FIELD OF THE INVENTION

The present invention relates to liquid pharmaceutical compositions of a VEGF antagonist for intravitreal administration comprising a histidine buffer or a dicarboxylic acidbuffer selected from malic acid buffer, succinate buffer and maleic acid buffer, an inorganic salt, carbohydrate and a surfactant. The composition may also contain methionine.

BACKGROUND OF THE INVENTION

Vascular endothelial growth factor (VEGF) is a protein that stimulates vasculogenesis (i.e. de novo formation of new blood vessels) and angiogenesis (i.e. formation of new blood vessels from pre-existing vessels). There are at least six subtypes of VEGF, i.e. VEGF- A, VEGF-B, VEGF-C, VEGF-D, virus VEGF-E and placental VEGF (PIGF). VEGF-A is associated with increases of vascular permeability and degeneration of the extracellular matrix. Four isomers of VEGF-A that arise from alternative splicing of mRNA have been reported in humans (VEGF 121, VEGF 165 , VEGF 184, VEGF206) (Ferrara and Davis Smyth, Endocr Rev, 1997, 18:1-22). Further, VEGF110 is produced from VEGF 165 by protease cleavage. VEGF-A binds to receptors VEGFr-1 and VEGFr-2 (Kajdaniuk et al., Endokrynol Pol, 2011, 62(5):444-55; Kajdaniuk et al, Endokrynol Pol, 2011, 62(5):456-64). The specificity of VEGF action for endothelial cells supports a key role in the process of abnormal blood vessel growth and vascular leakage. Anti-VEGF agents have demonstrated efficacy in reducing choroidal neovascularisation in both animal models and clinical trials (Okamoto et al. (1997) Am J Pathol 151 : 281-91 ; Adamis et al. (1996) Arch Ophthalmol, 114: 66-71). Specifically, anti-VEGF antibodies have been used for the treatment of treatments of intraocular neo vascular disorders. Currently available anti-VEGF antibodies are bevacizumab and ranibizumab. Bevacizumab is a full-length, humanized murine monoclonal antibody that recognizes all isoforms of VEGF. Ranibizumab is the Fab fragment of the humanized murine monoclonal antibody that is used to create bevacizumab and has been affinity-matured so that it binds VEGF-A with a significantly higher affinity than bevacizumab. Ranibizumab and bevacizumab appear to have similar efficacy profiles in the treatment of neovascular age-related macular degeneration although rare adverse events seem to occur more often with bevacizumab (Johnson and Sharma, Curr Opin Ophthalmol, 2013, 24(3):205-12).

Another class of VEGF antagonists is represented by fusion proteins of parts of the VEGF receptors and the Fc portion of human immunoglobulins. In particular, aflibercept, marketed under the name Eylea ^® , is a recombinant fusion protein consisting of the VEGF binding portion from the extracellular domains of human VEGF receptors 1 and 2 that are fused to the Fc portion of the human IgGl immunoglobulin. It is approved for the treatment of wet macular degeneration and some further ocular diseases.

For medical purposes stable pharmaceutical compositions are of great interest, in particular ready-to-use solutions which require no dissolution or reconstitution before use. A main problem of such a liquid composition is a decreasing content of the active ingredient due to the formation of insoluble particles during repeated freeze/thaw cycles during manufacturing or proteins being degraded and forming degradation products during long-term storage.

WO 2006/104852A2 discloses liquid pharmaceutical formulations of aflibercept for subcutaneous or intravenous delivery which comprise a histidine buffer, sodium chloride, sucrose and polysorbate 20.

In particular for pharmaceutical compositions which are intended to be delivered to the eye, such as pharmaceutical compositions for intravitreal injections, it is important to keep the amount of insoluble particles at a minimum level, since particles may cause irritation or inflammation when injected into the eye.

WO 2007/149334 A2 describes liquid pharmaceutical compositions of aflibercept comprising a sodium phosphate buffer, sodium chloride, sucrose and polysorbate 20 which formulations are suitable for ophthalmic use. WO 2015/071348 Al discloses liquid pharmaceutical formulations of ranibizumab for intravitreal injection comprising a buffer, a non-ionic surfactant, and, optionally, an inorganic salt, wherein the composition does not contain saccharides.

WO 2017/129685 Al discloses liquid pharmaceutical formulations of aflibercept comprising a histidine buffer, sodium chloride, sucrose and polysorbate 20 which formulations are suitable for ophthalmic use. Nevertheless, there is still a need for a pharmaceutical composition which has a low protein aggregate content and is therefore suitable for intravitreal injection and which is stable in liquid form. Preferably, such a composition is suitable for the treatment of AMD and formulated in a prefilled syringe. SUMMARY OF THE INVENTION

The inventors found that a liquid composition comprising a histidine buffer, a non-ionic surfactant, a VEGF antagonist, an inorganic salt and a carbohydrate and having a pH of 6.3 or 6.6 has a surprisingly low level of aggregates and is therefore particularly suitable for intravitreal injection and the treatment of neovascular intraocular diseases. If methionine is added to this formulation the oxidation of the protein is reduced.

Liquid compositions comprising a dicarboxylic acid buffer such as a malic acid, succinic acid or malic acid, a non-ionic surfactant, a VEGF antagonist, an inorganic salt and a carbohydrate and having a pH Of 6.0 to 6.2 may also be suitable for intravitreal injection.

A further advantage of the liquid pharmaceutical composition used in the present invention is that it does not require a lyophilisation step and is thus produced in a shorter time and with reduced costs. Another advantage is that the composition has a pH in the range of 5.8 to 6.6, i.e. a pH close to the physiological pH.

The object of the present invention is solved by the subject-matter of the independent claims. Preferred embodiments are apparent from the dependent claims. Accordingly, in one embodiment the present invention provides a pharmaceutical composition for use in the treatment of an intraocular neovascular disease comprising a) a histidine-containing buffer

b) a non-ionic surfactant

c) a VEGF antagonist

d) an inorganic salt, and

e) a carbohydrate,

wherein the pH of the composition is 6.3 or 6.6. The histidine-containing buffer may be present in a concentration of from 1 mM to 40 mM, preferably of 10 mM.

In one embodiment the pH of the composition is 6.3 and the pharmaceutical composition further comprises methionine. The methionine may be present in a concentration of from 1 mM to 40 mM, preferably of 10 mM.

In one embodiment the pH of the composition is 6.3 and the non-ionic surfactant is poloxamer 188. The poloxamer 188 may be present in a concentration of from 0.005 to 0.05% (w/v), preferably of 0.02% (w/v).

The present invention also relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease comprising

a) a dicarboxylic acid buffer,

b) a non-ionic surfactant,

c) a VEGF antagonist,

d) an inorganic salt, and

e) a carbohydrate.

The dicarboxylic acid buffer may be selected from malic acid buffer, succinate buffer and maleic acid buffer.

In one embodiment the dicarboxylic acid buffer is a malic acid buffer, and the pH of the composition is between 5.8 and 6.2, preferably 6.0. In one embodiment the dicarboxylic acid buffer is a succinate buffer, and the pH of the composition is between 5.8 and 6.2, preferably 6.0.

In one embodiment the dicarboxylic acid buffer is a maleic acid buffer, and the pH of the composition is between 6.0 and 6.4, preferably 6.2.

The dicarboxylic acid buffer may be present in a concentration of from 1 niM to 40 niM, preferably of 10 mM. The non-ionic surfactant may be polysorbate 20. The polysorbate 20 may be present in a concentration of from 0.01 to 0.08% (w/v), preferably of 0.03% (w/v).

The inorganic salt may be NaCl and/or may be present in a concentration of from 20 to 100 mM, preferably of 40 mM.

The VEGF antagonist may be an anti-VEGF antibody or an antigen-binding fragment of such antibody or a VEGF receptor fusion protein, preferably it may be aflibercept or ranibizumab. The VEGF antagonist may be present in a concentration of 6 to 45 mg/ml.

The carbohydrate may be sucrose and/or may be present in a concentration of 3-20% (w/v), preferably of 5% (w/v). In one embodiment the present invention relates to a liquid pharmaceutical

composition for use in the treatment of an intraocular neovascular disease consisting of histidine hydrochloride monohydrate/L-histidine, polysorbate 20, NaCl,

aflibercept, sucrose and water and having a pH of 6.3 or 6.6. Preferably, the liquid pharmaceutical composition consists of 10 mM histidine

hydrochloride monohydrate/L-histidine, 0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.3 or 6.6.

In another embodiment the present invention also relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease consisting of histidine hydrochloride monohydrate/L-histidine, polysorbate 20, NaCl, aflibercept, sucrose, methionine and water and having a pH of 6.3.

Preferably, the liquid pharmaceutical composition consists of 10 niM histidine hydrochloride monohydrate/L-histidine, 0.03% polysorbate 20 (w/v), 40 niM NaCl, 40 mg/ml aflibercept, 5% sucrose, 10 mM methionine and water and having a pH of 6.3.

In another embodiment the present invention also relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease consisting of histidine hydrochloride monohydrate/L-histidine, poloxamer 188, NaCl, aflibercept, sucrose and water and having a pH of 6.3.

Preferably, the liquid pharmaceutical composition consists of 10 mM histidine hydrochloride monohydrate/L-histidine, 0.02% poloxamer 188 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucrose, and water and having a pH of 6.3.

