WO2021050687A1 - Stable aqueous formulations of aflibercept - Google Patents

Stable aqueous formulations of aflibercept Download PDF

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Publication number
WO2021050687A1
WO2021050687A1 PCT/US2020/050141 US2020050141W WO2021050687A1 WO 2021050687 A1 WO2021050687 A1 WO 2021050687A1 US 2020050141 W US2020050141 W US 2020050141W WO 2021050687 A1 WO2021050687 A1 WO 2021050687A1
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weight percent
formulation
aqueous formulation
concentration
aflibercept
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PCT/US2020/050141
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French (fr)
Inventor
Jun Liu
Mark Manning
Sekhar R. Kanapuram
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Coherus Biosciences, Inc.
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Priority claimed from US16/566,847 external-priority patent/US11426446B2/en
Application filed by Coherus Biosciences, Inc. filed Critical Coherus Biosciences, Inc.
Publication of WO2021050687A1 publication Critical patent/WO2021050687A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • the present invention relates to stable aqueous pharmaceutical compositions of aflibercept, including compositions that are considered or intended to be a “biosimilar” of commercially available aflibercept products, methods of manufacture of the compositions, methods of their administration, and kits containing the same.
  • VEGF Vascular endothelial growth factor
  • VEGFR regulates angiogenesis.
  • Drugs that target VEGF-VEGFR interactions have demonstrated efficacy in treating ocular diseases. See Pozarowska et al. (2016) Cent Eur J Immunol 41(3): 311-316.
  • a soluble VEGF-specific fusion protein antagonist, termed a "VEGF trap” was developed that interferes with binding of VEGF with its receptors. See, Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99:11393-8.
  • Aflibercept developed by Regeneron Pharmaceuticals, Inc. is a
  • Aflibercept is a soluble decoy receptor that binds vascular endothelial growth factor-A (VEGF-A), VEGF-B, and placental growth factor (PIGF) with a greater affinity than the body’s native receptors.
  • VEGF-A vascular endothelial growth factor-A
  • VEGF-B vascular endothelial growth factor-B
  • PIGF placental growth factor
  • Aflibercept s high affinity for VEGF prevents the subsequent binding and activation of native VEGF receptors.
  • Reduced VEGF activity leads to decreased angiogenesis and vascular permeability.
  • Inhibition of VEGF-B may also aid in the treatment of angiogenic conditions.
  • VEGF-B overexpression has recently been connected with breakdown of the blood- retinal battier retinal angiogenesis.
  • Aflibercept products have been approved by the FDA and are commercially available under the brand names of Eylea® and Zaltrap®.
  • aflibercept Various formulations of aflibercept are known in the art. See, for example, Eylea®, Zaltrap®, U.S. Patents 7,608,261 ; 7,807,164; 8,092, 803; 8,481 ,046; 8,802,107; 9,340,594; and 9,580,489. There is still need for stable liquid formulations of aflibercept suitable for ophthalmic administration.
  • the invention provides stable aqueous formulations comprising aflibercept.
  • the invention provides several types of aflibercept formulations: buffer-free formulations; formulations free of an organic co solvent; and formulations that are free of both buffers and organic co-solvents.
  • aflibercept can be present at concentrations of about 1 mg/mL to about 200 mg/mL, e.g., a concentration of about 1 mg/mL to about 190 mg/mL, about 1 mg/mL to about 180 mg/mL, about 1 mg/mL to about 170 mg/mL, about 1 mg/mL to about 160 mg/mL, about 1 mg/mL to about 150 mg/mL, about 1 mg/mL to about 140 mg/mL, about 1 mg/mL to about 130 mg/mL, about 1 mg/mL to about 120 mg/mL, about 1 mg/mL to about 110 mg/mL, about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 90 mg/mL, about 1 mg/mL to about 80 mg/mL, about
  • 1 mg/mL to about 70 mg/mL about 1 mg/mL to about 60 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 45 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 35 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 15 mg/mL, about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 8 mg/mL, about 1 mg/mL to about 6 mg/mL, about 1 mg/mL to about 4 mg/mL, about 1 mg/mL to about
  • the invention provides an aqueous formulation comprising aflibercept, a stabilizer and an organic co-solvent, wherein the formulation is free of buffer.
  • the formulation has a pH of about 5.5 to about 7.0 (e.g., about 5.5 to about 6.9, about 5.5 to about 6.8, about 5.5 to about 6.7, about 5.5 to about 6.6, about 5.5 to about 6.5, about 5.5 to about 7.0
  • the pH is about 6.0 to about 6.5. In yet another embodiment, the pH is about 6.2. In another embodiment, the pH is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.
  • the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
  • the organic co-solvent is selected from the group consisting of a surfactant, polyethylene glycol (PEG), propylene glycol, a block copolymer, an alkyl saccharide (e.g., a ProTek alkyl saccharide, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000.
  • a surfactant polyethylene glycol (PEG), propylene glycol, a block copolymer
  • an alkyl saccharide e.g., a ProTek alkyl saccharide, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyrano
  • the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, sodium dodecyl sulfate (SDS), n-Dodecyl- -D-maltoside (DDM) and poloxamer 188 (Pluronic F-68).
  • the buffer-free formulations of the invention further comprise one or more salt(s).
  • the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, and potassium chloride.
  • Each of the one or more salt(s) may be present at a concentration of about 0.1 mM-100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 65 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 90
  • the stabilizer is a sugar. In another embodiment, the stabilizer is a sugar (e.g., sucrose). In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine.
  • a stabilizer e.g., any of the exemplary stabilizers described herein
  • a stabilizer can be present at a final concentration of about 0.01 % w/v to about 8.0% w/v (e.g., about 0.01 % w/v to about 7.5% w/v, about 0.01 % w/v to about 7.0% w/v, about 0.01 % w/v to about 6.5% w/v, about 0.01 % w/v to about 6.0% w/v, about 0.01 % w/v to about 5.5% w/v, about 0.01 % w/v to about 5.0% w/v, about 0.01 % w/v to about 4.5% w/v, about 0.01 % w/v to about 4.0% w/v, about 0.01 % w/v to about 3.5% w/v, about 0.01 % w/v to about 3.0% w/v, about 0.01 % w/v, about 0.01 %
  • the stabilizer is an amino acid (e.g., an aromatic amino acid, e.g., tyrosine, phenylalanine, and tryptophan).
  • the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof.
  • the amino acids may be present at a concentration of about 5-300 mM (e.g., about 5 mM to about 280 mM, about 5 mM to about 260 mM, about 5 mM to about 240 mM, about 5 mM to about 220 mM, about 5 mM to about 200 mM, about 5 mM to about 180 mM, about 5 mM to about 160 mM, about 5 mM to about 140 mM, about 5 mM to about 120 mM, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 60 mM, about 5 mM to about 40 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 300 mM, about 10 mM to about 280 mM, about 10 mM to about 260 mM, about 10 mM to about 240
  • the stabilizer is sucrose
  • the organic co solvent is polysorbate 20
  • the pH of the formulation is about 5.5-7.0 (e.g., about 5.5 to about 6.9, about 5.5 to about 6.8, about 5.5 to about 6.7, about
  • the formulation is about 5.5-7.0 (e.g., about 5.5 to about 6.9, about 5.5 to about 6.8, about 5.5 to about 6.7, about 5.5 to about 6.6, about 5.5 to about
  • the formulation can include one or more of: sodium ion at a final concentration of about 100 mM to about 200 mM (e.g., about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); potassium ion at a final concentration of about 1 mM to about 20 mM (e.g., about 1 mM
  • beta-hyrdoxybutyrate at a final concentration of about 0.01 mM to about 0.3 mM (e.g., about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about
  • the formulation is iso-osmolar relative to the vitreous.
  • Formulations Free of Organic Co-Solvent [0022]
  • the invention provides an aqueous formulation comprising aflibercept, a stabilizer and a buffer, wherein the formulation is free of an organic co-solvent.
  • the formulation has a pH of about 5.5 to about 7.0 (e.g., any of the subranges of described herein or about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
  • the pH is about 6.0 to about 6.5.
  • the pH is about 6.2.
  • the stabilizer is selected from the group consisting of amino acids, sugars (e.g., sucrose), polyols, polymers, block copolymers, alkyl saccharides (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000, or any combination thereof.
  • amino acids e.g., sucrose
  • polyols e.g., polymers, block copolymers
  • alkyl saccharides e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-
  • the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine. In some embodiments of any of the aqueous formulations described herein, a stabilizer (e.g., any of the exemplary stabilizers described herein) can be present at a final concentration of about 0.01 % w/v to about 8.0% w/v (e.g., about 0.01 % w/v to about 7.5% w/v, about 0.01 % w/v to about 7.0% w/v, about 0.01 % w/v to about 6.5% w/v, about 0.01 % w/v to about 6.0% w/v, about 0.01 % w/v to about 5.5% w/v, about 0.01 % w/v to about 5.0% w/v, about 0.01 % w/v to about 4.5% w/v, about 0.01 % w/v, about 0.01 %
  • the buffer is selected from the group consisting of acetate, histidine, phosphate, citrate, succinate, tartrate, imidazole, and maleate, and any combination thereof.
  • the buffer is present at a concentration of about 1 mM to 150 mM (e.g., about 1 mM to about 140 mM, about 1 mM to about 130 mM, about 1 mM to about 120 mM, about 1 mM to about 110 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to 150 mM (e.g
  • the buffer concentration is about 10 mM.
  • the stabilizer is a sugar. In another embodiment, the stabilizer is sucrose. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine.
  • a stabilizer e.g., any of the exemplary stabilizers described herein
  • a stabilizer can be present at a final concentration of about 0.01 % w/v to about 8.0% w/v (e.g., about 0.01 % w/v to about 7.5% w/v, about 0.01 % w/v to about 7.0% w/v, about 0.01 % w/v to about 6.5% w/v, about 0.01 % w/v to about 6.0% w/v, about 0.01 % w/v to about 5.5% w/v, about 0.01 % w/v to about 5.0% w/v, about 0.01 % w/v to about 4.5% w/v, about 0.01 % w/v to about 4.0% w/v, about 0.01 % w/v to about 3.5% w/v, about 0.01 % w/v to about 3.0% w/v, about 0.01 % w/v, about 0.01 %
  • the stabilizer is an amino acid (e.g., an aromatic amino acid, e.g., tyrosine, phenylalanine, and tryptophan).
  • the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof.
  • the amino acids may be present at a concentration of about 5-300 mM (e.g., about 5 mM to about 280 mM, about 5 mM to about 260 mM, about 5 mM to about 240 mM, about 5 mM to about 220 mM, about 5 mM to about 200 mM, about 5 mM to about 180 mM, about 5 mM to about 160 mM, about 5 mM to about 140 mM, about 5 mM to about 120 mM, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 60 mM, about 5 mM to about 40 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 300 mM, about 10 mM to about 280 mM, about 10 mM to about 260 mM, about 10 mM to about 240
  • the co-solvent-free formulations of the invention further comprise one or more salt(s).
  • the one or more salt(s) can be selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, and potassium chloride.
  • Each of the one or more salt(s) may be present at a concentration of about 10 mM-100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 65 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM
  • the formulation can include one or more of: sodium ion at a final concentration of about 100 mM to about 200 mM (e.g., about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); potassium ion at a final concentration of about 1 mM to about 20 mM (e.g., about 1 mM
  • beta-hyrdoxybutyrate at a final concentration of about 0.01 mM to about 0.3 mM (e.g., about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about
  • the formulation is iso-osmolar relative to the vitreous.
  • the invention provides an aqueous formulation comprising aflibercept and a stabilizer, wherein the formulation is free of both an organic co-solvent and a buffer.
  • the formulation has a pH of about 5.5 to about 7.0 (e.g., or any of the subranges of this range described herein, e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
  • the pH is about 6.0 to about 6.5.
  • the pH is about 6.2.
  • the stabilizer is selected from the group consisting of amino acids, sugars (e.g., sucrose), polyols, polymers, block copolymers, alkyl saccharides (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000, or any combination thereof.
  • amino acids e.g., sucrose
  • polyols e.g., polymers, block copolymers
  • alkyl saccharides e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-
  • the stabilizer is a block copolymer.
  • the block copolymer is poloxamer (a water-soluble nonionic A-B-A and B-A-B triblock copolymers, where A is polyethylene oxide) (PEO) and B is polypropylene oxide) (PPO)).
  • the poloxamer is one described in Bodratti AM, Alexandridis P. Formulation of Poloxamers for Drug Delivery. J Fund Biomater. 2018;9(1 ):11.
  • the stabilizer is a sugar. In another embodiment, the stabilizer is sucrose.
  • the stabilizer is an amino acid (e.g., an aromatic amino acid, e.g., tyrosine, phenylalanine, and tryptophan).
  • the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof.
  • the amino acids may be present at a concentration of about 5-300 mM (e.g., about 25 mM to about 280 mM, about 25 mM to about 260 mM, about 25 mM to about 240 mM, about 25 mM to about 220 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 95 mM, about 25 mM to about 90 mM, about 25 mM to about 85 mM, about 25 mM to about 80 mM, about 25 mM to about 75 mM, about 25 mM to about 70 mM, about 25 mM to about 65 mM, about 25 mM to about 60 mM, about 25 mM to about 55 mM
  • the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
  • the salts may be present at a concentration of about 10 mM-100 mM (e.g., or any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
  • the co-solvent-free formulations of the invention further comprise one or more salt(s).
  • the one or more salt(s) can be selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, and potassium chloride.
  • Each of the one or more salt(s) may be present at a concentration of about 10 mM-100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 65 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM
  • the formulation can include one or more of: sodium ion at a final concentration of about 100 mM to about 200 mM (e.g., about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); potassium ion at a final concentration of about 1 mM to about 20 mM (e.g., about 1 mM
  • beta-hyrdoxybutyrate at a final concentration of about 0.01 mM to about 0.3 mM (e.g., about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about
  • the formulation is iso-osmolar relative to the vitreous.
  • FIG. 1 is a graphic showing the effect of pH and acetate according to a PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
  • FIG. 2 is a graphic showing the effect of pH and acetate in the presence of 100 mM NaCI according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
  • FIG. 3 is a graphic showing the effect of pH and His according to the PLS1 model using monomer content after one week at 40 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 4 is a graphic showing the effect of sucrose and NaCI according to the PLS1 model using monomer content after one week at 40 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at
  • FIG. 5 is a graphic showing the effect of PS20 and PS80 according to the PLS1 model using monomer content after one week at 40 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at
  • FIG. 6 is a graphic showing the effect of glycerol and PEG 3350 according to the PLS1 model using monomer content after one week at 40 ° as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at
  • FIG. 7 is a graphic showing the effect of Gly and Pro according to the PLS1 model using monomer content after one week at 40 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at 5.5, and His at 0 mM.
  • FIG. 8 is a graphic showing the effect of trehalose and calcium chloride according to the PLS1 model using monomer content after one week at 40 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at 5.5, and His at 0 mM.
  • FIG. 9 is a graphic showing the effect of pH and His according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 10 is a graph showing the effect of pH and His in the presence of 100 mM NaCI according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 11 is a graph showing the effect of pH and acetate according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 12 is a graph showing the effect of NaCI and sucrose according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at 6.0, and the His at 20 mM.
  • FIG. 13 is a graphic showing the effect of PS20 and PS80 according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH 6.0, and His at 20 mM.
  • FIG. 14 is a graphic showing the effect of ArgHCI and PEG 3350 according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at 6.0, and His at 20 mM.
  • FIG. 15 is a graphic showing the effect of calcium chloride and glycine according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at 6.0, and His at 20 mM.
  • FIG. 16 is a graphic showing the effect of magnesium chloride and glycerol according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL, the pH at 6.0, and the His at 20 mM.
  • FIG. 17 is a graphic showing the effect of pH and His according to the PSL1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 18 is a graphic showing the effect of pH (down to pH 7.0) and His according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 19 is a graphic showing the effect of pH and acetate according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 20 is a graphic showing the effect of pH and Tris according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 21 is a graphic showing the effect of NaCI and sucrose according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL and pH at 6.0.
  • FIG. 22 is a graphic showing the effect of PS20 and PS80 according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL and pH at 6.0.
  • FIG. 23 is a graphic showing the effect of PEG 3360 and Gly according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL and pH 6.0.
  • FIG. 24 is a graphic showing the effect of ArgHCI and proline according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL and pH at 6.0.
  • FIG. 25 is a graphic showing the effect of magnesium chloride and calcium chloride according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL and pH at 6.0.
  • FIG. 26 is a graphic showing the effect of propylene glycol (PG) and glycerol according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • the protein concentration was fixed at 40 mg/mL and pH at 6.0.
  • FIG. 27 is a graphic showing the effect of pH and protein concentration according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
  • FIG. 28 is a graphic showing the effect of pH and His according to the PLS2 model using the total particle concentration (counts per ml_) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 29 is a graphic showing the effect of pH and acetate according to the PLS2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 30 is a graphic showing the effect of pH and phosphate according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 31 is a graphic showing the effect of sucrose and NaCI according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL.
  • FIG. 32 is a graphic showing the effect of PS20 and PS80 according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
  • FIG. 33 is a graphic showing the effect of glycine and ArgHCI according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
  • FIG. 34 is a graphic showing the effect of proline and PEG 3350 according to the PLS2 model using the total particle concentration (counts per ml_) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
  • FIG. 35 is a graphic showing the effect of Pluronic F-68 and magnesium chloride according to the PLS2 model using the total particle concentration (counts per ml_) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
  • FIG. 36 is a graphic showing the effect of trehalose and glycerol according to the PLS2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL and the pH set at pH 6.0.
  • FIG. 37 is a graphic showing the effect of propylene glycol (PG) and calcium chloride according to the PLS2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints.
  • the protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
  • FIG. 38 is a graphic showing the effect of pH and acetate according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL.
  • FIG. 39 is a graphic showing the e Effect of pH and His according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL.
  • FIG. 40 is a graphic showing the effect of pH and citrate according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL.
  • FIG. 41 is a graphic showing the effect of sucrose and NaCI according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
  • FIG. 42 is a graphic showing the effect of PS 20 and PS 80 according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
  • FIG. 43 is a graphic showing the effect of PEG 3350 and MgCh according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
  • FIG. 44 is a graphic showing the effect of Gly and Pro according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
  • FIG. 45 is a graphic showing the effect of glycerol and CaCh according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
  • FIG. 46 is a graphic showing the effect of ArgHCI and trehalose according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint.
  • the protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
  • Aflibercept is intended to be synonymous with the active pharmaceutical ingredient in Eylea® (Regeneron Pharmaceuticals,
  • Aflibercept is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF)-binding portions from the extracellular domains of human VEGF Receptors 1 and 2 fused to the Fc portion of the human lgG1 .
  • VEGF Vascular Endothelial Growth Factor
  • Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation, constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa.
  • Amino acid and nucleic acid sequences of aflibercept are known in the art. See, US 7,070,959 sequences 15 and 16 (figure 24).
  • Aflibercept encompasses the active pharmaceutical ingredient in products intended to be, or approved as, biosimilar, interchangeable, or bio-better product of commercially available Eylea® and Zaltrap® (also referred to ziv-aflibercept).
  • Eylea® and Zaltrap® also referred to ziv-aflibercept.
  • a biosimilar is a biological product that is highly similar to and has no clinically meaningful differences from an existing FDA-approved reference product. Therefore, a biosimilar for Eylea® or Zaltrap® may have essentially the same pharmacological effects as commercially available Eylea® or Zaltrap® even though the active pharmaceutical product in the biosimilar exhibits certain physical properties, such as glycosylation profile, that may be similar but not identical to Eylea® or Zaltrap®.
  • aflibercept also encompasses aflibercept with minor modifications in the amino acid structure (including deletions, additions, and/or substitutions of amino acids) or in the glycosylation properties, which do not significantly affect the function of the polypeptide.
