In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2%, LMWS at Ti month when stored at 25°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2% LMWS at T2 month when stored at 25°C.

In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2%, or less than about 1% LMWS at T3 months when stored at 25°C. In some embodiments, the aqueous formulation comprises less than about 5%, less than about 4%, less than about 3%, or less than about 2%, LMWS at T<> month when stored at 25°C.

[043] In some embodiments, the aqueous formulation comprises less than about 50%, less than about 40%, less than about 20%, or less than about 10%, LMWS at Ti month when stored at 40°C. In some embodiments, the aqueous formulation comprises less than about 50%, less than about 40%, less than about 20%, or less than about 10%, LMWS at T2 month when stored at 40°C. In some embodiments, the aqueous formulation comprises less than 50%, less than about 40%, less than about 20%, or less than about 10% LMWS at T3 months when stored at 40°C. In some embodiments, the aqueous formulation comprises 50%, less than about 40%, less than about 20%, or less than about 10% LMWS at Tt> month when stored at 40°C.

[044] In some embodiments, stability of the aflibercept in the disclosed formulations shows limited change over time in particle size, particle number, visual appearance, isoelectric point, HMWS, LMWS, isoelectric point, methionine oxidation (e.g. of M237, M192, or M10), asparagine deamination (e.g. of N84, N99, or N374), or truncation products as determined by methods known in the art.

[045] Aflibercept formulations in accordance with embodiments herein can be pre-packaged or pre-loaded in various useful containers and injection devices. Thus, the present disclosure includes containers and injection devices containing such formulations. In one embodiment, the container is a vial which may be sterile. In another embodiment, the container is a test tube which may be sterile. In another embodiment, an injection device (which may be sterile) is a syringe (e.g., a pre-filled syringe or autoinjector). In another embodiment, the injection device is an intravitreal implant, e.g., a refillable intravitreal implant. [046] A “pre-filled” syringe is a syringe which is filled with an aqueous formulation as disclosed herein prior to sale or use by a physician or patient. The pre-filled syringe is suitable for intra ocular or intravitreal injection.

[047] Syringes as used herein include barrels made, for example, of glass or polymer (for example, cycloolefin as described in U.S. Patent Publication No. 2017/0232199), a plunger and a needle. In some embodiments, the barrel and/or plunger of the syringe can be coated with silicone (e.g., silicone oil or baked- silicone (e.g., <40 pg or <100 pg)). In some embodiments, a container or injection device is substantially metal-free or substantially tungsten-free or low-tungsten.

[048] In some embodiments, the syringe contains one or more dose line graduations and/or is a dose metering system.

[049] Containers in accordance with embodiments herein can hold the aqueous formulations comprising aflibercept. In some aspects, the container or injection device can include a label stating indications of use. In some instances, the container or injection device herein can include package inserts with instructions for use as described throughout this specification.

[050] The container or pre-filled syringe may contain a volume of the intravitreal injection formulation that corresponds to a single dose of aflibercept. Typically, a single dose is between 4 and 10 mg of aflibercept. The volume in the container of a single dose of aflibercept may optionally further include a small amount of overfill volume, such as to facilitate extraction from the container, i.e., vial, into a syringe for injection or to remove air bubbles from a pre-filled syringe. Usually the overfill volume, if any, is about 5%-10% of the volume of intravitreal injection formulation.

[051] Syringe sizes can be, for example, 40 pl to 150 pl. Sterile syringes typically include a needle useful for ocular injection, typically about Yi inch, or 12.5 mm to 16 mm in length, and can be 29 gauge, 30 gauge, 31 gauge, 32 gauge or 33 gauge based on the patient and health care specialist preference. Other needle lengths and gauges can be used, as long as the needle is effective at accomplishing intravitreal injection.

[052] In one embodiment, a pre-filled syringe containing the intravitreal injection formulation of the present invention has a break free and glide force that does not exceed 10 N. That is, upon depression of the plunger to expel the formulation from the syringe through completion of the expulsion, the force needed does not exceed 10 N. In some embodiments, the break free and glide force does not exceed 8 N. The pre-filled syringe generally contains less than 0.1 ml of the formulation, typically 0.06 to 0.08 ml. This low force requirement is achieved by a combination of the syringe design (the friction of the plunger against the barrel of the syringe) and the low viscosity of the present formulation. Because of the low viscosity of the present formulation, a break free and glide force of less than 10 N can be achieved with commercially available syringes. This low break free and glide force ensures that medical practitioners can readily and rapidly complete the intravitreal injection. If the break free or glide force was too high, then a risk of injury to the patient, e.g. accidental injury to the retina, would be increased.