In one embodiment the present invention also relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease consisting of malic acid/malic acid disodium salt, polysorbate 20, NaCl, aflibercept, sucrose and water and having a pH of 6.0.

Preferably, the liquid pharmaceutical composition consists of 10 mM malic acid/malic acid disodium salt, 0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.0.

In another embodiment the present invention also relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease consisting of succinic acid/disodium succinate, polysorbate 20, NaCl, aflibercept, sucrose and water and having a pH of 6.0.

Preferably, the liquid pharmaceutical composition consists of 10 mM succinic acid/disodium succinate, 0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.0. In another embodiment the present invention also relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease consisting of maleic acid/sodium hydroxide, polysorbate 20, NaCl, aflibercept, sucrose and water and having a pH of 6.2.

Preferably, the liquid pharmaceutical composition consists of 10 mM maleic acid/sodium hydroxide (1 M), 0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.2. The present invention also relates to a liquid pharmaceutical composition consisting of 10 mM malic acid/malic acid disodium salt, 0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5% sucrose and water and has a pH of 6.0.

The present invention also relates to a liquid pharmaceutical composition consisting of 10 mM succinic acid/disodium succinate, 0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5% sucrose and water and has a pH of 6.0.

The present invention also relates to a liquid pharmaceutical composition consisting of 10 mM maleic acid/sodium hydroxide, 0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5% sucrose and water and has a pH of 6.2.

The present invention also relates to a liquid pharmaceutical composition consisting of 10 mM histidine hydrochloride monohydrate/L-histidine, 0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5% sucrose and water and having a pH of 6.3, or 6.6.

The present invention also relates to a liquid pharmaceutical composition consisting of 10 mM histidine hydrochloride monohydrate/L-histidine, 0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5% sucrose, 10 mM methionine and water and having a pH of 6.3.

The present invention also relates to a liquid pharmaceutical composition consisting of 10 mM histidine hydrochloride monohydrate/L-histidine, 0.02% poloxamer 188 (w/v), 40 mM NaCl, a recombinant protein, 5% sucrose, and water and having a pH of 6.3. The intraocular neovascular disease may be age-related macular degeneration

(AMD), visual impairment due to diabetic macular oedema (DME), visual

impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO), or visual impairment due to choroidal neovascularisation (CNV) secondary to pathologic myopia.

The present invention also relates to a prefilled syringe containing the pharmaceutical composition as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the invention will be described in the following with reference to the following drawings:

Figure 1: Detection of different aflibercept isoforms in samples incubated for one month at 40°C using isoelectric focussing.

Figure 2: Detection of different aflibercept isoforms in samples incubated for three months at 5°C using isoelectric focussing.

Figure 3: Detection of different aflibercept isoforms in samples incubated for three months at 25°C/60 RH using isoelectric focussing. Figure 4: Detection of different aflibercept isoforms in samples incubated for three months at 40°C/75 RH using isoelectric focussing.

DETAILED DESCRIPTION OF THE INVENTION The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments, but the invention is not limited thereto, but only by the claims. Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". If hereinafter e.g. a cell or organism is defined to be obtainable by a specific method, this is also to be understood to disclose a cell or organism which is obtained by this method.

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

The term "pharmaceutical composition" as used herein refers to any composition comprising a chemical substance or active ingredient which composition is intended for use in the medical cure, treatment, or prevention of disease and which is in such a form as to permit the active ingredient to be effective. In particular, a pharmaceutical composition does not contain excipients which are unacceptably toxic to a subject to which the composition is to be administered. The pharmaceutical compositions are sterile, i.e. aseptic and free from all living microorganisms and their spores. The pharmaceutical composition used in the present invention is liquid and stable. In a "liquid composition" the pharmaceutically active agent, e.g. the VEGF antagonist, can be combined with a variety of excipients to ensure a stable active medication following storage. The liquid pharmaceutical composition used in the invention is at no point lyophilised, i.e. the production method does not contain a lyophilisation step and the composition is not lyophilised for storage. Liquid compositions can be stored in vials, IV bags, ampoules, cartridges, and prefilled or ready-to-use syringes.

A "stable" liquid composition is one in which the VEGF antagonist contained therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage for a certain period. Preferably, the composition essentially retains upon storage its physical and chemical stability, as well as its biological activity. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed, Marcel Dekker, Inc, New York, New York, Pubs (1991) and Jones, Adv Drug Delivery Rev, 1993, 10:29-90. For example, stability can be measured at a selected temperature for a selected time period. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection), by assessing charge heterogeneity using cation exchange chromatography or capillary zone

electrophoresis, amino-terminal or carboxy-terminal sequence analysis, mass spectrometric analysis, SDS-PAGE analysis to detect aggregated or fragmented molecules, peptide map (for example tryptic or LYS-C) analysis, evaluating biological activity or binding of the antagonist, etc.

Preferably, the pharmaceutical composition is stable at a temperature of about 40°C for at least 1 to 2 weeks, and/or is stable at a temperature of about 5°C for at least 3 or 5 months, and/or is stable at a temperature of about 25°C for at least two weeks or one month.

Furthermore, the formulation is preferably stable following freezing (to, e.g., -20°C) and thawing of the formulation at 25 ^Q C as described in the examples herein, for example following 1, 2, 3, 4 or 5 cycles of freezing and thawing.

For example, in the pharmaceutical composition used in the present invention the percentage of high molecular weight species relative to the total amount of the VEGF antagonist as measured by size exclusion chromatography is not more than 5%, preferably not more than 4.5% or 4%, more preferably not more than 3.8% or 3.5% and most preferably not more than 3% or 2.8% after storage of the composition at 5°C for 1 month.

In the pharmaceutical composition used in the present invention the percentage of high molecular weight species relative to the total amount of the VEGF antagonist as measured by size exclusion chromatography is not more than 5%, preferably not more than 4.5% or 4%, more preferably not more than 3.8% or 3.5% and most preferably not more than 3% or 2.8% after storage of the composition at 5°C for 5 months.

In the pharmaceutical composition used in the present invention the percentage of high molecular weight species relative to the total amount of the VEGF antagonist as measured by size exclusion chromatography is not more than 8%, preferably not more than 7.5% or 7%, more preferably not more than 6.5% or 6% and most preferably not more than 5% after storage of the composition at 25°C for 5 months. A "buffer" is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or vice versa which resists changes in its pH and therefore keeps the pH at a nearly constant value. The buffer of the present invention preferably has a pH in the range from about 5.5 to about 7.0, preferably from about 5.8 to about 6.8, more preferably from about 5.9 to 6.7 and most preferably has a pH of about 6.0 to 6.6, such as a pH of 6.0, 6.2, 6.3 or 6.6.

The buffer used in the present invention is a dicarboxylic acid buffer A dicarboxylic acid is an organic acid having two carboxyl groups (-COOH). The term includes linear saturated dicarboxylic acids having the genereal formula HC C-CC fe CC H such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid and sebacic acid, preferably succinic acid. It also includes unsaturated dicarboxylic acids having at least one double bond such as maleic acid and fumaric acid as well as substituted dicarboxylic acids having at least one additional functional group such as malic acid, tartaric acid, cichoric acid and dimercaptosuccinic acid.

Preferably, the dicarboxylic acid is selected from succinic acid, malic acid and maleic acid. The malic acid buffer may comprise malic acid and malic acid disodium salt. The succinic acid buffer may comprise succinic acid and disodium succinate. The maleic acid buffer may comprise maleic acid and sodium hydroxide. The malic acid buffer has a pH of 5.8 to 6.2, preferably of 5.9 to 6.1 and most preferably of 6.0. The succinic acid buffer has a pH of 5.8 to 6.2, preferably of 5.9 to 6.1 and most preferably of 6.0. The maleic acid buffer has a pH of 6.0 to 6.4, preferably of 6.1 to 6.3 and most preferably of 6.2.

The terms "histidine-containing buffer" and "histidine buffer" are used interchangeably herein and refer to a buffer comprising histidine. Examples of histidine buffers include histidine chloride, histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulphate. The preferred histidine buffer of the invention further comprises L- histidine. Even more preferably the histidine buffer of the invention comprises histidine hydrochloride, most preferably it comprises histidine hydrochloride and L -histidine.

Preferably, the histidine buffer or histidine hydrochloride buffer or histidine hydrochloride/L-histidine buffer has a pH in the range from about 6.0 to about 7.0, preferably from about 6.1 to about 6.8, more preferably from about 6.0 to 6.5, even more preferably from about 6.2 to 6.5 and most preferably has a pH of about 6.2 or 6.5. In one embodiment, the malic acid buffer has a concentration in the range of 1 mM to 40 mM, preferably of 2 mM to 35 mM, more preferably of 3 mM to 30 mM, even more preferably of 5 mM to 20 mM and most preferably of 8 mM to 15 mM.

In another particular preferred embodiment the buffer is malic acid/ malic acid disodium salt with a concentration of 10 mM.

In another particular preferred embodiment the buffer is malic acid/malic acid disodium salt with a concentration of 10 mM and with a pH of 6.0. In one embodiment, the succinate buffer has a concentration in the range of 1 mM to 40 mM, preferably of 2 mM to 35 mM, more preferably of 3 mM to 30 mM, even more preferably of 5 mM to 20 mM and most preferably of 8 mM to 15 mM.