  • antibody refers to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • sugar refers to monosaccharides, disaccharides, and polysaccharides.
  • sugars include, but are not limited to, sucrose, glucose, dextrose, trehalose, lactose, and maltose.
  • polyol refers to an alcohol containing multiple hydroxyl groups.
  • examples of polyols include, but are not limited to, mannitol, sorbitol, glycerol, xylitol and inositol.
  • metal ion refers to a metal atom with a net positive or negative electric charge.
  • metal ion also includes sources of metal ions, including but not limited to metal salts.
  • stable is understood to mean that aflibercept contained in the formulation or pharmaceutical compositions does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the formulation or composition at the beginning of storage.
  • the term also should be understood to mean that the aflibercept formulations or compositions are at least comparable to, or better than commercially available aflibercept compositions, in terms of their stability and/or ability to resist formation of particulates during storage.
  • term is understood to mean the aflibercept formulations or compositions is stable when agitated, mixed, or handled.
  • Stability is also intended to denote the ability of the pharmaceutical aflibercept compositions disclosed herein to resist particulates formation such that the compositions, under storage conditions typical of protein therapeutics, exhibits levels and types of particulates that meet standard guidelines.
  • a reduced tendency to form particulates in formulations disclosed herein results in aflibercept formulations having reduced immunogenicity, and therefore reduced potential to cause harm to patients resulting from such immunogenicity.
  • long-term storage in connection with “formulation” or “pharmaceutical composition” is understood to mean that the formulation or pharmaceutical composition can be stored for three months or more, for six months or more, or for one year or more.
  • the composition is stored at about 2-8°C.
  • the composition is stored at about 0°C.
  • the composition is stored at about -20°C.
  • the composition is stored at about -40°C.
  • the composition is stored at about -70°C.
  • the composition is stored at about -80°C.
  • long term storage and “long term stability” further include stable storage durations that are at least comparable to or better than the stability of currently available commercial formulations of aflibercept, without losses in stability that would render the formulation unsuitable for its intended pharmaceutical application.
  • composition can be frozen and thawed more than once.
  • Stability of a protein in an aqueous formulation may also be defined as the percentage of monomer, aggregate, or fragment, or combinations thereof, of the protein in the formulation.
  • a protein “retains its physical stability” in a formulation if it shows substantially no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography.
  • a stable aqueous formulation is a formulation having less than about 10%, or less than about 5% of the protein being present as aggregate in the formulation.
  • a protein in an aqueous formulation can be stable at frozen storage temperatures, liquid storage temperatures such as refrigeration (e.g. 2-8°C) and room temperature (about 25°C), and under accelerated liquid stability testing temperatures (e.g. 40°C).
  • liquid storage temperatures such as refrigeration (e.g. 2-8°C) and room temperature (about 25°C)
  • accelerated liquid stability testing temperatures e.g. 40°C.
  • mammal includes, but is not limited to, a human.
  • ophthalmically suitable or “suitable for ophthalmic administration” means a composition as a whole, or the individual components (e.g. excipients) of a composition are safe for administration to a subject in, on, or around the subject’s eye. Examples of such excipients are found in products already approved by the FDA for administration to a person’s eye and are known to person of skill in the art.
  • a composition suitable for ophthalmic administration has a non-stinging formulation.
  • a composition suitable for ophthalmic administration is iso-osmolar relative to the vitreous.
  • a composition suitable for ophthalmic administration has an osmolality is between about 200 and about 350 mOsM.
  • a composition suitable for ophthalmic administration can be administered intravitreously.
  • a composition suitable for ophthalmic administration is administed by intravitreal injection.
  • a composition suitable for ophthalmic administration is administed topically.
  • a composition suitable for ophthalmic administration is administed via a port.
  • a composition suitable for ophthalmic administration has a volume of about 0.1 ml_ or less.
  • a composition suitable for ophthalmic administration has a volume of about 0.05 ml_ or less.
  • a composition suitable for ophthalmic administration has a volume of about 0.025 ml_ or less.
  • pharmaceutically acceptable carrier refers to a non toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • composition refers to a mixture of a protein, such as a fusion protein, e.g., aflibercept, together with one or more additional components.
  • the additional component is water or a buffer.
  • the additional components may include, e.g., one or more excipients, such as a stabilizer, tonicity modifier, surfactant, and the like, e.g., a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration to a subject for therapeutic, diagnostic, or prophylactic purposes.
  • the additional component is suitable for ophthalmic administration.
  • compositions / formulations according to the present invention may be aqueous formulation suitable for intravitreal administration.
  • substantially free of a particular substance means that either the substance is not present or only minimal, trace amounts of the substance are present which do not have any substantial impact on the properties of the composition. If reference is made to no amount of a substance (or that the substance is not present), it should be understood as "no detectable amount.”
  • iso-osmolar relative to the vitreous is meant that the formulation of interest has essentially the same osmotic pressure as vitreous.
  • buffer refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components.
  • a buffer consists of a weak acid and a conjugate salt or a weak base and a conjugate salt (e.g sodium acetate).
  • Buffers suitable for use in connection with this invention may have a pH in the range from about 4.0 to about 9.0; from about pH 4.0 to about 7.0; or from about pH 4.5 to about 6.5.
  • a pH of any point in between the above ranges is also contemplated.
  • buffer-free or “free of buffer” are synonymous and intend to exclude components traditionally used to introduce buffer capacity in the formulation in addition to any buffering capacity present in the formulation absent the buffer component.
  • proteins are known to provide buffering capacity to a formulation. Hence, these terms do not mean a formulation cannot have any buffering capacity.
  • a buffer-free protein formulation is self-buffering.
  • purified means that a molecule is present in a sample at a concentration of at least 95% by weight, or at least 98% by weight of the sample in which it is contained.
  • organic co-solvent is used herein in the same sense as it is used in U.S. 9,580,489. See, for example, column 2, lines 25-38.
  • U.S. 9,580,489 states “organic co-solvent may be polysorbate, for example, polysorbate 20 or polysorbate 80, polyethylene glycol (PEG), for example, PEG 3350, or propylene glycol, or a combination thereof.”
  • An organic co solvent is an organic (i.e. , carbon-based) substance that is added to composition primarily to keep the ingredients of the composition in solution (i.e. dissolved). This includes preventing precipitation, aggregation, and adsorption of composition ingredients.
  • Examples of compounds that are not an “organic co-solvent,” as used herein, include salt (e.g. sodium chloride, magnesium chloride), sugar (e.g. dextran or other polysaccharides), polyol (e.g. sorbitol, glycerol, mannitol), and amino acid (e.g. arginine, glycine, proline).
  • Additional examples of compounds that are not an organic co solvent include block copolymers, alkyl saccharides (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000, or any combination thereof.
  • the present invention provides aqueous formulations of aflibercept that allow stable storage of aflibercept, so that aflibercept is stable over the course of storage either in liquid or frozen states.
  • the provided formulations do not require any extra steps such as rehydrating.
  • Aflibercept suitable for storage in one of the present pharmaceutical compositions or formulations can be produced by standard methods known in the art.
  • U.S. Pat. No. 9,580,480 and WO 2006/104852 describe various methods that a skilled artisan could use to prepare aflibercept protein for use in the formulations of the present invention. These methods are incorporated by reference herein.
  • Aflibercept can also be produced by purifying and/or diluting from commercially-available Eylea® and/or Zaltrap® preparations using standard methods
  • Purification of the expressed aflibercept can be performed by standard methods known in the art.
  • aflibercept is produced intracellularly by host cells, the host cells are lysed and particulate debris is removed, for example, by centrifugation or ultrafiltration.
  • supernatants from such expression systems can be first concentrated using standard polypeptide concentration filters.
  • Protease inhibitors can also be added to inhibit proteolysis and antibiotics can be included to prevent the growth of microorganisms.
  • aflibercept can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography and affinity chromatography, and any combination of known or yet to be discovered purification techniques, including but not limited to Protein A chromatography, fractionation on an ion-exchange column, hydrophobic interaction chromatography (HIC), ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.
  • purification techniques including but not limited to Protein A chromatography, fractionation on an ion-exchange column, hydrophobic interaction chromatography (HIC), ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or
  • the formulations of the invention may include buffers, tonicity modifiers, excipients, pharmaceutically acceptable carriers and other commonly used inactive ingredients of the pharmaceutical compositions. However, formulations that indicate that an ingredient is excluded (e.g. buffer- free formulations) do not include such ingredient.
  • Buffers maintain pH in a desired range, e.g., between pH 4 and pH 9. Buffers may also serve to stabilize aflibercept by a variety of other mechanisms, meaning they may be used outside of the nominal buffer capacity range indicated by their respective pKa values.
  • Suitable buffers include acetate (e.g., at pH 4 to 5.5), citrate (e.g., at pH 5 to 6.5), histidine (e.g., at pH 5 to 7), phosphate (e.g., at about pH 6 and 8), Tris (e.g., at pH 7 to 8), and glycine (e.g., at pH 8 to 9).
  • Specific embodiments include, without limitation, sodium or potassium phosphate, sodium or potassium citrate, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), various forms of acetate and diethanolamine.
  • Other suitable buffers include succinate, histidine, tartrate, bicarbonate, borate, imidazole, and maleate.
  • the concentration of the buffer in the formulation is between about 1 mM to about 1 M (e.g., about 1 mM to about 950 mM, about 1 mM to about 900 mM, about 1 mM to about 850 mM, about 1 mM to about 800 mM, about 1 mM to about 750 mM, about 1 mM to about 700 mM, about 1 mM to about 650 mM, about 1 mM to about 600 mM, about 1 mM to about 550 mM, about 1 mM to about 500 mM, about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to
  • the concentration of the buffer is between 1 mM to about 150 mM. In one embodiment, the concentration of the buffer is about 5 mM to about 20 mM. In one embodiment, the concentration of the buffer is about 10 mM.
  • Buffers are well known in the art and are manufactured by known methods and available from commercial suppliers. The selection of an appropriate buffer may be informed by data on any interactions of specific buffers with other formulation components. For example, US Patent 8,349,321 discloses that degradation of polysorbate 80 (thought to be due to a metal catalyzed oxidation of polysorbate) is significantly impeded by replacing histidine buffer with phosphate buffer in a tested placebo formulation.
  • the pH of the pharmaceutical compositions of the invention is generally between 5 and 7 (e.g., about 5.0 to about 6.9, about 5.0 to about 6.8, about 5.0 to about 6.7, about 5.0 to about 6.6, about 5.0 to about 6.5, about 5.0 to about 6.4, about 5.0 to about 6.3, about 5.0 to about 6.2, about 5.0 to about 6.1 , about 5.0 to about 6.0, about 5.0 to about 5.9, about 5.0 to about 5.8, about 5.0 to about 5.7, about 5.0 to about 5.6, about 5.0 to about
  • a strong acid e.g. HCI
  • a strong base NaOH
  • Excipients Suitable for use in Aflibercept Formulations include components of a pharmaceutical formulation other than the active ingredient and are typically added during formulation development for a specific purpose, such as to stabilize the polypeptide while in solution, change viscosity of the formulation, adjust tonicity, and confer other desired properties to the formulation. Excipients are well known in the art and are manufactured by known methods and available from commercial suppliers.
  • Excipients may include, for example, tonicity modifiers (e.g., any of the exemplary tonicity modifiers described herein), stabilizers (e.g., any of the exemplary stabilizers described herein), salts (e.g., any of the exemplary salts described herein), chelating agents (e.g., any of the exemplary chelating agents described herein), sacrificial additives and surfactants (e.g., any of the exemplary additives or surfactants described herein), and miscellaneous excipients such as ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta- mannoglycerate, and the like.
  • tonicity modifiers e.g., any of the exemplary tonicity modifiers described herein
  • the concentration of an excipient in a formulation of the invention is between about 0.001 to 30 weight percent (e.g., about 0.001 to about 28 weight percent, about 0.001 to about 26 weight percent, about 0.001 to about 24 weight percent, about 0.001 to about 22 weight percent, about 0.001 to about 20 weight percent, about 0.001 to about 18 weight percent, about 0.001 to about 16 weight percent, about 0.001 to about 14 weight percent, about 0.001 to about 12 weight percent, about 0.001 to about 10 weight percent, about 0.001 to about 9 weight percent, about 0.001 to about 8 weight percent, about 0.001 to about 7 weight percent, about 0.001 to about 6 weight percent, about 0.001 to about 5 weight percent, about 0.001 to about 4.5 weight percent, about 0.001 to about 4.0 weight percent, about 0.001 to about 3.5 weight percent, about 0.001 to about 3.0 weight percent, about 0.001 to about 2.5 weight percent, about 0.001 to about 2.0 weight percent, about 0.001 to about
  • weight percent about 1.5 to about 9 weight percent, about 1 .5 to about 8 weight percent, about 1.5 to about 7 weight percent, about 1.5 to about 6 weight percent, about 1.5 to about about 5 weight percent, about 1.5 to about 4.5 weight percent, about 1 .5 to about 4.0 weight percent, about 1.5 to about
  • weight percent about 2.0 to about 9 weight percent, about 2.0 to about 8 weight percent, about 2.0 to about 7 weight percent, about 2.0 to about 6 weight percent, about 2.0 to about about 5 weight percent, about 2.0 to about
  • the concentration of the excipient is about 0.01 to 10 weight percent.
  • the concentration of an excipient in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM
  • a tonicity modifier is an ingredient that contributes to the osmolality of a solution.
  • a tonicity modifier may also provide some degree of conformational or colloidal stabilization as well.
  • the osmolality of a pharmaceutical composition is adjusted to minimize discomfort to the patient upon administration. Additionally, osmolality of a composition may impact the active ingredient's stability and can be adjusted to improve the stability. It is generally preferred that a pharmaceutical composition for intravitreal administration to a patient be isotonic with vitreous, i.e. , have the same or similar osmolality, which is achieved by addition of a tonicity modifier. However, hypertonic formulations which would then be diluted in an isotonic vehicle are also within the scope of this invention.
  • the osmolality of the provided formulations is from about 180 to about 500mOsM (e.g., about 180 to about 450 mOsM, about 180 to about 400 mOsM, about 180 to about 350 mOsM, about 180 to about 300 mOsM, about 180 to about 250 mOsM, about 180 to about 200 mOsM, about 200 to about 500 mOsM, about 200 to about 450 mOsM, about 200 to about 400 mOsM, about 200 to about 350 mOsM, about 200 to about 300 mOsM, about 200 to about 250 mOsM, about 250 to about 500 mOsM, about 250 to about 450 mOsM, about 250 to about 400 mOsM, about 250 to about 350 mOsM, about 250 to about 300 mOsM, about 300 to about 500 mOsM, about 300 to about 450 mOsM, about 300 to about 400 mOsM, about 250
  • the osmolality is between about 200 and about 300 mOsM. However, it is to be understood that the osmolality can be either higher or lower as specific conditions require.
  • tonicity modifiers suitable for modifying osmolality include, but are not limited to amino acids (e.g., cysteine, arginine, histidine and glycine), salts (e.g., sodium chloride, potassium chloride and sodium citrate) and/or nonelectrolytes (e.g., sugars or polyols, such as, for example, sucrose, glucose and mannitol).
  • amino acids e.g., cysteine, arginine, histidine and glycine
  • salts e.g., sodium chloride, potassium chloride and sodium citrate
  • nonelectrolytes e.g., sugars or polyols, such as, for example, sucrose, glucose and mannitol.
  • the concentration of the tonicity modifier in the formulation is between about 1 mM to about 1 M (e.g., any of the subranges of this range described herein). In another embodiment, the concentration is about 50 mM to about 500 mM.
  • the concentration of the tonicity modifier in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM
  • Tonicity modifiers are well known in the art and are manufactured by known methods and available from commercial suppliers.
  • Stabilizers are a class of excipients that may stabilize a composition, and the active pharmaceutical ingredient, in various way including providing physical stability, conformational stability, and chemical stability.
  • types of excipients that are used as a stabilizer include sugars, polymers, polyols, amino acids, a block copolymer, an alkyl saccharide (e.g., a ProTek alkyl saccharide, octyl glucoside, N-decyl- pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos.
  • the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine.
  • sugars include sucrose, dextrose, maltose, lactose, raffinose, and trehalose.
  • polyols include sorbitol, maltitol, xylitol, and mannitol.
  • amino acids include glycine, arginine, glutamate, and proline (either as the free base form or as a salt form).
  • the invention further includes specific combinations of stabilizers that have particularly desirable properties. Examples include combinations of amino acids, e.g., Arg and Glu, as well as combinations of one or more amino acids with one or more polyols (e.g., Gly together with sorbitol or mannitol).
  • Improvements in formulation stability may be observed over a period of hours, days, months, or may last for extended periods, e.g., 6 months, 9 months, a year, or up to two years or more.
  • Stabilizers may improve stability of a formulation overfree-thaw cycles.
  • the invention provides a stable aqueous formulation comprising aflibercept and a sugar.
  • the formulation may further comprise a polyol and/or an amino acid.
  • sugars include sucrose, lactose, maltose, trehalose, raffinose, and glucose.
  • the composition may be a buffer-free and/or free of an organic co-solvent.
  • a sugar in the formulation improves the physical stability and other properties of specific aflibercept formulations (e.g., reduce tendency of aflibercept to associate in an undesirable conformation, and therefore reduce aggregation in aflibercept formulations).
  • a sugar may stabilize aqueous pharmaceutical compositions containing aflibercept.
  • the concentration of a sugar (e.g., sucrose) in the provided formulations is between about 0.01% (w/v) to 40% (e.g., about 0.01 % to about 35% w/v, about 0.01 % to about 30% w/v, about 0.01 % to about 25% w/v, about 0.01 % to about 20% w/v, about 0.01 % to about 18% w/v, about 0.01% to about 16% w/v, about 0.01% to about 14% w/v, about 0.01 % to about 12% w/v, about 0.01 % to about 10% w/v, about 0.01 % to about 9% w/v, about 0.01 % to about 8% w/v, about 0.01 % to about 7% w/v, about 0.01 % to about 6% w/v, about 0.01 % to about 5% w/v, about 0.01 % to about 4% w/v, about 0.01 % to about 0.01 %
  • the concentration of the sugar in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about
  • a formulation of the invention comprises about 10 to about 200 mg/mL (e.g., any of the subranges of this range described herein) of aflibercept; about 10 mM to about 350 mM (e.g., any of the subranges of this range described herein) sucrose; about 0.01 mM to about 100 mM (e.g., any of the subranges of this range described herein) sodium chloride; about 0.0001 % to about 0.1% (e.g., about 0.0001 % to about 0.05%, about 0.0001 % to about 0.01 %, about 0.0001 % to about 0.0005%, about 0.0005% to about 0.1 %, about 0.0005% to about 0.05%, about 0.0005% to about 0.01%, about 0.01% to about 0.1 %, about 0.01 % to about 0.05%, or about 0.05% to about 0.1%) surfactant; and about 0.1 to about 5% (e.g., about 0.1%) surfactant; and about
  • sucrose can be replaced with another sugar such as trehalose (at about 10 mM to about 350 mM, or any of the subranges of this range described herein) in the formulation.
  • sodium chloride can be replaced with another salt such as magnesium chloride (at about 0.01 mM to about 100 mM, e.g., any of the subranges of this range described herein or about 0.01 mM, about 0.1 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about
  • a formulation of the invention comprises about 10 to about 200 mg/mL (e.g., any of the subranges of this range described herein) of aflibercept; about 10 mM to about 350 mM (e.g., any of the subranges of this range described herein) sucrose; about 0.01 mM to about 100 mM (e.g., any of the subranges of this range described herein) sodium chloride; about 0.01 mM to about 10 mM buffer (e.g., about 0.01 mM to about 8 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.1 mM
  • the formulations of the invention may also include buffering agents, tonicity modifiers, excipients, and other commonly used inactive ingredients of the pharmaceutical compositions.