[053] The intravitreal injection formulations of the invention can be used in the treatment or prevention of any angiogenic eye disorder by administration of a therapeutically effective amount of formulation comprising aflibercept to a subject in need thereof, e.g., by intravitreal injection. Angiogenic eye disorders herein refer to any disease of the eye which is caused by or associated with the growth or proliferation of blood vessels and/or by blood vessel leakage. Non-limiting examples of angiogenic eye disorders that are treatable or preventable using the formulations and methods herein, include:

• wet age-related macular degeneration (wet AMD),

• macular edema,

• macular edema following retinal vein occlusion,

• retinal vein occlusion (RVO),

• central retinal vein occlusion (CRVO),

• branch retinal vein occlusion (BRVO),

• diabetic macular edema (DME),

• choroidal neovascularization (CNV),

• iris neovascularization,

• neovascular glaucoma,

• post-surgical fibrosis in glaucoma,

• proliferative vitreoretinopathy (PVR),

• optic disc neovascularization,

• corneal neovascularization,

• retinal neovascularization,

• vitreal neovascularization, • pannus,

• pterygium,

• vascular retinopathy,

• diabetic retinopathies (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy; e.g., in a subject that does not suffer from DME) and

• Diabetic retinopathy in a patient who has diabetic macular edema (DME).

[054] Thus, the present disclosure provides methods for treating or preventing an angiogenic eye disorder in a subject in need thereof comprising administering a therapeutically effective amount of aflibercept, e.g., about 4 mg to about 10 mg aflibercept, more specifically, e.g., about 4, 6, or 8.0, 8.1 , 8.4 or 8.5 mg), e.g., in a pharmaceutical formulation according to the present invention, intraocularly, e.g., into the vitreous, of an eye of the subject. In an embodiment of the invention, both eyes are administered aflibercept. The amount and timing of the administration are generally known in the art and include administering 8 mg of aflibercept every 12-16 weeks, or more often if needed. An 8 mg dose is generally 0.06 ml to 0.08 ml of the intravitreal injection formulation of the present invention.

[055] Each dose of the aqueous formulations comprising aflibercept administered to a subject over the course of a treatment can contain the same or substantially the same amount of aflibercept. Alternatively, the quantity, and timing, of any one dose can be different or variable over a treatment course.

[056] Incorporated by reference: each of the patents and journal articles cited above are expressly incorporated herein by reference in their entirety. Additionally, the following are also incorporated by reference Hardman, et al. (2001 ) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y. [057] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.

EXAMPLES

Example 1: Formulation Screening

[058] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used, but some experimental errors and deviations should be accounted for. Any formulations set forth in these examples are part of the present disclosure.

[059] In these examples, when an experiment is conducted at 2- 8 °C, the temperature is targeted to 5°C with a tolerance of ±3°C variation.

Biophysical characterization of high-concentration Aflibercept formulations.

[060] A series of different formulations having high concentration of aflibercept, i.e., 114.3 mg/mL was tested for viscosity, folding stability, and colloidal stability. The commercial product EYLEA HD® (Regeneron) also has an aflibercept concentration of 114.3 mg/mL.

The components of each of the formulations are shown in Error! Reference source not found.. It should be noted that formulations denoted as “Eylea HD” are replications of the commercially available product, i.e., using the same amounts and excipients as the commercial product, but are not the EYLEA HD® product commercially sold by Regeneron Pharmaceuticals, Inc.

Table 1:

[061] Each of Formulations 1-8 was subjected to stability storage at 5°C, 25°C, and 40°C.

Stability was determined on each of the Formulations 1-8 and Eylea HD. Table 2 provides the biophysical results of Formulations 1-8 and Eylea HD.

Table 2

[062] Table 3 presents the results for formation of High Molecular Weight Species (HMWS), Eow Molecular Weight Species (EMWS) and capillary isoelectric focusing (cIEF). The formation of high molecular weight species was evaluated by SEC-HPEC, and degradation was evaluated by rCE-SDS. Charge stability and the stability of posttranslational modifications were determined by cIEF. Table 3

[063] Table 4 presents the amount of oxidation on each of the Formulations 1-8 and Eylea HD. Oxidation was determined by LC-MS. Table 4

[064] Table 5 presents the amount of deamidation and truncation on each of the

Formulations 1-8 and Eylea HD. Deamination, and truncation were determined by LC-MS. Table 5

[065] Table 6 presents the amount of VEGF binding and particle formation on each of the Formulations 1-8 and Eylea HD. The particle size and number of particles over 2 microns were assessed by dynamic light scattering and a Horizon particle analyzer, respectively.