In another particular preferred embodiment the buffer is succinic acid/disodium succinate with a concentration of 10 mM.

In another particular preferred embodiment the buffer is succinic acid/disodium succinate with a concentration of 10 mM and with a pH of 6.0. In a particular preferred embodiment, the maleic acid buffer has a concentration in the range of 1 mM to 40 mM, preferably of 2 mM to 35 mM, more preferably of 3 mM to 30 mM, even more preferably of 5 mM to 20 mM and most preferably of 8 mM to 15 mM.

In another particular preferred embodiment the buffer is maleic acid/sodium hydroxide with a concentration of 10 mM.

In another particular preferred embodiment the buffer is maleic acid/sodium hydroxide with a concentration of 10 mM and with a pH of 6.2. In a particular preferred embodiment, the histidine buffer has a concentration in the range of 1 niM to 40 niM, preferably of 2 mM to 35 niM, more preferably of 3 mM to 30 niM, even more preferably of 5 mM to 20 mM and most preferably of 8 mM to 15 mM. In another particular preferred embodiment the buffer is histidine hydrochloride/L-histidine with a concentration of 10 mM.

In another particular preferred embodiment the buffer is histidine hydrochloride/L-histidine with a concentration of 10 mM and with a pH of 6.3 or 6.6.

The pharmaceutical compositions of the present invention may be prepared by dissolving L- histidine, histidine hydrochloride, the carbohydrate, preferably sucrose, and the inorganic salt, preferably sodium chloride, in water before adding the non-ionic surfactant, preferably polysorbate 20 or poloxamer 188 and then adding the VEGF antagonist.

The pharmaceutical compositions of the present invention may be prepared by dissolving malic acid, malic acid disodium salt, the carbohydrate, preferably sucrose, and the inorganic salt, preferably sodium chloride, in water before adding the non-ionic surfactant, preferably polysorbate 20 and then adding the VEGF antagonist.

The pharmaceutical compositions of the present invention may be prepared by dissolving succinic acid, disodium succinate, the carbohydrate, preferably sucrose, and the inorganic salt, preferably sodium chloride, in water before adding the non-ionic surfactant, preferably polysorbate 20 and then adding the VEGF antagonist.

The pharmaceutical compositions of the present invention may be prepared by dissolving , or maleic acid, sodium hydroxide, the carbohydrate, preferably sucrose, and the inorganic salt, preferably sodium chloride, in water before adding the non-ionic surfactant, preferably polysorbate 20 and then adding the VEGF antagonist.

A "surfactant" as used herein refers to an amphiphilic compound, i.e. a compound containing both hydrophobic groups and hydrophilic groups which lowers the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. A "non-ionic surfactant" has no charged groups in its head. The formation of insoluble particles during freeze/thaw cycles of antibody-containing compositions can be remarkably inhibited by addition of surfactants. Examples of "non-ionic surfactants" include e.g. polyoxyethylene glycol alkyl ethers, such as octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether; polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers, such as decyl glucoside, lauryl glucoside, octyl glucoside; polyoxyethylene glycol octylphenol ethers, such as triton X-100; polyoxyethylene glycol alkylphenol ethers, such as nonoxynol- 9; glycerol alkyl esters, such as glyceryl laurate; polyoxyethylene glycol sorbitan alkyl esters, such as polysorbate; sorbitan alkyl esters, such as spans; cocamide MEA, cocamide DEA, dodecyldimethylamine oxide; block copolymers of polyethylene glycol and polypropylene glycol, such as poloxamer s; and polyethoxylated tallow amine (POEA). The pharmaceutical compositions of the present invention can contain one or more of these surfactants in combination.

Preferred non-ionic surfactants for use in the pharmaceutical compositions of the present invention are polysorbates such as polysorbate 20, 40, 60 or 80 or poloxamer 188, and especially polysorbate 20 (i.e. Tween 20) or poloxamer 188.

The concentration of the non-ionic surfactant is in the range of 0.01 to 0.08% (w/v), preferably in the range of 0.015 to 0.06% (w/v), more preferably in the range of 0.02 to 0.04% (w/v) and most preferably it is 0.03% (w/v) or 0.02% (w/v), relative to the total volume of the composition.

In a preferred embodiment, the non-ionic surfactant is polysorbate 20 with a concentration in the range of 0.015 to 0.06% (w/v), more preferably in the range of 0.02 to 0.04% (w/v) and most preferably it is 0.03% (w/v), relative to the total volume of the composition.

In another preferred embodiment, the non-ionic surfactant is poloxamer 188 with a concentration in the range of 0.01 to 0.06% (w/v), more preferably in the range of 0.015 to 0.03% (w/v) and most preferably it is 0.02% (w/v), relative to the total volume of the composition.

In a particularly preferred embodiment the non-ionic surfactant is polysorbate 20 with a concentration of 0.03% (w/v), relative to the total volume of the composition.

In another particularly preferred embodiment the non-ionic surfactant is poloxamer 188 with a concentration of 0.02% (w/v), relative to the total volume of the composition Herein, an "inorganic salt" refers to a ionic compound which has osmoregulatory properties. An inorganic salt such as sodium chloride (NaCl) can dissociate in solution into its constituent ions, i.e. NaCl dissociates into Na ⁺ and CI ^" ions, which both affect the osmotic pressure, i.e. the osmolality, of the solution. Preferred inorganic salts for use in the pharmaceutical formulation of the present invention are potassium chloride, calcium chloride, sodium chloride, sodium phosphate, potassium phosphate and sodium bicarbonate. Preferably the inorganic salt is a sodium salt, more preferably it is sodium chloride (NaCl). The concentration of the inorganic salt in the pharmaceutical composition used in the present invention is preferably in the range of 20 to 100 mM, more preferably in the range of 25 to 80 mM, even more preferably the inorganic salt has a concentration in the range of 30 to 60 mM or 35 to 45 mM, and most preferably the concentration is 40 mM. In a particular preferred embodiment, the inorganic salt is NaCl with a concentration in the range of 20 to 100 mM, more preferably in the range of 25 to 80 mM, even more preferably the inorganic salt has a concentration in the range of 30 to 60 mM or 35 to 45 mM, and most preferably the concentration is 40 mM. In a most preferred embodiment the inorganic salt is NaCl with a concentration of 40 mM.

In a further embodiment the pharmaceutical composition comprises an inorganic salt, preferably NaCl, preferably in a concentration of 40 mM, polysorbate 20 in a concentration of 0.03% (w/v), sucrose in a concentration of 5% (w/v) and a malic acid/malic acid disodium salt buffer with a concentration of 10 mM and a pH of 6.0.

In a further embodiment the pharmaceutical composition comprises an inorganic salt, preferably NaCl, preferably in a concentration of 40 mM, polysorbate 20 in a concentration of 0.03% (w/v), sucrose in a concentration of 5% (w/v) and a succinic acid/disodium succinate buffer with a concentration of 10 mM and a pH of 6.0.

In a further embodiment the pharmaceutical composition comprises an inorganic salt, preferably NaCl, preferably in a concentration of 40 mM, polysorbate 20 in a concentration of 0.03% (w/v), sucrose in a concentration of 5% (w/v) and a maleic acid/sodium hydroxide buffer with a concentration of 10 mM and a pH of 6.2. In a further embodiment the pharmaceutical composition comprises an inorganic salt, preferably NaCl, preferably in a concentration of 40 mM, polysorbate 20 in a concentration of 0.03% (w/v), sucrose in a concentration of 5% (w/v) and a histidine hydrochloride/L- histidine buffer with a concentration of 10 mM and a pH of 6.3, or a histidine

hydrochloride/L-histidine buffer with concentration of 10 mM and a pH of 6.6,.

In a further embodiment the pharmaceutical composition comprises an inorganic salt, preferably NaCl, preferably in a concentration of 40 mM, poloxamer 188 in a concentration of 0.02% (w/v), sucrose in a concentration of 5% (w/v) and a histidine hydrochloride /L- histidine buffer with a concentration of 10 mM and a pH of 6.3.

In a further embodiment the pharmaceutical composition comprises an inorganic salt, preferably NaCl, preferably in a concentration of 40 mM, polysorbate 20 in a concentration of 0.03% (w/v), sucrose in a concentration of 5% (w/v), methionine in a concentration of 10 mM and a histidine hydrochloride monohydrate/L-histidine buffer with a concentration of 10 mM and a pH of 6.3.

The term "VEGF antagonist" refers to a molecule which specifically interacts with VEGF and inhibits one or more of its biological activities, e.g. its mitogenic, angiogenic and/or vascular permeability activity. It is intended to include both anti-VEGF antibodies and antigen-binding fragments thereof and non-antibody VEGF antagonists.