  • buffering agents tonicity modifiers, excipients, and other commonly used inactive ingredients of the pharmaceutical compositions.
  • the invention provides a stable aqueous formulation comprising aflibercept and a polyol.
  • the formulation may further comprise a sugar and/or an amino acid.
  • polyols include glycerol, sorbitol, mannitol, xylitol, and maltitol.
  • the composition may be a buffer-free and/or free of an organic co-solvent.
  • the concentration of a polyol in the provided formulations is between about 0.1% to 30% (e.g., about 0.1% to about 28%, about 0.1% to about 26%, about 0.1% to about 24%, about 0.1 % to about 22%, about 0.1% to about 20%, about 0.1% to about 18%, about 0.1 % to about 16%, about 0.1% to about 14%, about 0.1% to about 12%, about 0.1% to about 10%, about 0.1% to about 8%, about 0.1% to about 6%, about 0.1% to about 4%, about 0.1% to about 2%, about 0.1% to about 1 %, about 0.1% to about 0.5%, about 0.5% to about 30%, about 0.5% to about 28%, about 0.5% to about 26%, about 0.5% to about 24%, about 0.5% to about 22%, about 0.5% to about 20%, about 0.5% to about 18%, about 0.5% to about 16%, about 0.5% to about 14%, about 0.5% to about 12%, about 0.5% to about 10%, about 0.5% to about 0.5% to about 0.5% to
  • the concentration of the polyol in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM
  • Amino acids e.g., proline, serine, sodium glutamic acid, glutamate, alanine, histidine, tryptophan, tyrosine, arginine, glycine, lysine, methionine, threonine, glutamic acid, aspartic acid, sarcosine, glycine betaine, and mixtures of the foregoing may be employed as stabilizers in certain aflibercept formulations.
  • Amino acids e.g., proline, serine, sodium glutamic acid, glutamate, alanine, histidine, tryptophan, tyrosine, arginine, glycine, lysine, methionine, threonine, glutamic acid, aspartic acid, sarcosine, glycine betaine, and mixtures of the foregoing may be employed as stabilizers in certain aflibercept formulations.
  • compositions may use the free base form of the amino acid or any conjugate acid form such as a hydrochloride salt.
  • amino acids are readily available from commercial suppliers.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept and one or more amino acids, wherein the amino acid(s) is selected from the group consisting of serine, proline, glycine, alanine, glutamate, arginine and combinations thereof.
  • the composition may further optionally include a sugar and/or polyol.
  • the composition may be a buffer-free and/or free of an organic co-solvent.
  • Such additives may improve the properties of the aflibercept formulations, e.g., by reducing aggregation or tendency for aflibercept to associate in undesired ternary or quaternary complexes.
  • amino acids such as serine, proline and glutamate are able to stabilize aqueous pharmaceutical compositions containing aflibercept because they are excluded from the surface of the protein, resulting in net conformation stabilization.
  • the concentration of the amino acids in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM,
  • the concentration of the amino acid(s) is between about 10 mM and about 350 mM; in related embodiments, the concentration of the amino acid(s) is about 50 mM, 100 mM, 150 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, 300 mM, 320 mM and 340 mM, for example, 50-100 mM, 100-150 mM, 150-200 mM, 200- 300 mM, 200-250 mM, 250-300 mM, and 300-350 mM.
  • an aflibercept formulation comprises a sugar and a polyol.
  • amino acids such as proline, serine, or glutamate maybe used together with a sugar to achieve a stability profile better than either excipient could provide on its own.
  • the ratio of a sugar to a polyol (or amino acid) in the formulation is between 5:1 and 1 :5 (e.g., about 5:1 , about 4:1 , about 3:1 , about 2:1 , about 1 :1 , about 1 :2, about 1 :3, about 1 :4, or about 1 :5).
  • a formulation with a combination of stabilizers has better stability than a formulation with just one of the stabilizers. In another embodiment, a formulation with a combination of stabilizers has better stability than a formulation with just one of the stabilizers where the formulation with the combination of stabilizers has a lower total amount of stabilizers than the formulation with the single stabilizer (e.g. a formulation with 100 mM of sucrose and 50 mM arginine has better stability than a formulation with 200 mM sucrose).
  • Salts are often part of protein (e.g., antibody) formulations.
  • salts such as NaCI, Na2SC>4, CaCh, MgCh, and KCI, together with specific buffer combinations as described above, can confer particularly advantageous stability properties.
  • Such salts may be used with as well as without a sugar added (e.g., sucrose and NaCI) or a polyol added (e.g., mannitol and NaCI).
  • concentrations as low as 20 to 50 mM can provide decreased levels of proteolysis (such as hinge region hydrolysis), oxidation, deamidation, or other chemical instabilities.
  • sodium chloride may be replaced with Na2SC>4, KCI, MgCh, CaCh, MgSC , ZnCh, or other physiologically- acceptable salts.
  • Surfactants can confer protection against agitation and freeze/thaw damage, as well as stabilizing a formulation during storage.
  • Surfactants which may be employed with aflibercept formulations of the present invention include Tween®-80 (polysorbate 80, PS 80), Tween®-20 (polysorbate 20, PS 20), SDS, polysorbate, polyoxyethylene copolymer, Brij 35, Triton X-10, poloxamer 188 (Pluronic F-68), Pluronic F127, and Maltosides, e.g., n-Decyl- -D-maltopyranoside (DM), n-Dodecyl- -D- maltopyranoside (DDM), and 6-Cyclohexyl-1-hexyl- -D-maltopyranoside (Cymal-6).
  • surfactants that may be particularly advantageous include PS 20, PS 40, PS 60, and PS 80 at different concentrations, as well as DMM and poloxamer 188 (Pluronic F-68). Other zwitterionic or nonionic surfactants may be used as well.
  • optimal formulation employing such surfactants may also employ specific buffers at specific pH ranges, and may optionally include other tonicity modifier such as NaCI, polyol, or amino acid(s).
  • Polymers such as dextrans, starches (e.g., hydroxyl ethyl starch (HETA)), polyethylene glycols (PEGs), e.g., PEG-3350 or PEG-4000, may also provide stabilization to aflibercept, presumably by being excluded from the surface of the protein due to steric effects arising from their higher molecular weight.
  • hydrophilic polymers such as polyethylene glycols (PEGs), polysaccharides, and inert proteins, and may be used to stabilize proteins and enhance protein assembly. Examples include dextran, hydroxyl ethyl starch (HETA), PEG-4000, and gelatin.
  • the concentration of polymer is between 0.01% and 40% (e.g., about 0.01% to about 35%, about 0.01 % to about 30%, about 0.01 % to about 25%, about 0.01 % to about 20%, about 0.01% to about 18%, about 0.01% to about 16%, about 0.01% to about 14%, about 0.01% to about 12%, about 0.01% to about 10%, about 0.01% to about 8%, about 0.01 % to about 6%, about 0.01 % to about 5%, about 0.01 % to about 4.5%, about 0.01% to about 4.0%, about 0.01% to about 3.5%, about 0.01 % to about 3.0%, about 0.01 % to about 2.5%, about 0.01 % to about 2.0%, about 0.01 % to about 1.5%, about 0.01 % to about 1
  • polymers cannot be present if they are organic co-solvents (for example, PEGs).
  • aflibercept may be stable even in the absence of surfactants, and/or may be stabilized with surfactants other than PS80 and at lower surfactant concentrations.
  • certain polymers e.g., PEG, can exhibit surfactant-like properties and may be employed to stabilize aflibercept formulations in the absence of surfactants according to the present invention.
  • Additional polymers which may be employed in specific aflibercept formulations include serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextrans (e.g., cyclodextrin, e.g., Captisol®), poly(vinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), and 2-Hydroxypropyl-beta-cyclodextrin (HP-beta-CD).
  • serum albumin bovine serum albumin (BSA), human SA or recombinant HA)
  • dextrans e.g., cyclodextrin, e.g., Captisol®
  • PVA poly(vinyl alcohol
  • HPMC hydroxypropyl methylcellulose
  • HPMC polyethyleneimine
  • gelatin polyvinylpyrrolidone
  • Chelating agents such as EDTA, DPTA, etc., and/or sacrificial additives (e.g., ascorbate, Met), may be employed at specific pH values and with and without buffers (that may also acts as chelating agents, e.g., citrate, phosphate) to enhance the formulation properties, especially in cases where there may be some level of oxidative damage (under certain conditions, certain metals can catalyze the degradation of antibodies, especially at the hinge region).
  • a chelating agent such as EDTA and DPTA, may be beneficial at improving the storage stability of aflibercept.
  • Such approaches may be employed to stabilize aflibercept formulations according to the present invention.
  • Certain buffers such as citrate, may also function as chelating agents and can serve multiple purposes in stabilization of aflibercept.
  • Sacrificial additives are well known to diminish certain oxidation events, such as oxidation of methionine residues. Addition of the free amino acid, methionine, or some derivative, can lead to decreased oxidation of aflibercept. Ascorbate and various thiol derivatives can serve the same purpose. Likewise, Trp and its derivatives can also serve as a sacrificial additive, even in the case of photolytic oxidation.
  • the formulations of the invention can be prepared by isolating or purifying aflibercept or ziv-aflibercept from commercially available Eylea® or Zaltrap® or by modifying commercial aflibercept (Eylea®) or ziv-aflibercept (Zaltrap®) formulations to arrive at the formulations of the invention.
  • the formulation is prepared with aflibercept purified from a host cell that produces aflibercept.
  • the formulation of the invention is a biosimilar of Eylea.
  • compositions of the invention may be prepared by combining aflibercept and an excipient such as a stabilizer (e.g. sugar, amino acid). Further, a buffer (except in buffer-free formulations), an organic co-solvent, such as a surfactant (except in formulations free of organic co-solvents), a tonicity modifier and an additional excipient may be added as needed.
  • a buffer except in buffer-free formulations
  • an organic co-solvent such as a surfactant (except in formulations free of organic co-solvents)
  • a tonicity modifier may be added as needed.
  • the buffer can be added first, middle or last
  • the tonicity modifier can also be added first, middle or last.
  • Eylea is a stable formulation of aflibercept.
  • Eylea has a pH of 6.2 and contains 40 mg/mL aflibercept, 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose. Since this formulation is stable, it is surprising if an aflibercept formulation that is free of at least one of the specific excipients in Eylea, or a class of excipients that one of the Eylea excipients belongs to, is stable.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a buffer.
  • the buffer is a phosphate buffer.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a tonicity agent.
  • the tonicty agent is a salt.
  • the salt is sodium chloride.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent.
  • the organic co-solvent is a surfactant.
  • the surfactant is polysorbate 20.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a stabilizer.
  • the stabilizer is a sugar.
  • the sugar is sucrose.
  • a stable aflibercept formulation contains an excipient, it is free of other functionally similar excipients.
  • a stable aflibercept formulation containing trehalose is free of sucrose.
  • a stable aflibercept formulation containing acetate buffer is free of phosphate buffer.
  • a stable aflibercept formulation containing acetate buffer is free of histidine buffer.
  • the formulations of the present invention can be free of one or more excipients.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition comprises at least 90% monomer aflibercept.
  • the amount of monomer aflibercept is at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%.
  • the amount of monomer aflibercept is determined by SEC.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the pH of the composition is stable. In one embodiment the pH does not change more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% during storage.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more total sub-visible particles than Eylea. In another embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more sub-visible particles > 10 pm in diameter than Eylea. In another embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more sub-visible particles > 25 pm in diameter than Eylea. In another embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more sub-visible particles > 50 pm in diameter than Eylea.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition meets the requirments of USP ⁇ 789>, which is incorporated herein by reference in its enterity.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 50 particles per ml_ of particles that are > 10 pm in diameter, wherein the particles are counted by a light obscuration test.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 5 particles per ml_ of particles that are > 25 pm in diameter, wherein the particles are counted by a light obscuration test.
  • the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 50 particles per mL of particles that are > 10 pm in diameter, wherein the particles are counted by a miscroscopic method. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 5 particles per mL of particles that are > 25 pm in diameter, wherein the particles are counted by a miscroscopic method. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 2 particles per mL of particles that are > 50 pm in diameter, wherein the particles are counted by a miscroscopic method.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a buffer.
  • the term "free of buffer” should be understood to allow inclusion of the inherent buffering effect of the protein itself.
  • Such “self-buffering” or “buffer-free” protein formulations comprise a protein, e.g., a pharmaceutical protein, and are buffered by the protein itself, i.e., the formulations do not require additional buffering agents to maintain a desired pH.
  • the protein is substantially the only buffering agent in such formulations (i.e., other ingredients, if any, do not act substantially as buffering agents in the formulation).
  • the buffer-free aflibercept composition further comprises a stabilizer, a tonicity agent, an organic co-solvent, or combinations thereof.
  • the buffer-free formulation has a pH of about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
  • the pH is about 5.0 to about 5.8.
  • the PH is about 5.5.
  • the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
  • the organic co-solvent is selected from the group consisting of a surfactant, polyethylene glycol (PEG), propylene glycol, and combinations thereof.
  • the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, SDS, DDM and poloxamer 188 (Pluronic F-68).
  • the buffer-free formulations of the invention may comprise a salt, which may be selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
  • the salt is present at a concentration of about 10 mM-100 mM (e.g., any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM,
  • the stabilizer is sucrose.
  • the stabilizer is an amino acid, which may be selected from the group consisting of glycine, arginine, and proline or combinations thereof.
  • the amino acids may be present at a concentration of about 25-300 mM (e.g., any of the subranges of this range described herein or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230
  • the stabilizer is sucrose
  • the organic co solvent is polysorbate 20
  • the pH of the formulation is about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
  • the pH is about 5.0 to about 5.8.
  • the PH is about 5.5.
  • the formulation is iso-osmolar relative to the vitreous.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a buffer and the composition has an osmolality of about 200 to 400 mOsm.
  • the composition is suitable for administration to a subject as a single dose.
  • the single dose is about 2mg of aflibercept.
  • the aflibercept is present at about 40 mg/mL.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is substantially free of a buffer the composition has an osmolality of about 200 to 400 mOsm, the composition is suitable for administration to a subject as a single dose, and the single dose is about 2mg of aflibercept.
  • the single dose of aflibercept has a volume of about 0.05 ml_.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent.
  • the co-solvent-free aflibercept composition further comprises a buffer, a stabilizer, a tonicity agent, or combinations thereof.
  • the formulation has a pH of about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
  • the pH is about 5.5 to about 6.3. In another embodiment, the PH is about 6.2.
  • the buffer is selected from the group consisting of acetate, histidine, phosphate, citrate, succinate, tartrate and maleate. In one embodiment, the buffer is present at a concentration of about 1 mM to 150 mM (e.g., any of the subranges of this range described herein).
  • the buffer is present at a concentration of about 5 mM to 20 mM. In another embodiment, the buffer is present at a concentration at about 10 mM.
  • the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof. [00198] In some organic co-solvent-free formulations, the stabilizer is sucrose.
  • the stabilizer is an amino acid, which may be selected from the group consisting of glycine, arginine, and proline or combinations thereof.
  • the amino acid may be present at a concentration of about 25-300 mM (e.g., any of the subranges of this range described herein or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 2
  • the formulation may comprise a salt, which may be selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
  • the salt is present at a concentration of about 10 mM-100 mM (e.g., any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95
  • the formulation is iso-osmolar relative to the vitreous.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent and the composition has an osmolality of about 200 to 400 mOsm.
  • the composition is suitable for administration to a subject as a single dose.
  • the single dose is about 2mg of aflibercept.
  • the aflibercept is present at about 40 mg/mL.
  • the invention provides a stable aqueous pharmaceutical composition
  • aflibercept wherein the composition is free or substantially free of an organic co-solvent, the composition has an osmolality of about 200 to 400 mOsm, the composition is suitable for administration to a subject as a single dose, and the single dose is about 2mg of aflibercept.
  • the single dose of aflibercept has a volume of about 0.05 ml_.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is substantially free of both an organic co-solvent and a buffer.
  • the buffer-free and co-solvent-free aflibercept composition further comprises a stabilizer, a tonicity agent, or combinations thereof.
  • the formulation has a pH of about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
  • the pH is about 5.0 to about 6.2. In another embodiment, the pH is about 5.5.
  • the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
  • the stabilizer is sucrose.
  • the stabilizer is an amino acid, which may be selected from the group consisting of glycine, arginine, and proline or combinations thereof.
  • the amino acids may be present at a concentration of about 25-300 mM (e.g., any of the subranges of this range described herein or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230
  • the formulation may comprise a salt, which may be selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
  • the salt is present at a concentration of about 10 mM-100 mM (e.g., any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95
  • the formulation is iso-osmolar relative to the vitreous.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of both a buffer and an organic co-solvent and the composition has an osmolality of about 200 to 400 mOsm.
  • the composition is suitable for administration to a subject as a single dose.
  • the single dose is about 2mg of aflibercept.
  • the aflibercept is present at about 40 mg/mL.
  • the single dose of aflibercept has a volume of about 0.05 ml_.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of both a buffer and an organic co-solvent, the composition has an osmolality of about 200 to 400 mOsm, the composition is suitable for administration to a subject as a single dose, and the single dose is about 2mg of aflibercept.
  • the invention provides a stable aqueous pharmaceutical composition
  • a stabilizer is a sugar, polyol, amino acid, salt, or a combination thereof.
  • the sugar is sucrose.
  • the salt is NaCI or CaCI2.
  • the amino acid is glycine.
  • the organic so-solvent is a surfactant.
  • the surfactant is a polysorbate.
  • the polysorbate is polysorbate 20 or polysorbate 80.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, sucrose, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, sucrose, a salt selected from the group consisting of NaCI and CaCI2, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, an amino acid, a salt selected from the group consisting of NaCI and CaCI2, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer.
  • the amino acid is glycine.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, sucrose, CaCI2, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer.
  • the composition is free of NaCI.
  • the invention provides a stable aqueous pharmaceutical composition
  • aflibercept a buffer, and a stabilizer
  • the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent.
  • the buffer is histidine or acetate.
  • the stabilizer is a sugar, polyol, amino acid, or a combination thereof.
  • the sugar is sucrose.
  • the amino acid is glycine.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, a buffer, and sucrose, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, a buffer, and an amino acid, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co solvent.
  • the amino acid is glycine.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, histidine, and sucrose, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent.
  • the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, histidine, and glycine, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent.
  • a co-solvent-free composition further comprises a salt.
  • the salt is selected from the group consisting of NaCI and CaCI2.
  • exemplary aflibercept formulations are listed in Table 1.1 below.
  • Formulations in Table 1.1 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co- solvent-free. All formulations Table 1.1 contain 40 mg/mL aflibercept.
  • the formulation can include aflibercept
  • a sugar e.g., sucrose
  • about 1 mM to about 60 mM e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 60 mM, about 10 mM to about 50
  • the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 0.01 % w/v to about 0.1 % w/v (e.g., about 0.01 % w/v to about 0.05% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., any of the polysorbates described herein); having a pH of about 5.5 to about 6.2 (e.g., about 6.0), and is
  • the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 0.1 mM to about 10 mM (e.g., about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 8
  • the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); and is free of both a buffer and an organic co-solvent.
  • aflibercept e.g., at any of the final concentrations described herein
  • about 0.5% w/v to about 6% w/v e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0%
  • the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM, about 5 mM, about
  • the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 5 mM, about
  • the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM, about 5 mM, about
  • exemplary aflibercept formulations are listed in Table 1 .2 below.
  • a surfactant e.g. polysorbate 80
  • Formulations in Table 1.2 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co-solvent-free All formulations Table 1.2 contain 40 mg/mL aflibercept.