Table 6

[066] Table 7 presents the photolytic degradation on each of the Formulations 1-8 and Eylea HD. A PharmaEvent-L photostability chamber illuminated with cool, white, fluorescent lamps was utilized to assess the photolytic stability in the study. The illumination and temperature in the chamber were at 100% and 25°C, respectively, resulting in an illumination intensity of approximately 18 klux per hour. Formulations were filled into 2R vials that were laid in a horizontal position inside the photo stability chamber to maximize the light exposure and positioned with sufficient interval to avoid interference of the light source. Once the target value of 1,200,000 lux*h was reached, the lights were automatically turned off and the samples were aliquoted into Eppendorf tubes and stored at -70 °C until they were analyzed.

Table 7 Example 2: Comparison of Histidine Buffered and Bufferless Formulations

[067] The following formulations as shown in Table 8 were prepared and tested.

Table 8 [068] The results are shown below in Table 9.

Table 9

[069] The osmolality results show, as expected, a reduction in osmolality upon removal of the histidine from the formulation (from 297 to 263 mOsm/kg for formulation 1 vs formulation 2). Trehalose concentration increased to 195 mM (formulation 2) resulted in a concurrent osmolality increase to 301 mOsm/kg. The reverse was true for the viscosity, where an increase in viscosity is observed upon removal of histidine from the formulation (formulation 1 vs formulation 2) and a further increase upon trehalose concentration increase (formulation 3). The slight increase in viscosity does however not increase the Max BLGF (measured at 100 mm/min) which ranged from 5.3-7.0 N for the three formulations (average value of the two syringes measured for each formulation). High variability is expected for the method and the difference from 5.3-7.0 observed is not considered significant for the method.

[070] The pH was measured at TO and after 1 month’s storage at 5 °C and 25 °C ( [071] Table 10

[072] ). The pH remained stable within 6.0-6.1 for all formulations.

Table 10

[073] The formulations were stored under various conditions and examined via SEC and the results are shown below in Tables 11 and 12.

Table 11

Table 12

[074] The differences between the histidine buffered formulation and the bufferless formulations are mild and the stability is considered comparable. Photo stability at 200 kLux*h yielded different results as the histidine buffer formulation had 2.3% HMW as determined by SEC while the two bufferless formulations were 3.8% HMW.

Claims

1. An intravitreal injection formulation comprising:

(i) 100 mg/mL to 125 mg/mL of aflibercept;

(ii) optionally a histidine buffer

(iii) a trehalose;

(iv) a methionine; and

2. The intravitreal injection formulation according to claim 1, wherein said histidine buffer is present and in a concentration in the range of 3 mM to 40 mM, preferably 5 mM to 20 mM, and more preferably 5 mM to 15 mM, such as 14.3 mM.

3. The intravitreal injection formulation according to claim 1 or 2, wherein said trehalose is present in a concentration in the range of 140 mM to 200 mM, preferably 145 mM to 180.

4. The intravitreal injection formulation according to any one of claims 1-3, wherein said methionine is present in a concentration in the range of 3 mM to 50 mM.

5. The intravitreal injection formulation according to any one of claims 1-4, wherein said methionine is present in a concentration in the range of 4 mM to 10 mM.

6. The intravitreal injection formulation according to any one of claims 1-5, which further comprises 0.02% (w/v) to 0.04% (w/v) of a poloxamer.

7. The intravitreal injection formulation according to any one of claims 1-6, which comprises

(i) about 114.3 mg/mL aflibercept; (ii) about 170 mM trehalose; (iii) about 5 mM L- methionine; (iv) about 0.03% poloxamer 188; (v) about 14.3 mM histidine buffer; and (vi) water.

8. The intravitreal injection formulation according to claim 1, wherein the viscosity of said formulation is in the range of 7.5 mPa*s to 8.5 mPa*s at 20°C.

9. The intravitreal injection formulation according to claim 1, wherein said formulation is bufferless.

10. The intravitreal injection formulation according to claim 9, wherein said trehalose is present in a concentration in the range of 140 mM to 200 mM, preferably 145 mM to 180.

11. The intravitreal injection formulation according to any one of claims 9 and 10, wherein said methionine is present in a concentration in the range of 3 mM to 50 mM.

12. The intravitreal injection formulation according to any one of claims 9-11, wherein said methionine is present in a concentration in the range of 4 mM to 10 mM.

13. The intravitreal injection formulation according to any one of claims 9-12, which further comprises 0.02% (w/v) to 0.04% (w/v) of a poloxamer.

14. The intravitreal injection formulation according to any one of claims 8-13, which comprises (i) about 114.3 mg/mL aflibercept; (ii) about 170 mM trehalose; (iii) about 5 mM L-methionine; (iv) about 0.03% poloxamer 188; and (v) water.

15. A pre-filled syringe for intravitreal injection, which comprises a syringe containing less than 0.1 ml of the formulation according to any one of claims 1-13, wherein said prefilled syringe exhibits a break free and glide force that does not exceed 10 N.