Non-antibody VEGF antagonists include aflibercept, pegaptanib and antibody mimetics. Preferably, the non-antibody VEGF antagonist is aflibercept. Aflibercept which is presently marketed under the name Eylea ^® and which is also known as VEGF-trap is a recombinant human soluble VEGF receptor fusion protein in which the second immunoglobulin-like domain of VEGF receptor 1 and the third immunoglobulin-like domain of VEGF receptor 2 are fused to the Fc portion of human IgGl (Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99(17): 11393-11398; WO 00/75319 Al). The CAS number of aflibercept is 862111-32-8. It has received a marketing authorization for the treatment of wet age-related macular degeneration, visual impairment due to diabetic macular oedema (DME) and diabetic retinopathy in patients with diabetic macular edema. The present commercial aflibercept formulation contains sodium phosphate, sodium chloride, polysorbate 20, sucrose and water for injection and is supplied in a concentration of 40 mg/ml. Pegaptanib which is presently marketed under the name Macugen " is a pegylated anti- vascular endothelial growth factor (VEGF) aptamer (Bell et al. (1999) In Vitro Cell Dev Biol Anim. 35(9): 533-42). Antibody mimetics which are VEGF antagonists include binding proteins comprising an ankyrin repeat domain that binds VEGF and inhibits its binding to the receptor, such as DARPin ^® MP0112 (see also WO 2010/060748 and WO 2011/135067).

The term„antibody" or "immunoglobulin" is used herein in the broadest sense and includes full length antibodies, genetically engineered antibodies, recombinant antibodies, multivalent antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, as well as fragments of such antibodies as long as they remain functional and exhibit the desired biological activity. The "Biological activity" of an antibody refers to the ability of the antibody to bind to antigen and result in a biological response which can be measured in vitro or in vivo.

A full length antibody comprises an antigen -binding variable region of the light (V _L ) and heavy chain (VH), a light chain constant region (CL) and heavy chain constant domains CHI , C _H 2 and C _H 3.

The term "antibody fragment" or "antigen-binding fragment" is used herein in the broadest sense and comprises a portion of a full length antibody, preferably comprising the antigen- binding or variable region thereof. An antibody fragment retains the original specificity of the parent immunoglobulin. Examples of antibody fragments include, e.g., Fab, Fab', F(ab') ₂ , and Fv fragments, diabodies, linear antibodies, single -chain antibody molecules, and multispecific antibodies formed from antibody fragment(s). Preferably, the antibody fragment is a Fab fragment.

A "monoclonal antibody" is an antibody that is specific for a single epitope of an antigen, i.e. directed against a single determinant on an antigen. Methods for producing monoclonal antibodies are known to the person skilled in the art.

The term "recombinant antibody" refers to all antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a transgenic host cell, such as e.g. a NS0 or CHO cell, or from an animal transgenic for immunoglobulin genes, or antibodies expressed using recombinant expression vectors transfected into a host cell, such as e.g. SP 2/0 mouse myeloma cells.

A "humanised antibody" is a human antibody wherein the antigen binding portion (CDR) derived from non-human species, such as a mouse, and thus has a different specificity compared to the parent immunoglobulin. The CDR protein sequences can be modified to increase their similarities to antibody variants produced naturally in humans.

The term "anti-VEGF antibody" refers to an antibody or antibody fragment such as a Fab or a scFV fragment that specifically binds to VEGF and inhibits one or more of its biological activities, e.g. its mitogenic, angiogenic and/or vascular permeability activity. Anti-VEGF antibodies act, e.g., by interfering with the binding of VEGF to a cellular receptor, by interfering with vascular endothelial cell activation after VEGF binding to a cellular receptor, or by killing cells activated by VEGF. Anti-VEGF antibodies include, e.g., antibodies A4.6.1, bevacizumab, ranibizumab, G6, B20, 2C3, and others as described in, for example, WO 98/45331 , US 2003/0190317, US 6,582,959, US 6,703,020, WO 98/45332, WO 96/30046, WO 94/10202, WO 2005/044853, EP 0 666 868 Bl, WO 2009/155724 and Popkov et al. (2004) J. Immunol. Meth. 288: 149-64. Preferably, the anti-VEGF antibody or antigen-binding fragment thereof present in the pharmaceutical composition used in the present invention is ranibizumab or bevacizumab. Most preferably, it is ranibizumab or an antigen-binding fragment thereof.

"Ranibizumab" is a humanised monoclonal Fab fragment directed against VEGF -A having the light and heavy chain variable domain sequences of Y0317 as described in SEQ ID Nos. 115 and 116 of WO 98/45331 and Chen et al. (1999) J. Mol. Biol. 293: 865-81. The CAS number of ranibizumab is 347396-82-1. Ranibizumab inhibits endothelial cell proliferation and neovascularisation and has been approved for the treatment of neovascular (wet) age- related macular degeneration (AMD), the treatment of visual impairment due to diabetic macular oedema (DME), the treatment of visual impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO), or treatment of visual impairment due to choroidal neovascularisation (CNV) secondary to pathologic myopia. Ranibizumab is related to bevacizumab and derived from the same parent mouse antibody as bevacizumab but it is much smaller than the parent molecule and has been affinity matured to provide stronger binding to VEGF- A. Ranibizumab is produced recombinantly in Escherichia coli, e.g. as described in WO 98/45331 A2. The present commercial ranibizumab formulation contains α,α-trehalose dihydrate, histidine hydrochloride monohydrate, histidine, polysorbate 20 and water for injection and is supplied in a concentration of 10 mg/ml. "Bevacizumab" is a full-length, humanized murine monoclonal antibody that recognizes all isoforms of VEGF and which is the parent antibody of ranibizumab. The CAS number of bevacizumab is 216974-75-3. Bevacizumab inhibits angiogenesis and is presently approved for the treatment of different cancer types. However, it is also used off-label in

ophthalmological diseases such as age-related macular degeneration. The present commercial bevacizumab formulation contains α,α-trehalose dihydrate, sodium phosphate, polysorbate 20 and water for injection and is supplied as a concentrate with a concentration of 25 mg/ml.

The concentration of the VEGF antagonist in the pharmaceutical compositions is typically 5- 80 mg/ml, preferably 7-60 mg/ml, more preferably 8-50 mg/ml and most preferably 10 or 40 mg/ml. If the VEGF antagonist is aflibercept, the concentration of the VEGF antagonist, i.e. aflibercept, is preferably 40 mg/ml. If the VEGF antagonist is ranibizumab, the concentration of the VEGF antagonist, i.e. ranibizumab, is preferably 6 or 10 mg/ml. The term "carbohydrate" refers to an organic compound comprising only carbon, hydrogen, and oxygen, usually with a hydrogemoxygen atom ratio of 2:1 and the empirical formula Cm(H20)n. The term "carbohydrate" includes mono-, di-, oligo- and polysaccharides.

Examples of carbohydrates include glucose, fructose, galactose, xylose, ribose, sucrose, mannose, lactose, maltose, trehalose, starch, and glycogen. Various other forms of sugars are known, e.g., sugar alcohols such as glycerol, mannitol, sorbitol, and xylitol; sugar acids, e.g. aldonic acids such as ascorbic acid, aldaric acids such as tartaric acid; reducing sugars, e.g. glucose, glycer aldehydes, galactose, lactose, and maltose; amino sugars, e.g. N- acetylglucosamine, galactosamine, glucosamine, and sialic acid; or sulfoquinovose, a sulphonic acid derivative of glucose.

The pharmaceutical composition used in the present invention may further contain diluents, solubilising agents, isotonising agents, excipients, pH-modifiers, soothing agents, buffers, sulphur-containing reducing agents, antioxidants or the like. The pharmaceutical composition used in the present invention does not contain PEG3350 and/or glycine. In one embodiment, the pharmaceutical composition used in the present invention contains histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, water and aflibercept and no further components or active substances, i.e. the pharmaceutical composition consists of histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, water and aflibercept. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 40 mg/ml aflibercept.

In one embodiment, the pharmaceutical composition used in the present invention contains histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, water and ranibizumab and no further components or active substances, i.e. the pharmaceutical composition consists of histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, water and

ranibizumab. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 10 mg/ml ranibizumab.

In one embodiment, the pharmaceutical composition used in the present invention contains histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, methionine, water and aflibercept and no further components or active substances, i.e. the pharmaceutical composition consists of histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, methionine, water and aflibercept. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, lOmM methionine, water and 40 mg/ml aflibercept.

In one embodiment, the pharmaceutical composition used in the present invention contains histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, methionine, water and ranibizumab and no further components or active substances, i.e. the pharmaceutical composition consists of histidine hydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, methionine, water and ranibizumab. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, 10 mM methionine, water and 10 mg/ml ranibizumab. In one embodiment, the pharmaceutical composition used in the present invention contains histidine hydrochloride/L-histidine, poloxamer 188, NaCl, sucrose, water and aflibercept and no further components or active substances, i.e. the pharmaceutical composition consists of histidine hydrochloride/L-histidine, poloxamer 188, NaCl, sucrose, water and aflibercept. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 niM NaCl, 5% (w/v) sucrose, water and 40 mg/ml aflibercept.

In one embodiment, the pharmaceutical composition used in the present invention contains histidine hydrochloride/L-histidine, poloxamer 188, NaCl, sucrose, water and ranibizumab and no further components or active substances, i.e. the pharmaceutical composition consists of histidine hydrochloride/L-histidine, poloxamer 188, NaCl, sucrose, water and ranibizumab. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 niM NaCl, 5% (w/v) sucrose, water and 10 mg/ml ranibizumab.