  • Captisol if present in the formulation, is present at about 0.1 % to about 2% w/v.
  • CaCh if present in the formulation, is present at about 1 to about 10 mM.
  • hydroxypropyl-beta-cyclodextrin if present in the formulation, is present at about 0.1% to about 2% w/v.
  • human serum albumin if present in the formulation, is present at about 0.01 % to about 2% w/v.
  • stable formulations comprise aflibercept and low concentration of specific salts (calcium chloride, magnesium chloride and potassium chloride) within the range present in human vitreous humor.
  • the formulation comprises a stabilizer such as a sugar (e.g. sucrose) or Human Serum Albumin (e.g. recombinant HAS).
  • the formulation is free of an organic co-solvent.
  • rHSA is the most common protein found in human plasma and is known for ligand binding and colloidal stabilization, properties that are also ideal for the stabilization of proteins and other biologies.
  • rHSA is a natural protein in the body, it is safer than alternative surfactant excipients, such as synthetic polymers, because it has a low risk for immunogenicity and there is a natural pathway for its elimination from the body.
  • exemplary aflibercept formulations are listed in Table 1.3 below.
  • Formulations in Table 1.3 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co- solvent-free. All formulations Table 1 .3 contain 40 mg/mL aflibercept.
  • exemplary aflibercept formulations are listed in Table 1.4 below.
  • Formulations in Table 1.4 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co- solvent-free. All formulations Table 1 .4 contain 40 mg/mL aflibercept and 5%w/v sucrose.
  • the chemical and physical stability of the aflibercept protein in a formulation is measured using, e.g., SEC, CEX, HPLC, RP, UV, pH, MFI, Flow-CAM, icIEF, CE-IEF and CE-SDS.
  • SEC chemical and physical stability of the aflibercept protein in a formulation
  • CEX chemical and physical stability of the aflibercept protein in a formulation
  • HPLC high-LC
  • RP UV
  • pH pH
  • MFI Flow-CAM
  • icIEF Flow-CAM
  • CE-IEF Flow-CAM
  • CE-IEF CE-IEF
  • CE-SDS CE-SDS
  • biophysical techniques such as those described by Jiskoot and Crommelin (Methods for Structural Analysis of Protein Pharmaceuticals, Springer, New York, 2005).
  • spectroscopic analyses e.g., second derivative ultraviolet spectroscopy, circular dichroism, Fourier Transform infrared spectroscopy, Raman spectroscopy, fluorescence and phosphorescence spectroscopy
  • thermal analyses e.g., differential scanning calorimetry
  • size-based analyses e.g., analytical ultracentrifuge, light scattering
  • Formulations of the present invention may be used in methods of treating an ocular condition in a subject in need thereof.
  • the subject is a human patient.
  • treating comprises administering a therapeutically effective amount of the formulations of the invention to the subject.
  • the subject has an ocular disease or disorder that can be beneficially treated with aflibercept, for example any ocular disease or disorder that can benefit from an anti-VEGF injection.
  • the ocular disease or disorder is a vascular eye disease or disorder.
  • Ocular diseases or disorders that can be treated with the formulations of the present invention includes, but is not limited to, those which can be treated with Eylea®, such as macular degeneration (AMD), diabetic macular edema (DME), retinal vein occlusion (RVO), diabetic retinopathy (DR).
  • Eylea® such as macular degeneration (AMD), diabetic macular edema (DME), retinal vein occlusion (RVO), diabetic retinopathy (DR).
  • the formulations of the present invention are administered intravitreally.
  • the terms “treat”, “treating”, or the like mean to alleviate symptoms, eliminate the causation of symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of an eye disease.
  • the present methods are useful for treating or ameliorating at least one symptom or indication including, but not limited to, retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 pm of the center of the fovea, hard, yellow exudates within 500 pm of the center of the fovea with adjacent retinal thickening, and at least 1 disc area of retinal thickening, any part of which is within 1 disc diameter of the center of the fovea, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
  • the term means that, from the initiation of treatment, the patient exhibits gain of one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) letters on the Early Treatment Diabetic Retinopathy Study (EDTRS) visual acuity chart.
  • ETRS Early Treatment Diabetic Retinopathy Study
  • the term means that, from initiation of treatment, vision loss of greater than or equal to 15 letters is prevented in the patient.
  • the terms “prevent”, “preventing”, or the like mean to prevent development of a symptom, indication or a complication of a vascular eye disease.
  • a vascular eye disease such as AMD or DME
  • the term means, from initiation of treatment, moderate or severe vision loss is prevented in a patient.
  • vascular eye disease or disorder refers to eye disease or disorders that affect blood vessels in the eye.
  • the diseases may be caused due to abnormal angiogenesis (formation of new blood vessels) or occlusion or blockage of blood vessels.
  • the term includes eye diseases or disorders associated with angiogenesis.
  • the term includes, but is not limited to, eye disease or disorder selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, and choroidal neovascularization.
  • the term "neovascular eye disease or disorder” may be used interchangeably with the term "eye disease or disorder associated with angiogenesis.”
  • the present invention includes methods for treating, preventing, or ameliorating at least one symptom or indication of an eye disease or disorder associated with angiogenesis in a subject, wherein the disease or disorder is selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, polypoidal choroidal vasculopathy, and choroidal neovascularization.
  • DME Diabetic Macular Edema
  • a serious eye condition that affects people with diabetes type 1 or 2.
  • Macular edema occurs when blood vessels in the retina leak into the macula and fluid and protein deposits collect on or under the macula of the eye (a yellow central area of the retina) and causes it to thicken and swell (edema). The swelling may distort a person's central vision, as the macula is near the center of the retina at the back of the eyeball.
  • the primary symptoms of DME include, but are not limited to, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
  • DME The pathology of DME is characterized by breakdown of the blood-retinal barrier, normally preventing water movement in the retina, thus allowing fluid to accumulate in the retinal tissue, and presence of retinal thickening.
  • DME is presently diagnosed during an eye examination consisting of a visual acuity test, which determines the smallest letters a person can read on a standardized chart, a dilated eye exam to check for signs of the disease, imaging tests such as optical coherence tomography (OCT) or fluorescein angiography (FA) and tonometry, an instrument that measures pressure inside the eye.
  • OCT optical coherence tomography
  • FA fluorescein angiography
  • tonometry an instrument that measures pressure inside the eye.
  • DME can be broadly characterized into two main categories-Focal and Diffuse.
  • Focal DME is characterized by specific areas of separate and distinct leakage in the macula with sufficient macular blood flow.
  • Diffuse DME results from leakage of the entire capillary bed surrounding the macula, resulting from a breakdown of the inner blood-retina barrier of the eye.
  • DME is also categorized based on clinical exam findings into clinically significant macular edema (CSME), non-CSME and CSME with central involvement (CSME-CI), which involves the fovea.
  • CSME clinically significant macular edema
  • CSME-CI central involvement
  • the present invention includes methods to treat the above-mentioned categories of DME.
  • Age-related macular degeneration refers to a serious eye condition when the small central portion of the retina, known as the macula, deteriorates.
  • the wet form of AMD is characterized by the growth of abnormal blood vessels from the choroid underneath the macula. This is called choroidal neovascularization. These blood vessels leak blood and fluid into the retina, causing distortion of vision that makes straight lines look wavy, as well as blind spots and loss of central vision. These abnormal blood vessels eventually scar, leading to permanent loss of central vision.
  • the symptoms of AMD include dark, blurry areas in the center of vision; and diminished or changed color perception. AMD can be detected in a routine eye exam.
  • One of the most common early signs of macular degeneration is the presence of drusen-tiny yellow deposits under the retina-or pigment clumping.
  • a subject in need thereof means a human or non-human mammal that exhibits one or more symptoms or indications of, and/or who has been diagnosed with an eye disease or disorder associated angiogenesis.
  • the term "a subject in need thereof” may also include, e.g., subjects who, prior to treatment, exhibit (or have exhibited) one or more indications of a neovascular eye disease such as, e.g., retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 pm of the center of the fovea, hard, yellow exudates within 500 pm of the center of the fovea with adjacent retinal thickening, and at least 1 disc area of retinal thickening, any part of which is within 1 disc diameter of the center of the fovea, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
  • a neovascular eye disease such as, e.g., retinal angiogenesis, neovascularization, vascular leak
  • a "subject in need thereof” also includes human or non-human mammal who has a vascular eye disease or disorder selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, and choroidal neovascularization.
  • a vascular eye disease or disorder selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, and choroidal neovascularization.
  • a subject in need thereof may include a subset of population which is more susceptible to DME or AMD or may show an elevated level of a DME-associated or an AMD- associated biomarker.
  • a subject in need thereof may include a subject suffering from diabetes for more than 10 years, have frequent high blood sugar levels or high fasting blood glucose levels.
  • the term "a subject in need thereof” includes a subject who, prior to or at the time of administration of the formulations of the invention, has or is diagnosed with diabetes.
  • the term “a subject in need thereof” includes a subject who, prior to or at the time of administration of the formulations of the invention, is more than 50 years old.
  • the term "a subject in need thereof” includes subjects who are smokers, or subjects with high blood pressure or high cholesterol.
  • the present invention includes methods for treating, preventing or reducing the severity of a vascular eye disease comprising administering a therapeutically effective amount of a formulation of the present invention to a subject in need thereof, wherein the formulation is administered to the subject as a single dose or in multiple doses, e.g., as part of a specific therapeutic dosing regimen.
  • the therapeutic dosing regimen may comprise administering multiple doses of the formulation to the subject at a frequency of about once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or less frequently.
  • the therapeutic dosing regimen may comprise administering multiple doses of the formulation to the subject at a frequency of once a day or 2 times a day or more.
  • methods of treating include administering to a subject in need thereof an aflibercept composition of the present invention according to the dosing and administration regimen for Eylea®. See, Eylea® package insert.
  • a subject with Neovascular (Wet) Age- Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 28 days, monthly) for the first 3 months, followed by 2 mg (0.05 ml_) via intravitreal injection once every 8 weeks (2 months).
  • a subject with Neovascular (Wet) Age-Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 25 days, monthly).
  • a subject with Neovascular (Wet) Age-Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 4 week (monthly) dosing after the first 12 weeks (3 months).
  • a subject with Neovascular (Wet) Age-Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 12 weeks after one year of effective therapy.
  • a subject with Macular Edema Following Retinal Vein Occlusion is administered 2 mg of aflibercept by intravitreal injection once every 4 weeks (approximately every 25 days, monthly).
  • a subject with Diabetic Macular Edema (DME) or Diabetic Retinopathy (DR) in Patients with Diabetic Macular Edema is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 28 days, monthly) for the first 5 injections followed by 2 mg via intravitreal injection once every 8 weeks (2 months).
  • a subject with Diabetic Macular Edema (DME) or Diabetic Retinopathy (DR) in Patients with Diabetic Macular Edema is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 25 days, monthly).
  • a subject with Diabetic Macular Edema (DME) or Diabetic Retinopathy (DR) in Patients with Diabetic Macular Edema is administered 2 mg of aflibercept by intravitreal injection every 4 week (monthly) dosing after the first 20 weeks (5 months).
  • the formulations of the invention may be administered in combination with other ocular therapy, for example, laser treatment.
  • other ocular therapy for example, laser treatment.
  • the phrase ' in combination with means that the formulations of the invention may be administered to the subject at the same time as, just before, or just after other therapy including laser treatment.
  • the present invention also includes methods for inhibiting or reducing or suppressing vascular leak in a subject.
  • the methods according to this aspect of the invention comprise administering to the subject one or more doses of a pharmaceutical formulation of the invention to reduce or inhibit vascular leak in the eye of a subject.
  • the methods comprise administering to the subject one or more doses of a formulation of the invention.
  • the vascular leak is inhibited for more than 3 weeks, more than 4 weeks, more than 8 weeks, or more than 10 weeks than in a subject who has not been administered the formulations of the invention.
  • the methods comprise administering an initial dose of the pharmaceutical composition, followed by one or more secondary doses, wherein each secondary dose is administered 1 to 4 weeks after the immediately preceding dose.
  • one or more tertiary doses of the pharmaceutical composition are administered, wherein each tertiary dose is administered 5 to 12 weeks after the immediately preceding dose.
  • each dose of the pharmaceutical composition may comprise about 2 mg of the VEGF antagonist.
  • the methods of the present invention demonstrate efficacy within 36 weeks of the initiation of the treatment regimen (with the initial dose administered at "week 0"), e.g., by the end of week 6, by the end of week 12, by the end of week 18, by the end of week 24, etc.
  • "efficacy” means that, from the initiation of treatment, the patient exhibits a loss of 10 or fewer letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart.
  • EDRS Early Treatment Diabetic Retinopathy Study
  • "efficacy” means a gain of one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) letters on the ETDRS chart from the time of initiation of treatment.
  • the formulations of the invention may be intravitreally administered to a subject (e.g., a human) with an aflibercept concentration of about 0.01 mg/mL to about 200 mg/mL. In another embodiment, the concentration of aflibercept is about 0.1 mg/mL to about 100 mg/mL. In another embodiment, the concentration of aflibercept is about 1 mg/mL to about 60 mg/mL. In another embodiment, the concentration of aflibercept is about 40 mg/mL.
  • the formulations of the invention may be prepared in a bulk formulation, and as such, the components of the bulk formulation are adjusted to be higher than would be required for administration and then diluted appropriately prior to administration.
  • the invention includes a container comprising a formulation of the present invention.
  • the container is a vial.
  • the container is a syringe.
  • the syringe is silicone free.
  • the internal surface of the syringe is coated with baked-on silicone.
  • the invention includes pre-filled syringes suitable for intravitreal administration wherein the pre-filled syringes comprise the formulations of the invention.
  • the pre-filled syringe is filled a volume of a formulation that delivers 2mg of aflibercept upon administration.
  • the 2mg of aflibercept is delivered in 0.05 ml_.
  • the syringe is over-filled.
  • the syringe is coupled to a needle.
  • the needle is a 30-gauge needle.
  • the pre-filled syringe is a glass syringe. In another embodiment, the pre-filled syringe is a plastic syringe. In another embodiment, the pre-filled syringe is a glass syringe with an internal non glass coating.
  • a formulation of the present invention is in a container that has no headspace.
  • a closed container with no headspace interfacial stress on the formulation is reduced and the formulation is stabilized.
  • Such containers and methods of preparing a container with no headspace that comprises a formulation are known in the art. See, US 20180043020 which is hereby incorporated by reference in its enterity.
  • the methods of administration include administering the formulations of the invention to a subject in need thereof every four weeks for the first three months, and then once every eight weeks.
  • dose is about 2 mg of aflibercept (for example 0.05 ml_ of an aqueous solution containing 40 mg/mL aflibercept).
  • T reatment for longer periods may be necessary to induce the desired degree of improvement.
  • the regimen may be continued indefinitely.
  • the pharmaceutical compositions may be administered as a sole therapeutic or in combination with additional therapies as needed.
  • the provided methods of treatment and/or prevention are used in combination with administering a therapeutically effective amount of another active agent.
  • the other active agent may be administered before, during, or after administering the pharmaceutical compositions of the present invention.
  • Another active agent may be administered either as a part of the provided compositions, or alternatively, as a separate formulation.
  • the formulations of the invention can be administered in ways other than intravitreal administration, for example parenteral, oral, buccal, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, and topical application.
  • compositions may, if desired, be presented in a vial, pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the dispenser device can comprise a syringe having a single dose of the liquid formulation ready for injection.
  • the syringe can be accompanied by instructions for administration.
  • the present invention is directed to a kit or container, which contains an aqueous pharmaceutical composition of the invention.
  • concentration of the polypeptide in the aqueous pharmaceutical composition can vary over a wide range, but is generally within the range of from about 0.05 to about 20,000 micrograms per milliliter (pg/ml) of aqueous formulation.
  • the kit can also be accompanied by instructions for use.
  • Aflibercept formulations may be prepared as described more fully below.
  • the formulations are typically grouped into blocks, where each block is described in the context of one Example or portion of one Example.
  • the formulations are exposed to storage conditions, for example, 1 week at 40° C, 2 weeks at 25° C, and 4 weeks at 5° C. Storage at 40°C is an accelerated model which indicates that a formulation will be stable at lower temperatures for longer period of times.
  • Aflibercept formulations of the present invention are compared to the aflibercept formulation for Elyea®.
  • Eylea® has a pH of 6.2 and contains 40 mg/mL aflibercept, 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose.
  • the chemical and physical stability of the aflibercept protein may be measured by, e.g., SEC, HIC, CEX, AF4, RP, UV, pH, CE-IEF and CE-SDS.
  • SEC chemical and physical stability of the aflibercept protein
  • Tween 20 was removed from Zaltrap (comcerically available aflibercept formulation) using a protein A column. The processed material was buffer exchanged into 10 mM sodium phosphate at pH 6.2. TFF was used to buffer exchange the bulk material and the concentrate sample. The parameters below were used to process the material by TFF for buffer exchange and concentration.
  • the TFF system was stored in 0.1% NaoH, this was washed out with the exchange buffer which was 10 mM sodium phosphate, pH 6.2. To ensure that sodium hydroxide was been flushed from the system and the lines, the pH was checked in the tank and the waste line. After flushing the system the material was added to the tank and the flow was changed to pass through the Pellicon ® 3.
  • the processed material was added the tank and diluted with the dialysis buffer to 500ml_. After buffer exchange the sample volume was reduced to 100 ml_, concentrating aflibercept.
  • the material was UF/DF vs 2000 mL of 10 mM phosphate, pH 6.2, using the following parameters: Stir 30%; Pump 30%; TMP target 14.1 pSI; DP target 28.1 pSI.
  • FMA is a method that quantifies polysorbates (Tweens) by the interaction of a fluorescence dye with the surfactant micelles.
  • PS20 polysorbate 20
  • the UF/DF processed bulk aflibercept material was formulated by dialysis using a Slide-A-Lyzer ® Mini Dialysis 20K MWCO, 2ML.
  • the Slide- A-Lyzer® Mini Dialysis was prepared by hydrating the membrane using the formulation buffer. After the membrane was hydrated the liquid was removed and replaced UF/DF processed bulk aflibercpt material. Slide-A-Lyzer then shacked using an orbital rotator set to 300 rpm for 48 to 72 hours at 5-8 °C. The dialysis buffer was changed out 3 times during this procedure at 4, 24 and 48 hours.
  • the sample was concentrated by spin concentrator.
  • the spin concentrator was first hydrated by 4 mL of water pass through the membrane. The sample was then loaded into the spin concentrator and rotated at 2,000 g for 7 to 13 minutes. The sample was concentrated above the target concentration of 40 mg/mL and was then diluted to be within 10% of the target protein concentration for the formulation.
  • the concentration of each formulation was measured using the Solo VP.
  • the concentration of the sample was measured by added 50 to 100 pl_ of material into a SoloVP small UV disposable vessel.
  • a new fibrette was installed and the sample absorbance was measured by the instrument, using an extinction coefficient of 1 .55 mL. mg 1 cm 1 , and correcting background scattering.
  • the sample is removed with a pipette the disposable vessel and fibrette are both disposed. This procedure is repeated for each sample.
  • the pH will be checked for each formulation and be within ⁇ 0.1 of the target pH.
  • the pH probe was calibrated with three pH standards ordered from fisher. The pH of the formulation will be measured by inserting the pH probe in to the sample and waiting until the measured value has stabilized, which can take up to 1 to 2 minutes. After the analysis the pH probe is washed with 18 W water for one minute and stored in the pH storage solution.
  • the osmotic analysis was performed using an Advanced Instruments, Osmo 1.
  • Osmo 1 Advanced Instruments
  • a reference standard at 290 mOsm is analyzed to insure the instrument is working properly.
  • the samples are then analyzed.
  • 20ul_ of material is removed and analyzed by the Osmo 1 , after analysis the chamber is cleared by a using a chamber cleaner. This procedure is repeated for each sample.