In one embodiment, the pharmaceutical composition used in the present invention contains malic acid/malic acid disodium, polysorbate 20, NaCl, sucrose, water and aflibercept and no further components or active substances, i.e. the pharmaceutical composition consists of malic acid/malic acid disodium, polysorbate 20, NaCl, sucrose, water and aflibercept. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM malic acid/malic acid disodium, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 40 mg/ml aflibercept. In one embodiment, the pharmaceutical composition used in the present invention contains malic acid/malic acid disodium, polysorbate 20, NaCl, sucrose, water and ranibizumab and no further components or active substances, i.e. the pharmaceutical composition consists of malic acid/malic acid disodium, polysorbate 20, NaCl, sucrose, water and ranibizumab. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM malic acid/malic acid disodium, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 10 mg/ml ranibizumab.

In one embodiment, the pharmaceutical composition used in the present invention contains succinic acid/disodium succinate, polysorbate 20, NaCl, sucrose, water and aflibercept and no further components or active substances, i.e. the pharmaceutical composition consists of succinic acid/disodium succinate, polysorbate 20, NaCl, sucrose, water and aflibercept. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 40 mg/ml aflibercept.

In one embodiment, the pharmaceutical composition used in the present invention contains succinic acid/disodium succinate, polysorbate 20, NaCl, sucrose, water and ranibizumab and no further components or active substances, i.e. the pharmaceutical composition consists of succinic acid/disodium succinate, polysorbate 20, NaCl, sucrose, water and ranibizumab. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 10 mg/ml ranibizumab.

In one embodiment, the pharmaceutical composition used in the present invention contains maleic acid/sodium hydroxide, polysorbate 20, NaCl, sucrose, water and aflibercept and no further components or active substances, i.e. the pharmaceutical composition consists of maleic acid/sodium hydroxide, polysorbate 20, NaCl, sucrose, water and aflibercept. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM maleic acid/32 niM sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 40 mg/ml aflibercept.

In one embodiment, the pharmaceutical composition used in the present invention contains maleic acid/sodium hydroxide, polysorbate 20, NaCl, sucrose, water and ranibizumab and no further components or active substances, i.e. the pharmaceutical composition consists of maleic acid/sodium hydroxide, polysorbate 20, NaCl, sucrose, water and ranibizumab. More preferably, the pharmaceutical composition used in the present invention consists of 10 niM maleic acid/32 niM sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, water and 10 mg/ml ranibizumab. An "intraocular neovascular disease" is a disease characterized by ocular neovascularisation. Examples of intraocular neovascular diseases include, e.g., proliferative retinopathies, choroidal neovascularisation (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathies, diabetic macular oedema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion (BRVO), corneal neovascularisation, and retinal neovascularisation. The term "age-related macular degeneration" refers to a medical condition which usually affects older adults and results in a loss of vision in the centre of the visual field (the macula) because of damage to the retina. If the VEGF antagonist present in the pharmaceutical composition used in the present invention is aflibercept, the pharmaceutical composition is preferably for use in the treatment of neo vascular (wet) age-related macular degeneration (AMD), visual impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO), visual impairment due to diabetic macular oedema (DME) or visual impairment due to myopic choroidal neovascularisation (myopic CNV).

If the VEGF antagonist present in the pharmaceutical composition used in the present invention is ranibizumab, the pharmaceutical composition is preferably for use in the treatment of neo vascular (wet) age-related macular degeneration (AMD), of visual impairment due to diabetic macular edema (DME), of visual impairment due to macular edema secondary to retinal vein occlusion (branch RVO or central RVO) or of visual impairment due to choroidal neovascularisation (CNV) secondary to pathologic myopia (PM). The term "intravitreal injection" refers to the administration of a pharmaceutical composition in which the substance is injected directly into the eye. More specifically, the substance is injected into the vitreous humour (also called vitreous body or simply vitreous) which is the clear gel that fills the space between the lens and the retina of the eyeball of humans and other vertebrates.

Pharmaceutical compositions of the present invention can be supplied in sealed and sterilized plastic, glass or other suitable containers having a defined volume such as vials, ampoules or syringes or a large volume such as bottles. It is preferred that the liquid pharmaceutical composition containing a VEGF antagonist, preferably aflibercept or ranibizumab, is supplied in a prefilled syringe. A "ready-to-use syringe" or "prefilled syringe" is a syringe which is supplied in a filled state, i.e. the pharmaceutical composition to be administered is already present in the syringe and ready for administration. Prefilled syringes have many benefits compared to separately provided syringe and vial, such as improved convenience, affordability, accuracy, sterility, and safety. The use of prefilled syringes results in greater dose precision, in a reduction of the potential for needle sticks injuries that can occur while drawing medication from vials, in pre- measured dosage reducing dosing errors due to the need to reconstituting and/or drawing medication into a syringe, and in less overfilling of the syringe helping to reduce costs by minimising drug waste.

In a preferred embodiment the pH of the liquid pharmaceutical composition used in the present invention is in the range from about 5.5 to about 7.0, preferably from about 5.8 to about 6.9, more preferably from about 5.9 to 6.8, even more preferably from about 6.0 to 6.6.

The liquid pharmaceutical composition used in the present invention is to be used in the treatment of an intraocular neovascular disease such as age-related macular degeneration (AMD), in the treatment of visual impairment due to diabetic macular oedema (DME), in the treatment of visual impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO), or in the treatment of visual impairment due to choroidal neovascularisation (CNV) secondary to pathologic myopia.

In particular, the invention relates to a liquid pharmaceutical composition for use in the treatment of an intraocular neovascular disease such as AMD comprising a histidine- containing or dicarboxylic acid buffer, a non-ionic surfactant, an inorganic salt, a carbohydrate and a VEGF antagonist.

In one embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises a histidine-containing or dicarboxylic acid buffer, a non-ionic surfactant, an inorganic salt, a carbohydrate and a VEGF antagonist.

In a preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises a histidine-containing or dicarboxylic acid buffer in a concentration of 1 mM to 40 mM, a non-ionic surfactant in a concentration of 0.01 to 0.08% (w/v), an inorganic salt in a concentration of 20 to 100 mM, a carbohydrate in a concentration of 3 to 20% (w/v) and a VEGF antagonist. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of ImM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of ImM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v), methionine in a concentration of ImM to 40 mM and aflibercept and has a pH of 6.3. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of ImM to 40 mM, poloxamer 188 in a concentration of 0.005 to 0.05% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept and has a pH of 6.3.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of 10 mM, polysorbate 20 in a concentration of 0.03% (w/v), NaCl in a concentration of 40 mM, sucrose in a concentration of 5% (w/v), methionine in a concentration of 10 mM and aflibercept and has a pH of 6.3.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of 10 mM, polpxamer 188 in a concentration of 0.02% (w/v), NaCl in a concentration of 40 mM, sucrose in a concentration of 5% (w/v), and aflibercept and has a pH of 6.3.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises malic acid/malic acid disodium salt in a concentration of 1 mM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and aflibercept. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.0. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises malic acid/malic acid disodium salt of 1 mM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM malic acid/malic acid disodium salt, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.0.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises succinic acid/disodium succinate in a concentration of 1 mM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.0.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises succinic acid/disodium succinate in a concentration of 1 niM to 40 niM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 niM, sucrose in a concentration of 3 to 20% (w/v) and ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose and ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 niM succinic acid/disodium succinate, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.0.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises maleic acid/sodium hydroxide in a concentration of 1 niM to 40 niM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.2.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises maleic acid/sodium hydroxide in a concentration of 1 mM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM maleic acid/sodium hydroxide, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.2.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of 1 mM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and aflibercept and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of lmM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and ranibizumab and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and ranibizumab and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.3 or 6.6.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.3 or 6.6. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of 1 niM to 40 niM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 niM, sucrose in a concentration of 3 to 20% (w/v), methionine in a concentration of 1 niM to 40 niM and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, 10 niM methionine and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, 10 niM methionine, and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 niM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, 10 niM methionine, and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 niM histidine hydrochloride monohydrate/L-histidine, 0.03% (w/v) polysorbate 20, 40 niM NaCl, 5% (w/v) sucrose, 10 mM methionine, and 40 mg/ml aflibercept and has a pH of 6.3.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of lmM to 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v), methionine in a concentration of lmM to 40 mM and ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose, 10 mM methionine and ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose, 10 mM methionine, and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose, 10 mM methionine, and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose, 10 mM methionine, and 6 or 10 mg/ml ranibizumab and has a pH of 6.3.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of 1 mM to 40 mM, poloxamer 188 in a concentration of 0.005 to 0.05% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.3.

In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises histidine hydrochloride/L-histidine in a concentration of lmM to 40 mM, poloxamer 188 in a concentration of 0.005 to 0.05% (w/v), NaCl in a concentration of 20 to 100 mM, sucrose in a concentration of 3 to 20% (w/v) and ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease such as AMD comprises 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease comprises 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 mM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab. In another preferred embodiment of the invention the liquid pharmaceutical composition for intravitreal administration for use in the treatment of an intraocular neovascular disease consists of 10 niM histidine hydrochloride/L-histidine, 0.02% (w/v) poloxamer 188, 40 mM NaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.3.

Moreover, the invention encompasses the intravitreal administration of the liquid pharmaceutical composition of the invention to a subject in an effective amount to treat an intraocular neovascular disease such as AMD. In a preferred embodiment, the liquid pharmaceutical composition of the invention for intravitreal administration is present in a prefilled syringe.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims.