  • the bulk processed aflibercept material was formulated following the procedures below.
  • the aflibercept material was buffered exchanged following the dialysis procedure. After dialysis the osmotic pressure and the pH was checked, if the pH of the sample was not within ⁇ 0.1 pH units, the formulation buffer was made more acidic or basic, by repeat dialysis or by addition of HCI or NaOH, until the pH target was reached. Following pH adjustment if needed, the sample was concentrated above the target formulation concentration. The pH, osmotic pressure and the protein concentration was then measured once more. The sample was then diluted with the formulation buffer to reached the target formulation protein concentration within 10%. The protein concentration was then measured once more to insure the diluted sample was within 10% of the target concentration. The last step was the addition of the 20% PS 20 (diluted in water) to the sample based on weight, if required.
  • SEC Size exclusion chromatography
  • Micro Flow Imaging analysis was conducted. The MFI analysis was performed on a MFI 5200 Protein Simple system following the procedure described below.
  • Sample Preparation Samples were placed in the BSC and allowed to equilibrate to room temperature for about 30 minutes. Using Neptune Barrier Tips (1000uL Barrier Tip, Cat#1000.96N), samples were diluted 1 :1 with MilliQ Water, specifically 190uL MilliQ water + 190uL Samples. All samples were degassed for 20min @ 70cmHg.
  • MFI Analysis Prior to analysis, approximately 10mL of MilliQ water was flushed through the MFI system using a 100uM FlowCell (FlowCell, 100uM, 1.6mm, Silane Coating). For each sample, 350 pL was pipetted and sample-filled pipette was placed in the MFI inlet port. 0.03-0.05 ml_ manual purge (by volume) was performed followed by selecting Optimize illumination” to calibrate and set the background. Analysis was started by selecting “Start analysis”. -A total of 180 ul of sample was analyzed for each measurement. After each measurement, 1-4ml of MilliQ water was flushed through the flow-cell.
  • SVPs subvisible particles
  • MFI micro-flow imaging
  • Imaged capillary isoelectric focusing was used to quantify the charge distribution for the formulations.
  • the icIEF analysis was performed on aniCE3 Protein Simple system following the procedure described below.
  • icIEF Matrix Preparation The icIEF reagents were removed from 5°C storage and placed at ambient temperatures for 30 minutes. The following icIEF Matrix preparation was prepared for N+1 samples and all preparations were thoroughly mixed.
  • icIEF Sample Preparation and icIEF Analysis For each sample and reference standard, 180uL of iciEF Matrix was added to 20uL of working sample. For blank preparation, 20uL of MilliQ water was combined with 180ul_ of icIEF matrix. Prepared sample was thoroughly mixed and centrifuge for at 3000g for 10-15 seconds. The sample was analyzed by the iCE3 instrument with a Pre-Focus of 1500V for 1 minute and a Focus of 3000V for 15minutes.
  • Formulations are filled into containers (e.g. a syringe) then packed into cardboard containers separated by corrugated cardboard. The containers are then shipped at least 1 ,000 miles by truck over paved roads. During shipment, the containers are kept at a constant temperature or within a suitable temperature range (e.g. +/- 10% of target temperature). The temperature may be 25°C, 2-8°C, 0°C, or freezing (i.e. below 0°C). After reaching their destination the formulations in the containers are inspected for particles (e.g. visible particles and sub-visible particles).
  • particles e.g. visible particles and sub-visible particles.
  • a drop test is performed.
  • Formulations are filled into containers (e.g. a syringe) then packed into cardboard containers separated by corrugated cardboard.
  • the containers are then subjected to a dropping test.
  • the dropping test may be conducted at 2-8°C or around room temperature (e.g. 25 °C).
  • the container is dropped in various orientations.
  • a cardboard container holding a pre-filled syringe containing a formulation is dropped a total of 16 times from a height of 36 inches.
  • the 16 drops include: 1) dropping the container on each of its 6 faces; 2) dropping the container on each of the 4 edges with the needle (or needle connector) pointing in the horizontal position (i.e. perpendicular to surface the container is being dropped on); 3) dropping the container on 1 edge with the needle pointing upward; 4) dropping the container on 1 edge with the needle pointing downward; 5) dropping the container on 2 separate corners with the needle generally pointing downward; and 6) dropping the container on 2 separate corners with the needle generally pointing upward.
  • the formulations in the containers are inspected for particles (e.g. visible particles and sub-visible particles).
  • Partial least squares regression was used to shed additional light on the effects of the various formulation parameters on stability of the formulations.
  • X-matrix For any large matrix of values, where there are a reasonable number of samples (together forming the so-called X-matrix), mathematical models can be constructed that explain the largest amount of variance in the dependent variable(s) of interest (the Y-matrix).
  • the best single description of the relationship between the variation in the X-matrix and the endpoint (the Y matrix) is called the first principal component, PC1 .
  • the next important (in terms of describing the variance in the Y-matrix) component is called the second principal component, PC2, and so on.
  • PCs are required to explain most of the variance in the Y-matrix.
  • Each of these PCs contains some contribution from each of the variables in the X-matrix. If a variable within the X- matrix contributes heavily to the construction of a given PC, then it is ranked as being significant.
  • regression coefficients can be calculated for each variable in the X-matrix for a given model, where a model is the composite of a certain number of PCs in order to provide an adequate description of the Y-matrix.
  • PLS takes information from the X- matrix, calculates the desired number of PCs, and constructs a suitable model.
  • the model that includes all of the samples is termed a calibration model.
  • the overall coefficient of determination (r 2 ) indicates the quality of the model. All PLS calculations were conducted using Unscrambler ® software (CAMO, Corvallis, OR). A PLS analysis done with a single variable in the Y-matrix is termed PLS1 analysis. Building a model that fits multiple variables in the Y-matrix is called PLS2 analysis.
  • a full cross validation was performed on all calibration models using standard techniques. Briefly, one sample is removed at a time, the data set is recalibrated, and a new model is constructed. This process is repeated until all of the calibration samples are removed once and quantified as a validation model. Therefore, the first set, containing all samples is referred to as the calibration set and the one after cross-validation as the validation set.
  • the jack-knife algorithm was used to determine statistical significance for any factor used in constructing the PLS models described above.
  • formulations may be maintained at an indicated temperature, e.g., 40° C, 25° C, 2-8° C, or a freezing temperature (e.g. 0 °C and below) for a period of time, e.g., days, weeks, or months, and assessed for stability and/or other parameters.
  • the pH, conductivity and/or viscosity of the samples may be measured at time zero and periodically throughout the experiment.
  • the material may be analyzed for stability and other properties using, e.g., chromatography or other methods; non-limiting examples include SEC, RP, HIC, CEX, AF4 and/or CE-SDS. These analyses are typically carried out at time zero and then periodically during the experiment.
  • the samples may also be assessed by starting the experiment at one temperature, e.g., 40° C, and then stepping or ramping to a different temperature, e.g., 25° C, and/or 2-8° C, for a selected period of time.
  • the samples may be stored at -20° C or 2 months; and/or at 40° C for 1 and 2 weeks; and/or at 25° C for 2 and 4 weeks, and/or at 2-8° C for 2 months.
  • a drop test can be carried out at 25° C.
  • Aflibercept may be prepared either de novo or through purification/isolation from a commercially available source. The steps below describe production of aflibercept by culturing cells transformed or transfected with a vector containing aflibercept nucleic acid. Alternative methods, which are well known in the art, may also be employed to prepare aflibercept. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et ai, Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)].
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of aflibercept may be chemically synthesized separately and combined using chemical or enzymatic methods to produce aflibercept.
  • DNA encoding aflibercept is inserted into an expression vector appropriate for the host cell to express aflibercept.
  • Host cells transfected or transformed with expression or cloning vectors described herein for aflibercept production is cultured in nutrient media modified as appropriate for inducing promoters, selecting transformants, and/or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, are selected by the skilled artisan using well-known approaches without undue experimentation. For example, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrookef a/., supra.
  • Aflibercept may be expressed in a mammalian cell line, such as a CHO cell and may be modified post-translationally.
  • Aflibercept is recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g Triton-X 100) or by enzymatic cleavage. Cells employed in expression of aflibercept can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • a suitable detergent solution e.g Triton-X 100
  • Cells employed in expression of aflibercept can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • aflibercept from recombinant cell proteins or polypeptides.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation- exchange resin such as DEAE; chromatofocusing; SD S-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG.
  • Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982).
  • the aflibercept composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.
  • the mixture comprising aflibercept and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5.
  • the HIC may be performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • aflibercept is purified from a commercially available preparation such as Eylea® or Zaltrap®.
  • Aflibercept is purified away from other formulation components in Eylea® or Zaltrap® using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, and/or any other applicable purification techniques, including but not limited to Protein A chromatography, fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.
  • compositions of the present invention are prepared by buffer exchange.
  • Eylea® or Zaltrap® is subjected to a buffer exchange wherein the ingredients, other than aflibercept, of the Eylea® or Zaltrap® formulation are removed and replaced with the ingredients of a formulation of the present invention.
  • Block A This example describes experiments to evaluate the effects of various pH levels and different buffering agents on aflibercept formulations (Block A). Block A formulations are listed in Table 2.
  • Block A formulations were evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 4-7 below.
  • Block A formulations were evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. The relative areas for each of the twelve features detected by icIEF are listed for samples from Block A. These samples were frozen and shipped before analysis. Results are reported in Table 12-15 below.
  • an aflibercept formulation may have a succinate buffer or a tartrate buffer at a pH of 3.2-5.2 or 2.0-5.4, respectively.
  • a stable formulation for aflibercept can include these buffers within the pH range.
  • an aflibercept formulation with a buffer may further include an excipient such as a stabilizer or a tonicity modifier.
  • An exemplary aflibercept formulation includes a buffer, sucrose, sodium chloride, and a surfactant.
  • This example describes experiments to evaluate aflibercept formulations that are buffer-free (Block B).
  • the target pH ranged from 5.5 to 7.0 and different levels of co-solvents (e.g. surfactants or water-miscible nonaqueous solvents) were included.
  • co-solvents e.g. surfactants or water-miscible nonaqueous solvents
  • Block B formulations The stability of the Block B formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Table 18-21 below.
  • Block B formulations were evaluated by measuring the particles present in the formulation after being subjected to storage conditions. Block B samples, along with those in Block C were shipped on blue ice. This means that they were exposed to interfacial stress during the shipping process. While Block B formulation do not contain a buffer, all of the formulations contain some co-solvent, usually a surfactant, which appears to have provided some level of protection. A couple formulations (e.g., B-10 and B-14) did display elevated levels of SVPs, likely due to shipping stress. Results are reported in Table 22-25 below.
  • an aflibercept formulation may include additional excipients, including but not limited to, sorbitol and/or mannitol.
  • Block C This example describes experiments to evaluate aflibercept formulations that are free of an organic co-solvent (Block C).
  • the design of Block C centered on removing the co-solvents (e.g., PS 20, PEG 3350) used in Block B, while using buffers, such as acetate, histidine (His), and phosphate across the same pH range of 5.5 to 7.0.
  • buffers such as acetate, histidine (His), and phosphate across the same pH range of 5.5 to 7.0.
  • Table 30 shows an exemplary set of such formulations.
  • Block C formulations The stability of the Block C formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 32-35 below.
  • Block C formulations were evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were shipped on blue ice pior to analysis. Results are reported in Tables 40-43 below.
  • This example describes experiments to evaluate buffer-free aflibercept formulations that have a stabilizer other than sucrose such as amino acids (Gly, Pro, ArgHCI) as well as electrolytes, such NaCI and MgCh. (Block D). In these samples, no surfactant or co-solvent was included. In addition, dextran, a large polymer was investigated. Table 44, below, shows an exemplary set of such formulations.
  • Block D formulations were evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 46-49 below.
  • Block D formulations The chemical stability of the Block D formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Table 54-57 below.
  • phosphate could be used instead of histidine buffer. It is also contemplated that CaC could be used instead of, or in addition to, MgCh.
  • Block E formulations The stability of the Block E formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Table 60-63 below.
  • Block E formulations were evaluated by measuring the particles present in the formulation after being subjected to storage conditions. As with Block D samples, they were shipped for MFI analysis frozen on dry ice. Results are reported in Table 64-67 below.
  • Block E formulations The chemical stability of the Block E formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Tables 68-71 below.
  • Block F formulations The stability of the Block F formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 74-77 below.
  • Block F formulations were evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Tables 82-85 below.
  • Block G The aflibercept formulations of Block F are physically and chemically stable.
  • Example 8 - Aflibercept Formulations [00367] This example describes experiments to evaluate aflibercept formulations with various excipient combinations (Block G).
  • Formulations in Block G include those that are free of a buffer; free of an organic co-solvent; include a sugar other than sucrose, include a polyol; include a surfactant other than polysorbate 20 (e.g. in buffer-free formulations); include CaCh as a stabilizer; and have a high concentration of aflibercept.
  • Table 86 shows an exemplary set of such formulations.
  • Block G formulations The chemical stability of the Block G formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Tables 96-99 below.
  • a PLS1 model used the monomer content after storage at 40° C for one week as the endpoint. Two of the 112 formulations (A-2 and F-13) were found to be outliers. The correlation coefficient for the calibration set is 0.89 and the r-value for the validation set is 0.74, which is reasonable for a data set that is this large and diverse. Significant factors were determined to be sucrose, NaCI, PS 20, PEG 3350, His, Gly, MgCh, CaCh, protein, and pH.
  • Example 10 Factors Contributing to SEC Stability at 25°C for 2 Weeks
  • a PLS1 model used the monomer content by after two weeks at 25° C as the endpoint. All of the 112 formulations were included and the model indicates that the significant factors are sucrose, PEG 3350, His, ArgHCI, CaCh, and pH.
  • the correlation coefficient for the calibration set is 0.82, while the r-value for the validation set is 0.73, so the model quality is similar, but slightly lower than the last model.
  • the PLS analysis of the 25° C SEC data indicates that the optimal pH for a buffer-free formulation is between about 5.0 and about 6.0. The range appears to broaden if one uses 20 mM His buffer. His appears to be superior to acetate as a buffer system in this model. Stability is maintained in the presence of sucrose concentrations up to about 180 mM. There appears to be storage stabilization by different co-solvents, like glycerol and polysorbates (but not PEG 3350). Of the amino acids, ArgHCI is the most effective stabilizer. Finally, increased stability is seen with the levels of CaCh evlauted (and higher amounts may also be stabilizing) and with increasing amounts of MgCh.
  • Example 11 - Factors Contributing to SEC Stability at 5°C for 4 Weeks A PLS1 model employed the monomer content after storage for four weeks at 5° C (a suitable storage condition of aflibecpt according to the Eylea package insert) as the endpoint. All formulations were included and the significant factors were determined to be sucrose, PS 20, PS 80, PEG 3350, tris, ArgHCI, Pro, MgCh, dextran, and pH. The r-value for the calibration set was 0.88, while the correlation coefficient for the validation set was 0.79.
  • the PLS1 model indicates that the optimal pH for a buffer-free formulation is 5.0 to 5.5 under these conditions, with a slightly extended range (maybe to 5.7) in the presence of 20 mM His (Figure 17).
  • the pH-His response surface is expended to remove the clearly unstable conditions > pH 7 ( Figure 18).
  • This zoomed in view suggests that the best pH conditions may be ⁇ 5.5.
  • With acetate as the buffer the profile is quite similar to that with His, suggesting that either buffer may be sufficient (Figure 19).
  • the model also predicts tris as having a significant effect, but this is in relation to stabilization at elevated pH, where the stability profile is the poorest ( Figure 20). It would not be suitable for a formulation between pH 5 and 6.
  • Sucrose is found to be a stabilizer, especially at concentrations between 50-200 mM, while destabilization is predicted for NaCI, especially at concentrations above 50 mM (Figure 21).
  • the effects of the two polysorbates, PS 20 and PS 80, are shown in Figure 22. The range is very small, so effectively neither excipient impacts the stability profile of aflibercept at 5° C.
  • the effect of PEG 3350 is again detrimental to storage stability ( Figure 23), while Gly has only a small effect, primarily at concentrations below 200 mM.
  • the effects of other amino acids are similar to what was seen for Gly ( Figure 24).
  • the PLS1 model for the 5°C SEC data on storage stability find the best stability is ⁇ pH 6.0, preferably below 5.5. Both His and acetate appears to be good buffer choices. Sucrose is an effective stabilizer, while salts and amino acids contribute less to stability. The one ionic compound that appears to be of value in stabilizing the protein is CaCh. Most of the co-solvents tested appear to provide stabilization, except PEG 3350, which is destabilizing, and polysorbates that have little effect.
  • Example 12 Factors Contributing to Particle Stability
  • a PLS2 model was constructed using the total particles concentration (counts per ml_) at all three time points (t1w/40° C, t2w/25° C, t4w/5° C). All of the formulations were considered, but a number were identified as outliers, especially within Block C, where many of the samples were damaged due to agitation upon shipping on blue ice.
  • the model has some scatter, with a correlation coefficient of 0.74 for the calibration set and 0.56 for the validation set, but it still shows a clear trend of particle levels as a function of formulation composition.
  • Example 13 Factors Contributing to Chemical Stability
  • the average measured isoelectric points for each of the 12 peaks in the icIEF data for the formulations in the blocks above is listed in Table 100 below.
  • a PLS1 model was constructed using the change or difference in average pi value for samples stored at 5° C as the endpoint.
  • the model quality was lower than for the previous models, but the results still are sufficient to allow some indication of the effect of various factors, especially pH and buffers. Likely, these would be the most influential on chemical processes leading to differences in pi value.
  • Figure 38 shows the response surface for pH and acetate.
  • a line has been drawn in to mark the line where no change in pi (D) value occurs.
  • D pi
  • the model predicts the optimal pH is close to 5.6.
  • 20 mM acetate this value shifts all the way to 7.4.
  • a similar trend is seen for using citrate buffer ( Figure 40).
  • His buffer the effect of pH on D is evened out, but does not reach 0 at a concentration of 20 mM ( Figure 39).
  • Example 14 Discussion of Formulations and Stability [00399] The stability of the formulations from the blocks were elvauated.
  • T able 101 List of fifteen most stable formulations after one week at 40° C based on monomer content by SEC
  • Table 102 List of fifteen most stable formulations after four weeks at 5° C based on monomer content by SEC
  • Sucrose appears to be potent stabilizer, while many of the co solvents tested appear to provide some stabilization as well in terms of recovery of monomer as measured by SEC upon storage.
  • PEG 3350 appears to be the least favorable, while PS 20, PS 80, and glycerol would be the better choices.
  • PEG provided stability at lower temperatures (e.g. 5 °C) indicating that it may suitable for aflibercept since Eylea requires storage at 2-8°C.
  • Trehalose may also be a good choice for a stabilizer.
  • Amino acids provide some degree of stabilization, with Gly being the most consistent in terms of stabilization performance.
  • CaCh at a concentration of 10 mM, appears to provide a measurable improvement in storage stability. Without being limited to any particular theory, this stability is a result of Ca 2+ binding to aflibercept and providing conformational stability.
  • the optimal pH range was from 5.5 to 7.0, more preferably at about 6.3. So, the range overlaps with optimal range determed by SEC analysis. Again, for buffered formulations, acetate and His appear to be favorable buffer choices. Co-solvents appear to reduce SVP formation upon storage, along with sucrose, trehalose, NaCI, and CaCI 2 .

Abstract

A stable aqueous pharmaceutical formulation comprising a therapeutically effective amount of aflibercept, wherein the formulation is free of organic co-solvent and/or free of buffer; methods for making such a formulation; and methods of using such a formulation.

Description

STABLE AQUEOUS FORMULATIONS OF AFLIBERCEPT
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims priority to U.S. Patent Application Serial
No. 16/566,847, filed September 10, 2019; the entire contents of each which are herein incorporated by reference.
FIELD OF THE INVENTION
[002] The present invention relates to stable aqueous pharmaceutical compositions of aflibercept, including compositions that are considered or intended to be a “biosimilar” of commercially available aflibercept products, methods of manufacture of the compositions, methods of their administration, and kits containing the same.
BACKGROUND OF THE INVENTION [003] Vascular endothelial growth factor (VEGF) and its receptors
(VEGFR) regulates angiogenesis. Drugs that target VEGF-VEGFR interactions have demonstrated efficacy in treating ocular diseases. See Pozarowska et al. (2016) Cent Eur J Immunol 41(3): 311-316. A soluble VEGF-specific fusion protein antagonist, termed a "VEGF trap" was developed that interferes with binding of VEGF with its receptors. See, Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99:11393-8.
[004] Aflibercept, developed by Regeneron Pharmaceuticals, Inc. is a
115 kDa recombinant fusion protein. It consists of an IgG backbone fused to extracellular Vascular Endothelial Growth Factor receptor sequences of the human VEGFR1 and VEGFR2 receptors. Aflibercept is a soluble decoy receptor that binds vascular endothelial growth factor-A (VEGF-A), VEGF-B, and placental growth factor (PIGF) with a greater affinity than the body’s native receptors. Aflibercept’s high affinity for VEGF prevents the subsequent binding and activation of native VEGF receptors. Reduced VEGF activity leads to decreased angiogenesis and vascular permeability. Inhibition of VEGF-B may also aid in the treatment of angiogenic conditions. VEGF-B overexpression has recently been connected with breakdown of the blood- retinal battier retinal angiogenesis. Aflibercept products have been approved by the FDA and are commercially available under the brand names of Eylea® and Zaltrap®.
[005] Various formulations of aflibercept are known in the art. See, for example, Eylea®, Zaltrap®, U.S. Patents 7,608,261 ; 7,807,164; 8,092, 803; 8,481 ,046; 8,802,107; 9,340,594; and 9,580,489. There is still need for stable liquid formulations of aflibercept suitable for ophthalmic administration.
BRIEF SUMMARY OF THE INVENTION [006] The invention provides stable aqueous formulations comprising aflibercept.
[007] In general, the invention provides several types of aflibercept formulations: buffer-free formulations; formulations free of an organic co solvent; and formulations that are free of both buffers and organic co-solvents.
[008] In all embodiments of the invention, aflibercept can be present at concentrations of about 1 mg/mL to about 200 mg/mL, e.g., a concentration of about 1 mg/mL to about 190 mg/mL, about 1 mg/mL to about 180 mg/mL, about 1 mg/mL to about 170 mg/mL, about 1 mg/mL to about 160 mg/mL, about 1 mg/mL to about 150 mg/mL, about 1 mg/mL to about 140 mg/mL, about 1 mg/mL to about 130 mg/mL, about 1 mg/mL to about 120 mg/mL, about 1 mg/mL to about 110 mg/mL, about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 90 mg/mL, about 1 mg/mL to about 80 mg/mL, about
1 mg/mL to about 70 mg/mL, about 1 mg/mL to about 60 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 45 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 35 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 15 mg/mL, about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 8 mg/mL, about 1 mg/mL to about 6 mg/mL, about 1 mg/mL to about 4 mg/mL, about 1 mg/mL to about
2 mg/mL, about 2 mg/mL to about 200 mg/mL, about 2 mg/mL to about 190 mg/mL, about 2 mg/mL to about 180 mg/mL, about 2 mg/mL to about 170 mg/mL, about 2 mg/mL to about 160 mg/mL, about 2 mg/mL to about 150 mg/ml_, about 2 mg/mL to about 140 mg/mL, about 2 mg/mL to about 130 mg/mL, about 2 mg/mL to about 120 mg/mL, about 2 mg/mL to about 110 mg/mL, about 2 mg/mL to about 100 mg/mL, about 2 mg/mL to about 90 mg/mL, about 2 mg/mL to about 80 mg/mL, about 2 mg/mL to about 70 mg/mL, about 2 mg/mL to about 60 mg/mL, about 2 mg/mL to about 50 mg/mL, about 2 mg/mL to about 45 mg/mL, about 2 mg/mL to about 40 mg/mL, about 2 mg/mL to about 35 mg/mL, about 2 mg/mL to about 30 mg/mL, about 2 mg/mL to about 25 mg/mL, about 2 mg/mL to about 20 mg/mL, about 2 mg/mL to about 15 mg/mL, about 2 mg/mL to about 10 mg/mL, about 2 mg/mL to about 8 mg/mL, about 2 mg/mL to about 6 mg/mL, about 2 mg/mL to about 4 mg/mL, about 4 mg/mL to about 200 mg/mL, about 4 mg/mL to about 190 mg/mL, about 4 mg/mL to about 180 mg/mL, about 4 mg/mL to about 170 mg/mL, about 4 mg/mL to about 160 mg/mL, about 4 mg/mL to about 150 mg/mL, about 4 mg/mL to about 140 mg/mL, about 4 mg/mL to about 130 mg/mL, about 4 mg/mL to about 120 mg/mL, about 4 mg/mL to about 110 mg/mL, about 4 mg/mL to about 100 mg/mL, about 4 mg/mL to about 90 mg/mL, about 4 mg/mL to about 80 mg/mL, about 4 mg/mL to about 70 mg/mL, about 4 mg/mL to about 60 mg/mL, about 4 mg/mL to about 50 mg/mL, about 4 mg/mL to about 45 mg/mL, about 4 mg/mL to about 40 mg/mL, about 4 mg/mL to about 35 mg/mL, about 4 mg/mL to about 30 mg/mL, about 4 mg/mL to about 25 mg/mL, about 4 mg/mL to about 20 mg/mL, about 4 mg/mL to about 15 mg/mL, about 4 mg/mL to about 10 mg/mL, about 4 mg/mL to about 8 mg/mL, about 4 mg/mL to about 6 mg/mL, about 6 mg/mL to about 200 mg/mL, about 6 mg/mL to about 190 mg/mL, about 6 mg/mL to about 180 mg/mL, about 6 mg/mL to about 170 mg/mL, about 6 mg/mL to about 160 mg/mL, about 6 mg/mL to about 150 mg/mL, about 6 mg/mL to about 140 mg/mL, about 6 mg/mL to about 130 mg/mL, about 6 mg/mL to about 120 mg/mL, about 6 mg/mL to about 110 mg/mL, about 6 mg/mL to about 100 mg/mL, about 6 mg/mL to about 90 mg/mL, about 6 mg/mL to about 80 mg/mL, about 6 mg/mL to about 70 mg/mL, about 6 mg/mL to about 60 mg/mL, about 6 mg/mL to about 50 mg/mL, about 6 mg/mL to about 45 mg/mL, about 6 mg/mL to about 40 mg/mL, about 6 mg/mL to about 35 mg/mL, about 6 mg/mL to about 30 mg/ml_, about 6 mg/mL to about 25 mg/mL, about 6 mg/mL to about 20 mg/mL, about 6 mg/mL to about 15 mg/mL, about 6 mg/mL to about 10 mg/mL, about 6 mg/mL to about 8 mg/mL, about 8 mg/mL to about 200 mg/mL, about 8 mg/mL to about 190 mg/mL, about 8 mg/mL to about 180 mg/mL, about 8 mg/mL to about 170 mg/mL, about 8 mg/mL to about 160 mg/mL, about 8 mg/mL to about 150 mg/mL, about 8 mg/mL to about 140 mg/mL, about 8 mg/mL to about 130 mg/mL, about 8 mg/mL to about 120 mg/mL, about 8 mg/mL to about 110 mg/mL, about 8 mg/mL to about 100 mg/mL, about 8 mg/mL to about 90 mg/mL, about 8 mg/mL to about 80 mg/mL, about 8 mg/mL to about 70 mg/mL, about 8 mg/mL to about 60 mg/mL, about 8 mg/mL to about 50 mg/mL, about 8 mg/mL to about 45 mg/mL, about 8 mg/mL to about 40 mg/mL, about 8 mg/mL to about 35 mg/mL, about 8 mg/mL to about 30 mg/mL, about 8 mg/mL to about 25 mg/mL, about 8 mg/mL to about 20 mg/mL, about 8 mg/mL to about 15 mg/mL, about 8 mg/mL to about 10 mg/mL, about 10 mg/mL to about 200 mg/mL, about 10 mg/mL to about 190 mg/mL, about 10 mg/mL to about 180 mg/mL, about 10 mg/mL to about 170 mg/mL, about 10 mg/mL to about 160 mg/mL, about 10 mg/mL to about 150 mg/mL, about 10 mg/mL to about 140 mg/mL, about 10 mg/mL to about 130 mg/mL, about 10 mg/mL to about 120 mg/mL, about 10 mg/mL to about 110 mg/mL, about 10 mg/mL to about 100 mg/mL, about 10 mg/mL to about 90 mg/mL, about 10 mg/mL to about 80 mg/mL, about 10 mg/mL to about 70 mg/mL, about 10 mg/mL to about 60 mg/mL, about 10 mg/mL to about 50 mg/mL, about 10 mg/mL to about 45 mg/mL, about 10 mg/mL to about 40 mg/mL, about 10 mg/mL to about 35 mg/mL, about 10 mg/mL to about 30 mg/mL, about 10 mg/mL to about 25 mg/mL, about 10 mg/mL to about 20 mg/mL, about 10 mg/mL to about 15 mg/mL, about 15 mg/mL to about 200 mg/mL, about 15 mg/mL to about 190 mg/mL, about 15 mg/mL to about 180 mg/mL, about 15 mg/mL to about 170 mg/mL, about 15 mg/mL to about 160 mg/mL, about 15 mg/mL to about 150 mg/mL, about 15 mg/mL to about 140 mg/mL, about 15 mg/mL to about 130 mg/mL, about 15 mg/mL to about 120 mg/mL, about 15 mg/mL to about 110 mg/mL, about 15 mg/mL to about 100 mg/mL, about 15 mg/mL to about 90 mg/mL, about 15 mg/mL to about 80 mg/mL, about 15 mg/mL to about 70 mg/ml_, about 15 mg/mL to about 60 mg/mL, about 15 mg/mL to about 50 mg/mL, about 15 mg/mL to about 45 mg/mL, about 15 mg/mL to about 40 mg/mL, about 15 mg/mL to about 35 mg/mL, about 15 mg/mL to about 30 mg/mL, about 15 mg/mL to about 25 mg/mL, about 15 mg/mL to about 20 mg/mL, about 20 mg/mL to about 200 mg/mL, about 20 mg/mL to about 190 mg/mL, about 20 mg/mL to about 180 mg/mL, about 20 mg/mL to about 170 mg/mL, about 20 mg/mL to about 160 mg/mL, about 20 mg/mL to about 150 mg/mL, about 20 mg/mL to about 140 mg/mL, about 20 mg/mL to about 130 mg/mL, about 20 mg/mL to about 120 mg/mL, about 20 mg/mL to about 110 mg/mL, about 20 mg/mL to about 100 mg/mL, about 20 mg/mL to about 90 mg/mL, about 20 mg/mL to about 80 mg/mL, about 20 mg/mL to about 70 mg/mL, about 20 mg/mL to about 60 mg/mL, about 20 mg/mL to about 50 mg/mL, about 20 mg/mL to about 45 mg/mL, about 20 mg/mL to about 40 mg/mL, about 20 mg/mL to about 35 mg/mL, about 20 mg/mL to about 30 mg/mL, about 20 mg/mL to about 25 mg/mL, about 25 mg/mL to about 200 mg/mL, about 25 mg/mL to about 190 mg/mL, about 25 mg/mL to about 180 mg/mL, about 25 mg/mL to about 170 mg/mL, about 25 mg/mL to about 160 mg/mL, about 25 mg/mL to about 150 mg/mL, about 25 mg/mL to about 140 mg/mL, about 25 mg/mL to about 130 mg/mL, about 25 mg/mL to about 120 mg/mL, about 25 mg/mL to about 110 mg/mL, about 25 mg/mL to about 100 mg/mL, about 25 mg/mL to about 90 mg/mL, about 25 mg/mL to about 80 mg/mL, about 25 mg/mL to about 70 mg/mL, about 25 mg/mL to about 60 mg/mL, about 25 mg/mL to about 50 mg/mL, about 25 mg/mL to about 45 mg/mL, about 25 mg/mL to about 40 mg/mL, about 25 mg/mL to about 35 mg/mL, about 25 mg/mL to about 30 mg/mL, about 30 mg/mL to about 200 mg/mL, about 30 mg/mL to about 190 mg/mL, about 30 mg/mL to about 180 mg/mL, about 30 mg/mL to about 170 mg/mL, about 30 mg/mL to about 160 mg/mL, about 30 mg/mL to about 150 mg/mL, about 30 mg/mL to about 140 mg/mL, about 30 mg/mL to about 130 mg/mL, about 30 mg/mL to about 120 mg/mL, about 30 mg/mL to about 110 mg/mL, about 30 mg/mL to about 100 mg/mL, about 30 mg/mL to about 90 mg/mL, about 30 mg/mL to about 80 mg/mL, about 30 mg/mL to about 70 mg/mL, about 30 mg/mL to about 60 mg/mL, about 30 mg/mL to about 50 mg/mL, about 30 mg/mL to about 45 mg/ml_, about 30 mg/mL to about 40 mg/mL, about 30 mg/mL to about 35 mg/mL, about 35 mg/mL to about 200 mg/mL, about 35 mg/mL to about 190 mg/mL, about 35 mg/mL to about 180 mg/mL, about 35 mg/mL to about 170 mg/mL, about 35 mg/mL to about 160 mg/mL, about 35 mg/mL to about 150 mg/mL, about 35 mg/mL to about 140 mg/mL, about 35 mg/mL to about 130 mg/mL, about 35 mg/mL to about 120 mg/mL, about 35 mg/mL to about 110 mg/mL, about 35 mg/mL to about 100 mg/mL, about 35 mg/mL to about 90 mg/mL, about 35 mg/mL to about 80 mg/mL, about 35 mg/mL to about 70 mg/mL, about 35 mg/mL to about 60 mg/mL, about 35 mg/mL to about 50 mg/mL, about 35 mg/mL to about 45 mg/mL, about 35 mg/mL to about 40 mg/mL, about 40 mg/mL to about 200 mg/mL, about 40 mg/mL to about 190 mg/mL, about 40 mg/mL to about 180 mg/mL, about 40 mg/mL to about 170 mg/mL, about 40 mg/mL to about 160 mg/mL, about 40 mg/mL to about 150 mg/mL, about 40 mg/mL to about 140 mg/mL, about 40 mg/mL to about 130 mg/mL, about 40 mg/mL to about 120 mg/mL, about 40 mg/mL to about 110 mg/mL, about 40 mg/mL to about 100 mg/mL, about 40 mg/mL to about 90 mg/mL, about 40 mg/mL to about 80 mg/mL, about 40 mg/mL to about 70 mg/mL, about 40 mg/mL to about 60 mg/mL, about 40 mg/mL to about 50 mg/mL, about 40 mg/mL to about 45 mg/mL, about 45 mg/mL to about 200 mg/mL, about 45 mg/mL to about 190 mg/mL, about 45 mg/mL to about 180 mg/mL, about 45 mg/mL to about 170 mg/mL, about 45 mg/mL to about 160 mg/mL, about 45 mg/mL to about 150 mg/mL, about 45 mg/mL to about 140 mg/mL, about 45 mg/mL to about 130 mg/mL, about 45 mg/mL to about 120 mg/mL, about 45 mg/mL to about 110 mg/mL, about 45 mg/mL to about 100 mg/mL, about 45 mg/mL to about 90 mg/mL, about 45 mg/mL to about 80 mg/mL, about 45 mg/mL to about 70 mg/mL, about 45 mg/mL to about 60 mg/mL, about 45 mg/mL to about 50 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 190 mg/mL, about 50 mg/mL to about 180 mg/mL, about 50 mg/mL to about 170 mg/mL, about 50 mg/mL to about 160 mg/mL, about 50 mg/mL to about 150 mg/mL, about 50 mg/mL to about 140 mg/mL, about 50 mg/mL to about 130 mg/mL, about 50 mg/mL to about 120 mg/mL, about 50 mg/mL to about 110 mg/mL, about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 90 mg/mL, about 50 mg/mL to about 80 mg/ml_, about 50 mg/mL to about 70 mg/mL, about 50 mg/mL to about 60 mg/mL, about 60 mg/mL to about 200 mg/mL, about 60 mg/mL to about 190 mg/mL, about 60 mg/mL to about 180 mg/mL, about 60 mg/mL to about 170 mg/mL, about 60 mg/mL to about 160 mg/mL, about 60 mg/mL to about 150 mg/mL, about 60 mg/mL to about 140 mg/mL, about 60 mg/mL to about 130 mg/mL, about 60 mg/mL to about 120 mg/mL, about 60 mg/mL to about 110 mg/mL, about 60 mg/mL to about 100 mg/mL, about 60 mg/mL to about 90 mg/mL, about 60 mg/mL to about 80 mg/mL, about 60 mg/mL to about 70 mg/mL, about 70 mg/mL to about 200 mg/mL, about 70 mg/mL to about 190 mg/mL, about 70 mg/mL to about 180 mg/mL, about 70 mg/mL to about 170 mg/mL, about 70 mg/mL to about 160 mg/mL, about 70 mg/mL to about 150 mg/mL, about 70 mg/mL to about 140 mg/mL, about 70 mg/mL to about 130 mg/mL, about 70 mg/mL to about 120 mg/mL, about 70 mg/mL to about 110 mg/mL, about 70 mg/mL to about 100 mg/mL, about 70 mg/mL to about 90 mg/mL, about 70 mg/mL to about 80 mg/mL, about 80 mg/mL to about 200 mg/mL, about 80 mg/mL to about 190 mg/mL, about 80 mg/mL to about 180 mg/mL, about 80 mg/mL to about 170 mg/mL, about 80 mg/mL to about 160 mg/mL, about 80 mg/mL to about 150 mg/mL, about 80 mg/mL to about 140 mg/mL, about 80 mg/mL to about 130 mg/mL, about 80 mg/mL to about 120 mg/mL, about 80 mg/mL to about 110 mg/mL, about 80 mg/mL to about 100 mg/mL, about 80 mg/mL to about 90 mg/mL, about 90 mg/mL to about 200 mg/mL, about 90 mg/mL to about 190 mg/mL, about 90 mg/mL to about 180 mg/mL, about 90 mg/mL to about 170 mg/mL, about 90 mg/mL to about 160 mg/mL, about 90 mg/mL to about 150 mg/mL, about 90 mg/mL to about 140 mg/mL, about 90 mg/mL to about 130 mg/mL, about 90 mg/mL to about 120 mg/mL, about 90 mg/mL to about 110 mg/mL, about 90 mg/mL to about 100 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 190 mg/mL, about 100 mg/mL to about 180 mg/mL, about 100 mg/mL to about 170 mg/mL, about 100 mg/mL to about 160 mg/mL, about 100 mg/mL to about 150 mg/mL, about 100 mg/mL to about 140 mg/mL, about 100 mg/mL to about 130 mg/mL, about 100 mg/mL to about 120 mg/mL, about 100 mg/mL to about 110 mg/mL, about 110 mg/mL to about 200 mg/mL, about 110 mg/mL to about 190 mg/mL, about 110 mg/mL to about 180 mg/mL, about 110 mg/mL to about 170 mg/mL, about 110 mg/mL to about 160 mg/mL, about 110 mg/mL to about 150 mg/mL, about 110 mg/mL to about 140 mg/mL, about 110 mg/mL to about 130 mg/mL, about 110 mg/mL to about 120 mg/mL, about 120 mg/mL to about 200 mg/mL, about 120 mg/mL to about 190 mg/mL, about 120 mg/mL to about 180 mg/mL, about 120 mg/mL to about 170 mg/mL, about 120 mg/mL to about 160 mg/mL, about 120 mg/mL to about 150 mg/mL, about 120 mg/mL to about 140 mg/mL, about 120 mg/mL to about 130 mg/mL, about 130 mg/mL to about 200 mg/mL, about 130 mg/mL to about 190 mg/mL, about 130 mg/mL to about 180 mg/mL, about 130 mg/mL to about 170 mg/mL, about 130 mg/mL to about 160 mg/mL, about 130 mg/mL to about 150 mg/mL, about 130 mg/mL to about 140 mg/mL, about 140 mg/mL to about 200 mg/mL, about 140 mg/mL to about 190 mg/mL, about 140 mg/mL to about 180 mg/mL, about 140 mg/mL to about 170 mg/mL, about 140 mg/mL to about 160 mg/mL, about 140 mg/mL to about 150 mg/mL, about 150 mg/mL to about 200 mg/mL, about 150 mg/mL to about 190 mg/mL, about 150 mg/mL to about 180 mg/mL, about 150 mg/mL to about 170 mg/mL, about 150 mg/mL to about 160 mg/mL, about 160 mg/mL to about 200 mg/mL, about 160 mg/mL to about 190 mg/mL, about 160 mg/mL to about 180 mg/mL, about 160 mg/mL to about 170 mg/mL, about 170 mg/mL to about 200 mg/mL, about 170 mg/mL to about 190 mg/mL, about 170 mg/mL to about 180 mg/mL, about 180 mg/mL to about 200 mg/mL, about 180 mg/mL to about 190 mg/mL, or about 190 mg/mL to about 200 mg/mL, or a concentration of about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL, about 21 mg/mL, about 22 mg/mL, about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26 mg/mL, about 27 mg/mL, about 28 mg/mL, about 29 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, about 100 mg/ml_, about 105 mg/mL, about 110 mg/mL, about 115 mg/mL, about 120 mg/mL, about 125 mg/mL, about 130 mg/mL, about 135 mg/mL, about 140 mg/mL, about 145 mg/mL, about 150 mg/mL, about 155 mg/mL, about 160 mg/mL, about 165 mg/mL, about 170 mg/mL, about 175 mg/mL, about 180 mg/mL, about 185 mg/mL, about 190 mg/mL, about 195 mg/mL, or 200 mg/mL.