The detailed description is merely exemplary in nature and is not intended to limit application and uses. The following examples further illustrate the present invention without, however, limiting the scope of the invention thereto. Various changes and modifications can be made by those skilled in the art on the basis of the description of the invention, and such changes and modifications are also included in the present invention. EXAMPLES

1. Sample preparation

Aflibercept from the EU marketed product Zaltrap ^® was transferred by 3-step-dialysis into 9 different formulations containing

(a) 10 mM sodium dihydrogen phosphate/ disodium hydrogen phosphate, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 % (w/v) polysorbate 20, pH 6.2

(b) 10 mM histidine/ histidine chloride, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 % (w/v) polysorbate 20, pH 6.2 (c) 10 mM histidine/ histidine chloride, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 %

(w/v) polysorbate 20, pH 6.3

d) 10 mM histidine/ histidine chloride, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 % (w/v) polysorbate 20, pH 6.6

e) 10 mM histidine/ histidine chloride, 40 mM sodium chloride, 5 % (w/v) sucrose, 10 mM Methionine, 0.03 % (w/v) polysorbate 20, pH 6.3

f) 10 mM histidine/ histidine chloride, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.02 (w/v) Poloxamer 188, pH 6.3

g) 10 mM malic acid/ malic acid disodium salt, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 % (w/v) polysorbate 20, pH 6.0

h) 10 mM succinic acid/ di-sodium succinate, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 % (w/v) polysorbate 20, pH 6.0

i) 10 mM maleic acid/ 32 mM sodium hydroxide, 40 mM sodium chloride, 5 % (w/v) sucrose, 0.03 % (w/v) polysorbate 20, pH 6.2 as shown in Table 1.

Dialyzed aflibercept was adjusted to 40 mg/niL ± 10 % and stored at 5 °C or 25° CI 60 % relative humidity for up to 1, 2, 3 and 5 months and at 40 °C/ 75 % relative humidity for 1 and 3 months. Additionally, aflibercept in the different formulations was stressed by three or five freeze/ thaw cycles. Afibercept in the Eylea ^® formulation buffer system was included in the stability program as control sample (a).

Table 1 : Detailed information of all formulations used in this study

No. Aflibercept Buffer sucrose Sodium Other PolysorpH system chloride excipient bate 20

(a) 40 mg/niL 10 mM 5 % 40 mM - 0.03 % pH 6.2 sodium (w/v) (w/v) dihydro- gen

phosphate/

disodium

hydrogen

phosphate

(b) 40 mg/niL 10 mM L- 5 % 40 mM - 0.03 % pH 6.2 histidine/ (w/v) (w/v) histidine/

HC1

(c) 40 mg/mL lO mM L- 5 % 40 mM 0.03 % pH 6.3 histidine/ (w/v) (w/v) histidine/

HC1

(d) 40 mg/mL lO mM L- 5 % 40 mM 0.03 % pH 6.6 histidine/ (w/v) (w/v) histidine/

HC1

(e) 40 mg/mL lO mM L- 5 % 40 mM 10 mM 0.03 % pH 6.3 histidine/ (w/v) methio(w/v) histidine/ nine

HC1

(f> 40 mg/mL lO mM L- 5 % 40 mM 0.02 (w/v) pH 6.3 histidine/ (w/v) poloxa- histidine/ mer 188

HC1

(g) 40 mg/mL lO mM 5 % 40 mM 0.03 % pH 6.0 malic (w/v) (w/v) acid/

malic acid

disodium

salt

(h) 40 mg/mL 10 mM 5 % 40 mM 0.03 % pH 6.0 succinic (w/v) (w/v) acid/ disodium

succinate

(i) 40 mg/mL 10 mM 5 % 40 mM 0.03 % pH 6.2 maleic (w/v) (w/v) acid/

sodium

hydroxide Afterwards the samples according to Table 1 were analyzed by UV-Vis spectroscopy at 280 nm for protein concentration, by size exclusion chromatography (SEC) for the presence of high molecular weight species (HMWS) and by reduced-/ non-reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the presence of fragments and HMWS. Chemical modifications like methionine oxidation and deamidation were quantified by reduced peptide mapping. Isoelectric focusing was used to detect modifications leading to charge heterogeneities include e.g. glycosylation, C-terminal Lysine variations and deamidation of the protein. The activity of aflibercept was determined by Potency ELISA.

2. Analysis of protein content by UV-Vis

Table 2: Protein concentrations of all formulation determined via UV280 method

Concentration

Condition Formulation

[mg/niL]

(a) 39.7

(b) 40

(c) 39.3

(d) 40.2

TO (e) 39.1

(f) 39.7

(g) 40.2

(h) 40.2

(i) 40.7

(a) 40.3

(b) 40.7

(c) 39.7

(d) 41

1M 5°C (e) 40.2

(f) 40.1

(g) 41.1

(h) 39.9

(i) 40.7 Concentration

Condition Formulation

[mg/niL]

(a) 39.1

(b) 39.9

(c) 39.1

(d) 38.8

2M 5°C (e) 39.3

(f) 39.4

(g) 39.6

(h) 38.4

(i) 39.6

(a) 39.2

(b) 40.1

(c) 36.5

(d) 37.3

3M 5°C (e) 37.5

(f) 39.2

(g) 37.8

(h) 38.7

(i) 40.2

(a) 39.2

(b) 40.1

(c) 39.8

(d) 40.3

5M 5°C (e) 39.2

(f) 39.3

(g) 39.6

(h) 39.4

(i) 39.6

There was no significant change in protein concentration (spectrophotometric quantification at 280nm; n =3) and appearance (visible particles, change in color) detectable in all samples. 3. Analysis of high molecular weight species (HMWS) by SEC

The protein samples of the stability study were loaded onto a TSKgel G3000SWXL, (Tosoh, 300 x 7.8 mm, 5 μιη) column to detect high molecular weight species of aflibercept. The protein was eluted by isocratic elution using 0.02 M sodium phosphate (pH 6.0) and 0.8 M sodium chloride at a flow rate of 1.0 mL/min at 25 °C. Eluted species were detected at a wavelength of 214 nm and displayed on a graph showing the concentration of the eluted species vs. time. The elution profile showed a main peak with the non-aggregated protein and some further peaks of the protein representing higher molecular weight forms of the protein. The area of all peaks was determined. Table 3 shows the percentage of peak area for the aggregates in relation to the total peak area of the eluted species for the samples of Table 1. Each sample was examined in duplicate measurements.

Table 3: Overview of HMW species determined by SEC

Condition Formulation HMWS [%] SD [%]

(a) 2.11 0.03

(b) 2.01 0.01

(c) 1.94 0.00

(d) 2.12 0.02

TO (e) 1.98 0.02

(f) 2.01 0.02

(g) 2.11 0.01

(h) 2.12 0.02

(i) 2.13 0.02

(a) 2.15 0.00

(b) 2.10 0.01

(c) 2.12 0.00

(d) 2.24 0.00

1M 5°C (e) 2.08 0.01

(f) 2.09 0.01

(g) 2.14 0.01

(h) 2.13 0.01

(i) 2.16 0.00 Condition Formulation HMWS [%] SD [%]

(a) 2.24 n.d.

(b) 2.20 n.d.

(c) 2.19 n.d.

(d) 2.35 n.d.

2M 5°C (e) 2.19 n.d.

(f) 2.21 n.d.

(g) 2.24 n.d.

(h) 2.22 n.d.

(i) 2.25 n.d.

(a) 2.35 n.d.

(b) 2.24 n.d.

(c) 2.30 n.d.

(d) 2.48 n.d.

3M 5°C (e) 2.27 n.d.

(f) 2.36 n.d.

(g) 2.36 n.d.

(h) 2.36 n.d.

(i) 2.41 n.d.

(a) 2.58 n.d.

(b) 2.54 n.d.

(c) 2.57 n.d.

(d) 2.72 n.d.

5M 5°C (e) 2.57 n.d.

(f) 2.62 n.d.

(g) 2.50 n.d.

(h) 2.41 n.d.

(i) 2.49 n.d. Condition Formulation HMWS [%] SD [%]

(a) 2.51 0.01

(b) 2.30 0.02

(c) 2.33 0.00

(d) 2.53 0.01

1M 25°C (e) 2.26 0.01

(f) 2.28 0.00

(g) 2.48 0.01

(h) 2.48 0.01

(i) 2.55 0.01

(a) 2.94 n.d.

(b) 2.58 n.d.

(c) 2.59 n.d.

(d) 2.89 n.d.

2M 25 °C (e) 2.60 n.d.

(f) 2.61 n.d.

(g) 3.01 n.d.

(h) 3.02 n.d.

(i) 3.07 n.d.

(a) 3.38 n.d.

(b) 2.86 n.d.

(c) 2.85 n.d.

(d) 3.16 n.d.

3M 25°C (e) 2.88 n.d.

(f) 2.89 n.d.

(g) 3.55 n.d.

(h) 3.53 n.d.

(i) 3.56 n.d. Condition Formulation HMWS [%] SD [%]

(a) 4.21 n.d.

(b) 3.23 n.d.

(c) 3.37 n.d.

(d) 3.83 n.d.