Buffer-Free Formulations
[009] In one embodiment, the invention provides an aqueous formulation comprising aflibercept, a stabilizer and an organic co-solvent, wherein the formulation is free of buffer.
[0010] In one embodiment, the formulation has a pH of about 5.5 to about 7.0 (e.g., about 5.5 to about 6.9, about 5.5 to about 6.8, about 5.5 to about 6.7, about 5.5 to about 6.6, about 5.5 to about 6.5, about 5.5 to about
6.4, about 5.5 to about 6.3, about 5.5 to about 6.2, about 5.5 to about 6.1 , about 5.5 to about 6.0, about 5.5 to about 5.9, about 5.5 to about 5.8, about
5.5 to about 5.7, about 5.6 to about 7.0, about 5.6 to about 6.9, about 5.6 to about 6.8, about 5.6 to about 6.7, about 5.6 to about 6.6, about 5.6 to about
6.5, about 5.6 to about 6.4, about 5.6 to about 6.3, about 5.6 to about 6.2, about 5.6 to about 6.1 , about 5.6 to about 6.0, about 5.6 to about 5.9, about
5.6 to about 5.8, about 5.7 to about 7.0, about 5.7 to about 6.9, about 5.7 to about 6.8, about 5.7 to about 6.7, about 5.7 to about 6.6, about 5.7 to about
6.5, about 5.7 to about 6.4, about 5.7 to about 6.3, about 5.7 to about 6.2, about 5.7 to about 6.1 , about 5.7 to about 6.0, about 5.7 to about 5.9, about
5.8 to about 7.0, about 5.8 to about 6.9, about 5.8 to about 6.8, about 5.8 to about 6.7, about 5.8 to about 6.6, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1 , about 5.8 to about 6.0, about 5.9 to about 7.0, about 5.9 to about 6.9, about
5.9 to about 6.8, about 5.9 to about 6.7, about 5.9 to about 6.6, about 5.9 to about 6.5, about 5.9 to about 6.4, about 5.9 to about 6.3, about 5.9 to about 6.2, about 5.9 to about 6.1 , about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about 6.1 to about 7.0, about 6.1 to about 6.9, about 6.1 to about 6.8, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.1 to about 6.4, about 6.1 to about 6.3, about 6.2 to about 7.0, about 6.2 to about 6.9, about 6.2 to about 6.8, about 6.2 to about 6.7, about 6.2 to about 6.6, about 6.2 to about 6.5, about 6.2 to about 6.4, about 6.3 to about 7.0, about 6.3 to about 6.9, about 6.3 to about 6.8, about 6.3 to about 6.7, about 6.3 to about 6.6, about 6.3 to about 6.5, about 6.4 to about 7.0, about 6.4 to about 6.9, about 6.4 to about 6.8, about 6.4 to about 6.7, about 6.4 to about 6.6, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.0, about 6.6 to about 6.9, about 6.6 to about 6.8, about 6.7 to about 7.0, about 6.7 to about 6.9, or about 6.8 to about 7.0).
[0011] In another embodiment, the pH is about 6.0 to about 6.5. In yet another embodiment, the pH is about 6.2. In another embodiment, the pH is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.
[0012] In one embodiment, the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
[0013] In one embodiment, the organic co-solvent is selected from the group consisting of a surfactant, polyethylene glycol (PEG), propylene glycol, a block copolymer, an alkyl saccharide (e.g., a ProTek alkyl saccharide, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000.
[0014] In one embodiment, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, sodium dodecyl sulfate (SDS), n-Dodecyl- -D-maltoside (DDM) and poloxamer 188 (Pluronic F-68).
[0015] In another embodiment, the buffer-free formulations of the invention further comprise one or more salt(s). In one embodiment, the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, and potassium chloride. Each of the one or more salt(s) may be present at a concentration of about 0.1 mM-100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 65 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 10 mM to about 95 mM, about 10 mM to about 90 mM, about 10 mM to about 85 mM, about 10 mM to about 80 mM, about 10 mM to about 75 mM, about 10 mM to about 70 mM, about 10 mM to about 65 mM, about 10 mM to about 60 mM, about 10 mM to about 55 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 100 mM, about 12 mM to about 95 mM, about 12 mM to about 90 mM, about 12 mM to about 85 mM, about 12 mM to about 80 mM, about 12 mM to about 75 mM, about 12 mM to about 70 mM, about 12 mM to about 65 mM, about 12 mM to about 60 mM, about 12 mM to about 55 mM, about 12 mM to about 50 mM, about 12 mM to about 45 mM, about 12 mM to about 40 mM, about 12 mM to about 35 mM, about 12 mM to about 30 mM, about 12 mM to about 25 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 100 mM, about 14 mM to about 95 mM, about 14 mM to about 90 mM, about 14 mM to about 85 mM, about 14 mM to about 80 mM, about 14 mM to about 75 mM, about 14 mM to about 70 mM, about 14 mM to about 65 mM, about 14 mM to about 60 mM, about 14 mM to about 55 mM, about 14 mM to about 50 mM, about 14 mM to about 45 mM, about 14 mM to about 40 mM, about 14 mM to about 35 mM, about 14 mM to about 30 mM, about 14 mM to about 25 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 100 mM, about 16 mM to about 95 mM, about 16 mM to about 90 mM, about 16 mM to about 85 mM, about 16 mM to about 80 mM, about 16 mM to about 75 mM, about 16 mM to about 70 mM, about 16 mM to about 65 mM, about 16 mM to about 60 mM, about 16 mM to about 55 mM, about 16 mM to about 50 mM, about 16 mM to about 45 mM, about 16 mM to about 40 mM, about 16 mM to about 35 mM, about 16 mM to about 30 mM, about 16 mM to about 25 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, about 18 mM to about 100 mM, about 18 mM to about 95 mM, about 18 mM to about 90 mM, about 18 mM to about 85 mM, about 18 mM to about 80 mM, about 18 mM to about 75 mM, about 18 mM to about 70 mM, about 18 mM to about 65 mM, about 18 mM to about 60 mM, about 18 mM to about 55 mM, about 18 mM to about 50 mM, about 18 mM to about 45 mM, about 18 mM to about 40 mM, about 18 mM to about 35 mM, about 18 mM to about 30 mM, about 18 mM to about 25 mM, about 18 mM to about 20 mM, about 20 mM to about 100 mM, about 20 mM to about 95 mM, about 20 mM to about 90 mM, about 20 mM to about 85 mM, about 20 mM to about 80 mM, about 20 mM to about 75 mM, about 20 mM to about 70 mM, about 20 mM to about 65 mM, about 20 mM to about 60 mM, about 20 mM to about 55 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 100 mM, about 25 mM to about 95 mM, about 25 mM to about 90 mM, about 25 mM to about 85 mM, about 25 mM to about 80 mM, about 25 mM to about 75 mM, about 25 mM to about 70 mM, about 25 mM to about 65 mM, about 25 mM to about 60 mM, about 25 mM to about 55 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 100 mM, about 30 mM to about 95 mM, about 30 mM to about 90 mM, about 30 mM to about 85 mM, about 30 mM to about 80 mM, about 30 mM to about 75 mM, about 30 mM to about 70 mM, about 30 mM to about 65 mM, about 30 mM to about 60 mM, about 30 mM to about 55 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 100 mM, about 35 mM to about 95 mM, about 35 mM to about 90 mM, about 35 mM to about 85 mM, about 35 mM to about 80 mM, about 35 mM to about 75 mM, about 35 mM to about 70 mM, about 35 mM to about 65 mM, about 35 mM to about 60 mM, about 35 mM to about 55 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 100 mM, about 40 mM to about 95 mM, about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 100 mM, about 45 mM to about 95 mM, about 45 mM to about 90 mM, about 45 mM to about 85 mM, about 45 mM to about 80 mM, about 45 mM to about 75 mM, about 45 mM to about 70 mM, about 45 mM to about 65 mM, about 45 mM to about 60 mM, about 45 mM to about 55 mM, about 45 mM to about 50 mM, about 50 mM to about 100 mM, about 50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, about 50 mM to about 55 mM, about 55 mM to about 100 mM, about 55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, about 55 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM, about 60 mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, about 60 mM to about 65 mM, about 65 mM to about 100 mM, about 65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM, about 65 mM to about 80 mM, about 65 mM to about 75 mM, about 65 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM, about 70 mM to about 80 mM, about 70 mM to about 75 mM, about 75 mM to about 100 mM, about 75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM, about 75 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 95 mM, about 80 mM to about 90 mM, about 80 mM to about 85 mM, about 85 mM to about 100 mM, about 85 mM to about 95 mM, about 85 mM to about 90 mM, about 90 mM to about 100 mM, about 90 mM to about 95 mM, or about 95 mM to about 100 mM).
[0016] In one embodiment, the stabilizer is a sugar. In another embodiment, the stabilizer is a sugar (e.g., sucrose). In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine. In some embodiments of any of the aqueous formulations described herein, a stabilizer (e.g., any of the exemplary stabilizers described herein) can be present at a final concentration of about 0.01 % w/v to about 8.0% w/v (e.g., about 0.01 % w/v to about 7.5% w/v, about 0.01 % w/v to about 7.0% w/v, about 0.01 % w/v to about 6.5% w/v, about 0.01 % w/v to about 6.0% w/v, about 0.01 % w/v to about 5.5% w/v, about 0.01 % w/v to about 5.0% w/v, about 0.01 % w/v to about 4.5% w/v, about 0.01 % w/v to about 4.0% w/v, about 0.01 % w/v to about 3.5% w/v, about 0.01 % w/v to about 3.0% w/v, about 0.01 % w/v to about 2.5% w/v, about 0.01 % w/v to about 2.0% w/v, about 0.01 % w/v to about 1 .5% w/v, about 0.01 % w/v, to about 1.0% w/v, about 0.01 % w/v to about 0.5% w/v, about 0.1 % w/v to about 8.0% w/v, about 0.1 % w/v to about 7.5% w/v, about 0.1 % w/v to about 7.0% w/v, about 0.1 % w/v to about 6.5% w/v, about 0.1 % w/v to about 6.0% w/v, about 0.1 % w/v to about 5.5% w/v, about 0.1 % w/v to about 5.0% w/v, about 0.1 % w/v to about 4.5% w/v, about 0.1 % w/v to about 4.0% w/v, about 0.1 % w/v to about 3.5% w/v, about 0.1 % w/v to about 3.0% w/v, about 0.1 % w/v to about 2.5% w/v, about 0.1 % w/v to about 2.0% w/v, about 0.1 % w/v to about 1.5% w/v, about 0.1 % w/v, to about 1.0% w/v, about 0.1 % w/v to about 0.5% w/v, about 0.5% w/v to about 8.0% w/v, about 0.5% w/v to about 7.5% w/v, about 0.5% w/v to about 7.0% w/v, about 0.5% w/v to about 6.5% w/v, about 0.5% w/v to about 6.0% w/v, about 0.5% w/v to about 5.5% w/v, about 0.5% w/v to about 5.0% w/v, about 0.5% w/v to about 4.5% w/v, about 0.5% w/v to about 4.0% w/v, about 0.5% w/v to about 3.5% w/v, about 0.5% w/v to about 3.0% w/v, about 0.5% w/v to about 2.5% w/v, about 0.5% w/v to about 2.0% w/v, about 0.5% w/v to about 1 .5% w/v, about 0.5% w/v to about 1.0% w/v, about 1.0% w/v to about 8.0% w/v, about 1.0% w/v to about 7.5% w/v, about 1.0% w/v to about 7.0% w/v, about 1.0% w/v to about 6.5% w/v, about 1.0% w/v to about 6.0% w/v, about 1.0% w/v to about 5.5% w/v, about 1 .0% w/v to about 5.0% w/v, about 1.0% w/v to about 4.5% w/v, about 1.0% w/v to about 4.0% w/v, about 1 .0% w/v to about 3.5% w/v, about 1.0% w/v to about 3.0% w/v, about 1.0% w/v to about 2.5% w/v, about 1.0% w/v to about 2.0% w/v, about 1.0% w/v to about 1.5% w/v, about 1 .5% w/v to about 8.0% w/v, about 1.5% w/v to about 7.5% w/v, about 1.5% w/v to about 7.0% w/v, about 1.5% w/v to about 6.5% w/v, about 1.5% w/v to about 6.0% w/v, about 1 .5% w/v to about 5.5% w/v, about 1.5% w/v to about 5.0% w/v, about 1.5% w/v to about 4.5% w/v, about 1 .5% w/v to about 4.0% w/v, about 1.5% w/v to about 3.5% w/v, about 1.5% w/v to about 3.0% w/v, about 1 .5% w/v to about 2.5% w/v, about 1.5% w/v to about 2.0% w/v, about 2.0% w/v to about 8.0% w/v, about 2.0% w/v to about 7.5% w/v, about 2.0% w/v to about 7.0% w/v, about 2.0% w/v to about 6.5% w/v, about 2.0% w/v to about 6.0% w/v, about 2.0% w/v to about 5.5% w/v, about 2.0% w/v to about 5.0% w/v, about 2.0 w/v to about 4.5% w/v, about 2.0% w/v to about 4.0% w/v, about 2.0% w/v to about 3.5% w/v, about 2.0% w/v to about 3.0% w/v, about 2.0% w/v to about 2.5% w/v, about 2.5% w/v to about 8.0% w/v, about 2.5% w/v to about 7.5% w/v, about 2.5% w/v to about 7.0% w/v, about 2.5% w/v to about 6.5% w/v, about 2.5% w/v to about 6.0% w/v, about 2.5% w/v to about 5.5% w/v, about 2.5% w/v to about 5.0% w/v, about 2.5% w/v to about 4.5% w/v, about 2.5% w/v to about 4.0% w/v, about 2.5% w/v to about 3.5% w/v, about 2.5% w/v to about 3.0% w/v, about 3.0% w/v to about 8.0% w/v, about 3.0% w/v to about 7.5% w/v, about 3.0% w/v to about 7.0% w/v, about 3.0% w/v to about 6.5% w/v, about 3.0% w/v to about 6.0% w/v, about 3.0% w/v to about 5.5% w/v, about 3.0% w/v to about 5.0% w/v, about 3.0% w/v to about 4.5% w/v, about 3.0% w/v to about 4.0% w/v, about 3.0% w/v to about 3.5% w/v, about 3.5% w/v to about 8.0% w/v, about 3.5% w/v to about 7.5% w/v, about 3.5% w/v to about 7.0% w/v, about 3.5% w/v to about 6.5% w/v, about 3.5% w/v to about 6.0% w/v, about 3.5% w/v to about 5.5% w/v, about 3.5% w/v to about 5.0% w/v, about 3.5% w/v to about 4.5% w/v, about 3.5% w/v to about 4.0% w/v, about 4.0% w/v to about 8.0% w/v, about 4.0% w/v to about 7.5% w/v, about 4.0% w/v to about 7.0% w/v, about 4.0% w/v to about 6.5% w/v, about 4.0% w/v to about 6.0% w/v, about 4.0% w/v to about 5.5% w/v, about 4.0% w/v to about 5.0% w/v, about 4.0% w/v to about 4.5% w/v, about 4.5% w/v to about 8.0% w/v, about 4.5% w/v to about 7.5% w/v, about 4.5% w/v to about 7.0% w/v, about 4.5% w/v to about 6.5% w/v, about 4.5% w/v to about 6.0% w/v, about 4.5% w/v to about 5.5% w/v, about 4.5% w/v to about 5.0% w/v, about 5.0% w/v to about 8.0% w/v, about 5.0% w/v to about 7.5% w/v, about 5.0% w/v to about 7.0% w/v, about 5.0% w/v to about 6.5% w/v, about 5.0% w/v to about 6.0% w/v, about 5.0% w/v to about 5.5% w/v, about 5.5% w/v to about 8.0% w/v, about 5.5% w/v to about 7.5% w/v, about 5.5% w/v to about 7.0% w/v, about 5.5% w/v to about 6.5% w/v, about 5.5% w/v to about 6.0% w/v, about 6.0% w/v to about 8.0% w/v, about 6.0% w/v to about 7.5% w/v, about 6.0% w/v to about 7.0% w/v, about 6.0% w/v to about 6.5% w/v, about 6.5% w/v to about 8.0% w/v, about 6.5% w/v to about 7.5% w/v, about 6.5% w/v to about 7.0% w/v, about 7.0% w/v to about 8.0% w/v, about 7.0% w/v to about 7.5% w/v, or about 7.5% w/v to about 8.0% w/v).
[0017] In one embodiment, the stabilizer is an amino acid (e.g., an aromatic amino acid, e.g., tyrosine, phenylalanine, and tryptophan). In another embodiment, the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof. The amino acids may be present at a concentration of about 5-300 mM (e.g., about 5 mM to about 280 mM, about 5 mM to about 260 mM, about 5 mM to about 240 mM, about 5 mM to about 220 mM, about 5 mM to about 200 mM, about 5 mM to about 180 mM, about 5 mM to about 160 mM, about 5 mM to about 140 mM, about 5 mM to about 120 mM, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 60 mM, about 5 mM to about 40 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 300 mM, about 10 mM to about 280 mM, about 10 mM to about 260 mM, about 10 mM to about 240 mM, about 10 mM to about 220 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 40 mM, about 10 mM to about 20 mM, about 20 mM to about 300 mM, about 20 mM to about 280 mM, about 20 mM to about 260 mM, about 20 mM to about 240 mM, about 20 mM to about 220 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 40 mM, about 40 mM to about 300 mM, about 40 mM to about 280 mM, about 40 mM to about 260 mM, about 40 mM to about 240 mM, about 40 mM to about 220 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 40 mM, about 60 mM to about 300 mM, about 60 mM to about 280 mM, about 60 mM to about 260 mM, about 60 mM to about 240 mM, about 60 mM to about 220 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 150 mM to about 300 mM, about 150 mM to about 250 mM, about 150 mM to about 200 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, or about 250 mM to about 300 mM).