5M 25°C (e) 3.27 n.d.

(f) 3.37 n.d.

(g) 4.73 n.d.

(h) 4.49 n.d.

(i) 4.67 n.d.

(a) 16.87 0.00

(b) 13.85 0.00

(c) 13.04 0.03

(d) 12.00 0.01

1M 40°C (e) 12.78 0.00

(f) 13.01 0.03

(g) 19.83 0.03

(h) 19.01 0.00

(i) 19.21 0.01

(a) 27.58 n.d.

(b) 23.42 n.d.

(c) 21.83 n.d.

(d) 20.75 n.d.

2M 40°C (e) 21.45 n.d.

(f) 21.59 n.d.

(g) 32.48 n.d.

(h) 30.94 n.d.

(i) 32.94 n.d. Condition Formulation HMWS [%] SD [%]

(a) 36.38 n.d.

(b) 31.45 n.d.

(c) 30.23 n.d.

(d) 29.38 n.d.

3M 40°C (e) 29.30 n.d.

(f) 29.74 n.d.

(g) 41.17 n.d.

(h) 37.98 n.d.

(i) 41.79 n.d.

(a) 2.10 0.00

(b) 1.94 0.01

(c) 1.97 0.01

(d) 2.01 0.01

3x F/T (e) 1.93 0.00

(f) 1.94 0.01

(g) 2.11 0.02

(h) 2.13 0.01

(i) 2.12 0.00

(a) 2.11 0.01

(b) 1.99 0.02

(c) 2.02 0

(d) 2.05 0.01

5x F/T (e) 2.00 0.01

(f) 1.99 0.01

(g) 2.19 0.01

(h) 2.14 0.04

(i) 2.25 0.01

The generation of HMWS determined by SEC was comparable for the incubation parameters 5°C for 1 month and after application of 3 cycles freeze/ thaw as well as 5 cycles freeze/ thaw. Both the identities of the higher molecular weight species and the temperature dependent kinetics were comparable. For all samples incubated at 25 °C for 1 month there was a slight increase of the amount of HMWS up to 2.5% in comparison to the 5°C data at 1 month.

In contrast to that there was a clear trend detectable after storage of the samples at 40°C for 1 month. As an overall trend over all samples there was an increase in the percentage of HMWS. With about 12-13% HMWS the content of aggregates was comparable for all histidine buffered formulations, but lower than the content of aggregates in the Eylea ^® buffer system which showed about 16-17% HMWS. All other buffer systems (malate, succinate and maleate) showed a significantly higher content of HMWS than the histidine and Eylea ^® formulations.

In the non-reduced SDS-PAGE analysis of all samples incubated for three months at 40 °C/ 75 % relative humidity bands representing fragments and higher molecular weight species of aflibercept were visible. The generation of fragments and HMWS during the 3 months incubation was comparable in the kinetics and the identity of the impurities in all tested formulations shown in Table 1.

The generation of HMWS was lowest when using histidine buffered solutions for aflibercept. The stability of aflibercept in the formulation (f) poloxamer 188 was comparable to formulations containing polysorbate 20 as surfactant as regards the generation of HMWS.

4. Detection of modifications by reduced peptide mapping

By reduced peptide mapping the purity of aflibercept with regard to asparagine deamidation and methionine oxidation was analyzed after digestion with trypsin and liquid

chromatography coupled to mass spectrometry (LC-MS). After reduction and alkylation, the protein was submitted to enzymatic cleavage with trypsin. The resulting peptides were analyzed by RP-UPLC-MS. During chromatography the peptides were eluted by changing the mobile phase from highly polar (trifluoroacetic acid in water) to less polar

(trifluoroacetic acid in acetonitrile) and analyzed by mass spectrometry (Xevo G2-XS

QTOF). The peptide data were processed and compared with the theoretical protein sequence and a reference sample to detect oxidations and deamidations. Samples shown in Table 1 were analyzed as single measurement after 3 months incubation at 5 °C, 25 °C/ 60 % relative humidity or 40 °C/ 75 % relative humidity or after 5 freeze/ thaw cycles and compared to the starting material tO. Samples were diluted with denaturation buffer (50 mM Tris(hydroxymethyl)aminomethane) to a aflibercept concentration of 1.25 mg/niL. 80 μΐ of the diluted samples were mixed with 10 μΐ of 0.5 % RapiGest (from Waters, solved in 50 mM Tris(hydro- xymethyl)aminomethane) and incubated for 5 minutes at 95 °C. 4.5 μΐ of 0.02 M DTT

(dissolved in 50 mM Tris(hydroxymethyl)-aminomethane) were added for reduction and the samples were incubated for 30 minutes at 37 °C. For aflibercept digestion 5 μΐ of a 1 mg/niL Trypsin solution (solved in 50 mM acetic acid) were added and incubated for further 3 hours at 37°C. The reaction was stopped with 20 μΐ of 2% (v/v) trifluoroacetic acid and an incubation for 30 minutes at 37°C. The supernatant was diluted to 0.125 mg/mL with 50 mM Tris(hydroxymethyl)-aminomethane for analysis of the peptides.

UPLC Parameters:

The digested protein samples from the syringes were loaded onto an AC QUIT Y UPLC-CSH C-18 column from Waters, 100 mm x 2.1 mm, 1.7 μιη. 0.25 μg of the digested samples were eluted at 65°C with a gradient of eluent A (water), eluent B (acetonitrile), eluent C (0.25 % trifluoroacetic acid) and D (n-propanol) according to the following Table:

Table 4: Parameters used for UPLC

Time [minutes] Eluent A [ ] Eluent B [%] Eluent C [%] Eluent D [ ]

0.0 89.0 1.0 10.0 0.0

2.5 89.0 1.0 10.0 0.0

5.0 80.0 8.0 10.0 2.0

50.0 57.5 26.0 10.0 6.5

52.0 0.0 72.0 10.0 18.0

54.0 0.0 72.0 10.0 18.0

56.0 89.0 1.0 10.0 0.0

60.0 89.0 1.0 10.0 0.0 Method parameters for mass spectrometry:

Ionisation type: ESI Polarity: Positive

Analyser mode: Sensitivity Experiment type: MS

Start Mass: 50 m/z Cone Gas Flow: 30 L/h

End Mass: 2000 m/z Desolvation Gas Flow: 1000 L/h

Source Temperature: 120 °C Scan Time: 0.5 s

Desolvation Temperature: 450 °C Capillary Voltage: 3.0 kV

Cone Voltage: 35 V

LockSpray Profile

Reference Compound: Leucine Enkephalin

MS Lock mass: 556.2766 m/z

Scan Time: 0.5 s

Interval: 30 s

5 oxidated methionines in aflibercept could be identified (AA 20; AA 163; AA 192, AA 237, AA 413) and all values from AA 20; AA 163; AA 195, AA 237 were summed up for evaluation of the total methionine oxidation (see Table 5). 5 deamidations of asparagine could be identified (AA 84; AA 91 ; AA 99; AA 271 ; AA 300) and were summed up for evaluation of the total deamidation (see Table 5)

Table 5:

Total methionine Total deamidations

Condition Formulation

oxidations [%] [ ]

(a) 18.1 32.0

(b) 17.1 27.7

(c) 17.3 32.3

(d) 16.8 29.1

TO (e) 16.7 29.5

(f) 17.4 31.1

(g) 17.6 30.3

(h) 17.2 32.6

(i) 17.5 32.7 Total methionine Total deamidations

Condition Formulation

oxidations [%] [ ]

(a) 15.7 26.5

(b) 14.3 28.0

(c) 14.7 27.4

(d) 15.1 29.0

3 M 5°C (e) 15.4 27.2

(f) 14.8 27.4

(g) 16.4 27.6

(h) 16.3 27.7

(i) 15.3 29.5

(a) 18.9 35.9

(b) 18.4 35.5

(c) 17.6 35.6

(d) 17.9 36.0

5 M 5°C (e) 16.8 36.1

(f) 16.1 37.6

(g) 18.8 35.7

(h) 17.7 35.1

(i) 16.3 36.1

(a) 17.0 37.6

(b) 16.8 38.1

(c) 19.7 37.8

(d) 18.6 48.9

3 M 25°C (e) 17.1 44.9

(f) 16.8 39.1

(g) 16.6 37.0

(h) 18.8 35.9

(i) 16.2 38.5 Total methionine Total deamidations

Condition Formulation

oxidations [%] [ ]

(a) 19.6 46.2

(b) 20.4 49.9

(c) 20.4 53.3

(d) 21.2 59.1

5 M 25°C (e) 18.0 53.8

(f) 19.8 52.5

(g) 18.6 47.3

(h) 18.6 46.0

(i) 18.5 47.5

(a) 15.9 46.5

(b) 15.8 48.6

(c) 16.8 48.8

(d) 16.4 60.4

1 M 40°C (e) 13.4 51.1

(f) 15.7 50.4

(g) 15.4 47.6

(h) 15.6 44.4

(i) 18.9 54

(a) 17.8 83.6

(b) 21.0 87.2

(c) 20.7 91.6

(d) 21.8 101.8

3 M 40°C (e) 15.5 84.2

(f) 20.9 93.8

(g) 19.3 90.1

(h) 16.7 84.5

(i) 16.6 82.9 Total methionine Total deamidations

Condition Formulation

oxidations [%] [ ]

(a) 17.4 23.1

(b) 15.9 22.5

(c) 16.2 24.6

(d) 16.3 25.2

5 x F/T (e) 16.0 27.0

(f) 15.7 24.9

(g) 17.2 26.8

(h) 16.8 25.6

(i) 16.2 26.3

Formulations shown in Table 5 comprise a comparable stability with regard to methionine oxidation and deamidation of asparagine. Whereas in all temperature conditions just slight increases of methionine oxidations in the different formulations were detected, the increase of deamidations was significantly temperature dependent. Comparing the oxidation results from the 40°C 1 month samples a slight decrease could be detected for the formulation containing 10 mM methionine. Oxidation was lowest when using 10 mM methionine within the formulation. Taking a closer look on the deamidation data of these samples the histidine formulation with the highest pH 6.6 showed an increased deamidation level. Freeze/ thaw did not show an influence on both methionine oxidation and asparagine deamidations in all tested formulations.

5; Determination of the relative potency of aflibercept The relative potency of aflibercept was determined by an ELISA (enzyme linked immunosorbent assay). This sandwich ELISA is based on the binding of aflibercept to the Vascular Endothelial Growth Factor (VEGF). Aflibercept binds to Vascular Endothelial Growth Factor (VEGF) and thereby neutralizes its biological activity. This potency ELISA (Enzyme Linked Immunosorbent Assay) uses the principle of the sandwich ELISA technique. Increasing amounts of FYB203 reference standard as well as test samples were mixed with a defined constant concentration of VEGF-A165 (VEGF) and incubated overnight at 4 °C. Thereby, aflibercept binds to VEGF. These pre-incubated mixtures were then transferred to a microtiter plate, coated with an anti-VEGF antibody. Only non-neutralized VEGF binds to the coated anti-VEGF antibody, while the VEGF/RP or VEGF/test samples complexes are washed away. Binding of VEGF was detected by addition of an additional biotinylated polyclonal anti-VEGF antibody and visualized using a Streptavidin-HRP (Horseradish peroxidase) conjugate via the oxidation of 3,3',5,5'-tetramethylbenzidine. The colorimetric reaction was stopped by sulfuric acid and the absorption was measured at a wavelength of 450 nm. To determine the relative potencies of the samples, they were compared to the reference standard. All test samples were analyzed as duplicates.

Table 6: Overview of the Potency-ELISA results

Condition Formulation Potency result [%] Relative CV [%]

(a) 0.992 96.6 -103.5 (6.8%)

(b) 1.001 96.3%-103.9% (7.6%)

(c) 1.137 96.2%-103.9% (7.7%)

(d) 1.049 91.6%-109.1% (17.5%)

TO (e) 1.054 90.5%-110.5% (20.1%)

(f) 1.044 90.6%-110.4% (19.8%)

(g) 1.076 90.7%-110.2% (19.5%)

(h) 1.027 89.8%-111.4% (21.7%)

(i) 1.037 92.3%-108.4% (16.1%)

(a) 1.130 90.3%-110.0% (19.1 %)

(b) 1.050 95.5%-104.7% (9.2%)

(c) 1.174 94.5%-105.8% (11.3%)

(d) 1.105 95.3%-105.0% (9.7%)

3M 5°C (e) 1.140 91.9%-108.9% (17.0%)

(f) 1.020 89.3%-112.0% (22.7%)

(g) 1.003 87.3%-114.6% (27.3%)

(h) 1.011 90.0%-l l l. l% (21.1%)

(i) 1.055 87.2%-114.7% (27.5%) Condition Formulation Potency result [%] Relative CV [%]

(a) 1.090 90.9%-l 10.0% (19.0%)

(b) 1.097 90.5%-l 10.5% (20.0%)

(c) 1.084 89.6%-l 11.5% (21.9%)

(d) 1.115 90.6%-110.4% (19.8%)

5M 5°C (e) 1.127 92.0%-108.7% (16.8%)

(f) 1.093 90.5%-l 10.5% (20.0%)

(g) 1.014 95.1%-105.1% (10.0%)

(h) 1.070 92.2%-108.4% (16.2%)

(i) 1.090 92.0%-108.7% (16.7%)

(a) 0.980 89.6%-111.6% (22.1%)

(b) 1.004 88.4%-113.1% (24.6%)

(c) 1.003 90.4%-110.6% (20.1%)

(d) 1.009 90.6%-110.4% (19.8%)

3M 25°C (e) 1.090 93.8%-106.6% (12.7%)

(f) 1.095 94.4%-106.0% (11.6%)

(g) 1.061 95.1%-105.2% (10.1%)

(h) 1.057 95.7%-104.5% (8.8%)

(i) 1.017 93.7% -106.7% (12.9%)

(a) 1.057 91.8%-109.0% (17.2%)

(b) 0.997 91.4%-109.4% (18.0%)

(c) 1.056 91.7%-109.1% (17.4%)

(d) 0.993 95.0%-105.2% (10.2%)

5M 25°C (e) 0.986 96.4%-103.7% (7.3%)

(f) 0.985 96.0%-104.1% (8.1%)

(g) 0.849 92.8%-107.7% (14.9%)

(h) 1.021 94.8%-105.5% (10.6%)

(i) 1.066 90.5%-110.4% (19.9%) Condition Formulation Potency result [%] Relative CV [%]

(a) 0.925 96.5%-103.6% (7.1%)

(b) 0.945 92.9 -107.7 (14.8%)

(c) 0.907 90.7%-110.2% (19.1%)

(d) 1.054 90.5%-110.5% (20.1%)

1 M 40°C (e) 0.924 91.8%-109.0% (17.2%)

(f) 0.930 90.7%-110.2% (19.5%)

(g) 0.747 89.9%-111.2% (21.3%)

(h) 0.827 92.0%-108.7% (16.8%)

(i) 0.886 92.5%-108.1% (15.6%)

(a) 0.633 92.3%-108.4% (16.1%)

(b) 0.876 90.7%-110.2% (19.5%)

(c) 0.846 92.3%-108.4% (16.1%)

(d) 0.884 92.2%-108.5% (16.3%)

3M 40°C (e) 0.866 90.9%-110.0% (19.1%)

(f) 0.873 92.4%-108.3% (15.9%)

(g) 0.532 92.5%-108.1% (15.6%)

(h) 0.669 92.2%-108.4% (16.2%)

(i) 0.668 93.1%-107.4% (14.3%)

Incubation at 40 °C/ 75 % relative humidity for 1 month showed no change in potency in comparison to the tO values for all histidine based formulations. A slight decrease in binding could be detected for the malic acid based formulation (g) and for the succinate based formulation (h). The maleic acid formulation (i) showed comparable binding results for the tO and the sample after incubation.

Incubation at 40 °C/ 75 % relative humidity for three months led to decreasing relative potencies especially in the samples (a), (g), (h) and (i). All other samples retained their relative potency during the 5 months incubation at both 5 °C and 25 °C/ 60 % relative humidity and showed only a small decrease after storage at 40 °C/ 75 % relative humidity. 6. Detection of aflibercept isoforms by isoelectric focussing (IEF)

Isoelectric focusing (IEF) is a technique for separation of different isoforms of a molecule due to differences in their isoelectric points (pi). The IEF gel (FocusGel) contains a pH gradient within the gel mixing different am-pholytes in a pH range from 6-11. The FocusGel is ready to use. After application, proteins migrate due to their net charge in the pH gradient un-till they reach the pH equivalent to their isoelectric point. The pi of the unknown sample is determined by comparison to reference proteins with known isoelectric points.

Modifications leading to charge heterogeneities include e.g. glycosylation, C-terminal Lysine variations and deamidation of the protein. All samples were applied to the gel in duplicates.

Table 7: Quantification results of tO samples

Table 8: Quantification results of 40°C, 1 month samples

In the IEF gels there was a comparable pattern of bands for the formulation a-h for the tO samples as well as for the samples incubated for up to 3 months at 40°C. Only the maleic buffer system formulation (i) showed additional bands in the lower pi range.

7. Detection of HMWS and fragments by non-reduced SDS -PAGE

The strongly anionic detergent sodium dodecyl sulfate (SDS) is used in combination with heat to denature the proteins before they are loaded onto the gel. The denatured proteins are enclosed by SDS, become negatively charged and exhibit a consistent charge-to-mass ratio regardless of protein type. The amount of bound SDS is proportional to the molecular mass of the protein and independent of its sequence.

The movement of the particles is retarded by interactions with the sur -rounding gel matrix which acts as a molecular sieve. Thus, SDS -protein micelles migrate through polyacrylamide gels with mobility dependent on the size of the protein. The electrophoretic mobility of the resultant detergent-protein complexes all assume the same functional relationship to their molecular masses. SDS micelles migrate towards the anode in a predictable manner, with low-molecular-mass complexes migrating faster than larger ones. The apparent molecular mass of a protein can therefore be estimated from its relative mobility in calibrated SDS- PAGE and the occurrence of a single band in such a gel is a criterion of purity.

Table 9: Overview of all results determined via non-reduced SDS-PAGE

All samples show the same band pattern. For the samples incubated at 40°C/ 75 % RH for 1 month a slight decrease in the main band could be determined. Differences between the formulations were not detectable.