[0018] In some embodiments, the stabilizer is sucrose, the organic co solvent is polysorbate 20 and the pH of the formulation is about 5.5-7.0 (e.g., about 5.5 to about 6.9, about 5.5 to about 6.8, about 5.5 to about 6.7, about
5.5 to about 6.6, about 5.5 to about 6.5, about 5.5 to about 6.4, about 5.5 to about 6.3, about 5.5 to about 6.2, about 5.5 to about 6.1 , about 5.5 to about 6.0, about 5.5 to about 5.9, about 5.5 to about 5.8, about 5.5 to about 5.7, about 5.6 to about 7.0, about 5.6 to about 6.9, about 5.6 to about 6.8, about
5.6 to about 6.7, about 5.6 to about 6.6, about 5.6 to about 6.5, about 5.6 to about 6.4, about 5.6 to about 6.3, about 5.6 to about 6.2, about 5.6 to about 6.1 , about 5.6 to about 6.0, about 5.6 to about 5.9, about 5.6 to about 5.8, about 5.7 to about 7.0, about 5.7 to about 6.9, about 5.7 to about 6.8, about
5.7 to about 6.7, about 5.7 to about 6.6, about 5.7 to about 6.5, about 5.7 to about 6.4, about 5.7 to about 6.3, about 5.7 to about 6.2, about 5.7 to about 6.1 , about 5.7 to about 6.0, about 5.7 to about 5.9, about 5.8 to about 7.0, about 5.8 to about 6.9, about 5.8 to about 6.8, about 5.8 to about 6.7, about
5.8 to about 6.6, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1 , about 5.8 to about 6.0, about 5.9 to about 7.0, about 5.9 to about 6.9, about 5.9 to about 6.8, about 5.9 to about 6.7, about 5.9 to about 6.6, about 5.9 to about 6.5, about
5.9 to about 6.4, about 5.9 to about 6.3, about 5.9 to about 6.2, about 5.9 to about 6.1 , about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about
6.1 to about 7.0, about 6.1 to about 6.9, about 6.1 to about 6.8, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.1 to about
6.4, about 6.1 to about 6.3, about 6.2 to about 7.0, about 6.2 to about 6.9, about 6.2 to about 6.8, about 6.2 to about 6.7, about 6.2 to about 6.6, about
6.2 to about 6.5, about 6.2 to about 6.4, about 6.3 to about 7.0, about 6.3 to about 6.9, about 6.3 to about 6.8, about 6.3 to about 6.7, about 6.3 to about 6.6, about 6.3 to about 6.5, about 6.4 to about 7.0, about 6.4 to about 6.9, about 6.4 to about 6.8, about 6.4 to about 6.7, about 6.4 to about 6.6, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.0, about 6.6 to about 6.9, about 6.6 to about 6.8, about 6.7 to about 7.0, about 6.7 to about 6.9, or about 6.8 to about 7.0, or about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
[0019] In some embodiments of any of the formulations described herein, the formulation is about 5.5-7.0 (e.g., about 5.5 to about 6.9, about 5.5 to about 6.8, about 5.5 to about 6.7, about 5.5 to about 6.6, about 5.5 to about
6.5, about 5.5 to about 6.4, about 5.5 to about 6.3, about 5.5 to about 6.2, about 5.5 to about 6.1 , about 5.5 to about 6.0, about 5.5 to about 5.9, about 5.5 to about 5.8, about 5.5 to about 5.7, about 5.6 to about 7.0, about 5.6 to about 6.9, about 5.6 to about 6.8, about 5.6 to about 6.7, about 5.6 to about 6.6, about 5.6 to about 6.5, about 5.6 to about 6.4, about 5.6 to about 6.3, about 5.6 to about 6.2, about 5.6 to about 6.1 , about 5.6 to about 6.0, about
5.6 to about 5.9, about 5.6 to about 5.8, about 5.7 to about 7.0, about 5.7 to about 6.9, about 5.7 to about 6.8, about 5.7 to about 6.7, about 5.7 to about 6.6, about 5.7 to about 6.5, about 5.7 to about 6.4, about 5.7 to about 6.3, about 5.7 to about 6.2, about 5.7 to about 6.1 , about 5.7 to about 6.0, about
5.7 to about 5.9, about 5.8 to about 7.0, about 5.8 to about 6.9, about 5.8 to about 6.8, about 5.8 to about 6.7, about 5.8 to about 6.6, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1 , about 5.8 to about 6.0, about 5.9 to about 7.0, about 5.9 to about 6.9, about 5.9 to about 6.8, about 5.9 to about 6.7, about 5.9 to about 6.6, about 5.9 to about 6.5, about 5.9 to about 6.4, about 5.9 to about
6.3, about 5.9 to about 6.2, about 5.9 to about 6.1 , about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about 6.1 to about 7.0, about 6.1 to about 6.9, about 6.1 to about 6.8, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.1 to about 6.4, about 6.1 to about 6.3, about 6.2 to about 7.0, about 6.2 to about 6.9, about 6.2 to about 6.8, about 6.2 to about 6.7, about 6.2 to about 6.6, about 6.2 to about 6.5, about 6.2 to about
6.4, about 6.3 to about 7.0, about 6.3 to about 6.9, about 6.3 to about 6.8, about 6.3 to about 6.7, about 6.3 to about 6.6, about 6.3 to about 6.5, about 6.4 to about 7.0, about 6.4 to about 6.9, about 6.4 to about 6.8, about 6.4 to about 6.7, about 6.4 to about 6.6, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.0, about 6.6 to about 6.9, about 6.6 to about 6.8, about 6.7 to about 7.0, about
6.7 to about 6.9, or about 6.8 to about 7.0, or about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
[0020] In some embodiments of any of the formulations described herein, the formulation can include one or more of: sodium ion at a final concentration of about 100 mM to about 200 mM (e.g., about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); potassium ion at a final concentration of about 1 mM to about 20 mM (e.g., about 1 mM to about 18 mM, about 1 mM to about 16 mM, about 1 mM to about 14 mM, about 1 mM to about 12 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 20 mM, about 2 mM to about 18 mM, about 2 mM to about 16 mM, about 2 mM to about 14 mM, about 2 mM to about 12 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 20 mM, about 4 mM to about 18 mM, about 4 mM to about 16 mM, about 4 mM to about 14 mM, about 4 mM to about 12 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 20 mM, about 6 mM to about 18 mM, about 6 mM to about 16 mM, about 6 mM to about 14 mM, about 6 mM to about 12 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 20 mM, about 8 mM to about 18 mM, about 8 mM to about 16 mM, about 8 mM to about 14 mM, about 8 mM to about 12 mM, about 8 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, or about 18 mM to about 20 mM); chloride ion at a final concentration of about 10 mM to about 200 mM (e.g., about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 40 mM, about 10 mM to about 20 mM, about 50 mM to about 200 mM, 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); cacium ion at a final concentration of about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM); magnesium ion at a final concentration of about 0.1 mM to about 3 mM (e.g., about 0.1 mM to about 2.5 mM, about 0.1 mM to about 2.0 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 1 .0 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 0.5 mM to about 1 .5 mM, about 0.5 mM to about 1.0 mM, about 1.0 mM to about 3.0 mM, about 1.0 mM to about 2.5 mM, about 1.0 mM to about 2.0 mM, about 1.0 mM to about 1.5 mM, about 1.5 mM to about 3.0 mM, about 1 .5 mM to about 2.5 mM, about 1 .5 mM to about 2.0 mM, about 2.0 mM to about 3.0 mM, about 2.0 mM to about 2.5 mM, or about 2.5 mM to about 3.0 mM); sugar (e.g., glucose) at a final concentration of about 0.5 mM to about 10 mM (e.g., about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); lactate at a final concentration of about 1 mM to about 10 mM (e.g., about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about
2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); beta-hyrdoxybutyrate at a final concentration of about 0.01 mM to about 0.3 mM (e.g., about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about
3 mM, about 0.1 mM to about 0.2 mM, or about 0.2 mM to about 0.3 mM); copper ion at a concentration of about 0.1 mM to about 1.0 mM (e.g., about 0.1 mM to about 0.8 mM, about 0.1 mM to about 0.6 mM, about 0.1 mM to about 0.4 mM, or about 0.1 mM to about 0.2 mM); zinc ion at a final concentration of about 0.1 mM to about 10 mM (e.g., about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, or about 0.1 mM to about 2 mM); selenium ion at a final concentration of about 0.01 mM to about 0.2 mM (e.g., about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.10 mM, or about 0.01 mM to about 0.5 mM); iron ion at a final concentration of about 0.01 mM to about 8 mM (e.g., about 0.01 mM to about 6 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 0.05 mM); ferritin at a final concentration of about 0.1 mM to about 50 mM (e.g., 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 1 mM); and transferrin at final concentration of about 0.01 mM to about 0.2 mM (e.g., about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.10 mM, about 0.01 mM to about 0.05 mM).
[0021] In one embodiment, the formulation is iso-osmolar relative to the vitreous. Formulations Free of Organic Co-Solvent [0022] In one embodiment, the invention provides an aqueous formulation comprising aflibercept, a stabilizer and a buffer, wherein the formulation is free of an organic co-solvent.
[0023] In one embodiment, the formulation has a pH of about 5.5 to about 7.0 (e.g., any of the subranges of described herein or about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0). In another embodiment, the pH is about 6.0 to about 6.5. In yet another embodiment, the pH is about 6.2.
[0024] In one embodiment, the stabilizer is selected from the group consisting of amino acids, sugars (e.g., sucrose), polyols, polymers, block copolymers, alkyl saccharides (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000, or any combination thereof.
[0025] In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine. In some embodiments of any of the aqueous formulations described herein, a stabilizer (e.g., any of the exemplary stabilizers described herein) can be present at a final concentration of about 0.01 % w/v to about 8.0% w/v (e.g., about 0.01 % w/v to about 7.5% w/v, about 0.01 % w/v to about 7.0% w/v, about 0.01 % w/v to about 6.5% w/v, about 0.01 % w/v to about 6.0% w/v, about 0.01 % w/v to about 5.5% w/v, about 0.01 % w/v to about 5.0% w/v, about 0.01 % w/v to about 4.5% w/v, about 0.01 % w/v to about 4.0% w/v, about 0.01 % w/v to about 3.5% w/v, about 0.01 % w/v to about 3.0% w/v, about 0.01 % w/v to about 2.5% w/v, about 0.01 % w/v to about 2.0% w/v, about 0.01 % w/v to about 1 .5% w/v, about 0.01 % w/v, to about 1.0% w/v, about 0.01 % w/v to about 0.5% w/v, about 0.1 % w/v to about 8.0% w/v, about 0.1 % w/v to about 7.5% w/v, about 0.1 % w/v to about 7.0% w/v, about 0.1 % w/v to about 6.5% w/v, about 0.1 % w/v to about 6.0% w/v, about 0.1 % w/v to about 5.5% w/v, about 0.1 % w/v to about 5.0% w/v, about 0.1 % w/v to about 4.5% w/v, about 0.1 % w/v to about 4.0% w/v, about 0.1 % w/v to about 3.5% w/v, about 0.1 % w/v to about 3.0% w/v, about 0.1 % w/v to about 2.5% w/v, about 0.1 % w/v to about 2.0% w/v, about 0.1 % w/v to about 1.5% w/v, about 0.1 % w/v, to about 1.0% w/v, about 0.1 % w/v to about 0.5% w/v, about 0.5% w/v to about 8.0% w/v, about 0.5% w/v to about 7.5% w/v, about 0.5% w/v to about 7.0% w/v, about 0.5% w/v to about 6.5% w/v, about 0.5% w/v to about 6.0% w/v, about 0.5% w/v to about 5.5% w/v, about 0.5% w/v to about 5.0% w/v, about 0.5% w/v to about 4.5% w/v, about 0.5% w/v to about 4.0% w/v, about 0.5% w/v to about 3.5% w/v, about 0.5% w/v to about 3.0% w/v, about 0.5% w/v to about 2.5% w/v, about 0.5% w/v to about 2.0% w/v, about 0.5% w/v to about 1.5% w/v, about 0.5% w/v to about 1 .0% w/v, about 1 .0% w/v to about 8.0% w/v, about 1 .0% w/v to about 7.5% w/v, about 1.0% w/v to about 7.0% w/v, about 1.0% w/v to about 6.5% w/v, about 1.0% w/v to about 6.0% w/v, about 1.0% w/v to about 5.5% w/v, about 1.0% w/v to about 5.0% w/v, about 1 .0% w/v to about 4.5% w/v, about 1.0% w/v to about 4.0% w/v, about 1.0% w/v to about 3.5% w/v, about 1 .0% w/v to about 3.0% w/v, about 1.0% w/v to about 2.5% w/v, about 1.0% w/v to about 2.0% w/v, about 1 .0% w/v to about 1.5% w/v, about 1 .5% w/v to about 8.0% w/v, about 1.5% w/v to about 7.5% w/v, about 1.5% w/v to about 7.0% w/v, about 1.5% w/v to about 6.5% w/v, about 1.5% w/v to about 6.0% w/v, about 1.5% w/v to about 5.5% w/v, about 1 .5% w/v to about 5.0% w/v, about 1.5% w/v to about 4.5% w/v, about 1.5% w/v to about 4.0% w/v, about 1 .5% w/v to about 3.5% w/v, about 1.5% w/v to about 3.0% w/v, about 1.5% w/v to about 2.5% w/v, about 1 .5% w/v to about 2.0% w/v, about 2.0% w/v to about 8.0% w/v, about 2.0% w/v to about 7.5% w/v, about 2.0% w/v to about 7.0% w/v, about 2.0% w/v to about 6.5% w/v, about 2.0% w/v to about 6.0% w/v, about 2.0% w/v to about 5.5% w/v, about 2.0% w/v to about 5.0% w/v, about 2.0 w/v to about 4.5% w/v, about 2.0% w/v to about 4.0% w/v, about 2.0% w/v to about 3.5% w/v, about 2.0% w/v to about 3.0% w/v, about 2.0% w/v to about 2.5% w/v, about 2.5% w/v to about 8.0% w/v, about 2.5% w/v to about 7.5% w/v, about 2.5% w/v to about 7.0% w/v, about 2.5% w/v to about 6.5% w/v, about 2.5% w/v to about 6.0% w/v, about 2.5% w/v to about 5.5% w/v, about 2.5% w/v to about 5.0% w/v, about 2.5% w/v to about 4.5% w/v, about 2.5% w/v to about 4.0% w/v, about 2.5% w/v to about 3.5% w/v, about 2.5% w/v to about 3.0% w/v, about 3.0% w/v to about 8.0% w/v, about 3.0% w/v to about 7.5% w/v, about 3.0% w/v to about 7.0% w/v, about 3.0% w/v to about 6.5% w/v, about 3.0% w/v to about 6.0% w/v, about 3.0% w/v to about 5.5% w/v, about 3.0% w/v to about 5.0% w/v, about 3.0% w/v to about 4.5% w/v, about 3.0% w/v to about 4.0% w/v, about 3.0% w/v to about 3.5% w/v, about 3.5% w/v to about 8.0% w/v, about 3.5% w/v to about 7.5% w/v, about 3.5% w/v to about 7.0% w/v, about 3.5% w/v to about 6.5% w/v, about 3.5% w/v to about 6.0% w/v, about 3.5% w/v to about 5.5% w/v, about 3.5% w/v to about 5.0% w/v, about 3.5% w/v to about 4.5% w/v, about 3.5% w/v to about 4.0% w/v, about 4.0% w/v to about 8.0% w/v, about 4.0% w/v to about 7.5% w/v, about 4.0% w/v to about 7.0% w/v, about 4.0% w/v to about 6.5% w/v, about 4.0% w/v to about 6.0% w/v, about 4.0% w/v to about 5.5% w/v, about 4.0% w/v to about 5.0% w/v, about 4.0% w/v to about 4.5% w/v, about 4.5% w/v to about 8.0% w/v, about 4.5% w/v to about 7.5% w/v, about 4.5% w/v to about 7.0% w/v, about 4.5% w/v to about 6.5% w/v, about 4.5% w/v to about 6.0% w/v, about 4.5% w/v to about 5.5% w/v, about 4.5% w/v to about 5.0% w/v, about 5.0% w/v to about 8.0% w/v, about 5.0% w/v to about 7.5% w/v, about 5.0% w/v to about 7.0% w/v, about 5.0% w/v to about 6.5% w/v, about 5.0% w/v to about 6.0% w/v, about 5.0% w/v to about 5.5% w/v, about 5.5% w/v to about 8.0% w/v, about 5.5% w/v to about 7.5% w/v, about 5.5% w/v to about 7.0% w/v, about 5.5% w/v to about 6.5% w/v, about 5.5% w/v to about 6.0% w/v, about 6.0% w/v to about 8.0% w/v, about 6.0% w/v to about 7.5% w/v, about 6.0% w/v to about 7.0% w/v, about 6.0% w/v to about 6.5% w/v, about 6.5% w/v to about 8.0% w/v, about 6.5% w/v to about 7.5% w/v, about 6.5% w/v to about 7.0% w/v, about 7.0% w/v to about 8.0% w/v, about 7.0% w/v to about 7.5% w/v, or about 7.5% w/v to about 8.0% w/v).
[0026] In one embodiment, the buffer is selected from the group consisting of acetate, histidine, phosphate, citrate, succinate, tartrate, imidazole, and maleate, and any combination thereof. In another embodiment, the buffer is present at a concentration of about 1 mM to 150 mM (e.g., about 1 mM to about 140 mM, about 1 mM to about 130 mM, about 1 mM to about 120 mM, about 1 mM to about 110 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 18 mM, about 1 mM to about 16 mM, about 1 mM to about 14 mM, about 1 mM to about 12 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 150 mM, about 2 mM to about 140 mM, about 2 mM to about
130 mM, about 2 mM to about 120 mM, about 2 mM to about 110 mM, about
2 mM to about 100 mM, about 2 mM to about 90 mM, about 2 mM to about 80 mM, about 2 mM to about 70 mM, about 2 mM to about 60 mM, about 2 mM to about 50 mM, about 2 mM to about 45 mM, about 2 mM to about 40 mM, about 2 mM to about 35 mM, about 2 mM to about 30 mM, about 2 mM to about 25 mM, about 2 mM to about 20 mM, about 2 mM to about 18 mM, about 2 mM to about 16 mM, about 2 mM to about 14 mM, about 2 mM to about 12 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 150 mM, about 4 mM to about 140 mM, about 4 mM to about 130 mM, about 4 mM to about 120 mM, about 4 mM to about 110 mM, about 4 mM to about
100 mM, about 4 mM to about 90 mM, about 4 mM to about 80 mM, about 4 mM to about 70 mM, about 4 mM to about 60 mM, about 4 mM to about 50 mM, about 4 mM to about 45 mM, about 4 mM to about 40 mM, about 4 mM to about 35 mM, about 4 mM to about 30 mM, about 4 mM to about 25 mM, about 4 mM to about 20 mM, about 4 mM to about 18 mM, about 4 mM to about 16 mM, about 4 mM to about 14 mM, about 4 mM to about 12 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 150 mM, about 6 mM to about 140 mM, about 6 mM to about 130 mM, about 6 mM to about 120 mM, about 6 mM to about 110 mM, about 6 mM to about 100 mM, about 6 mM to about 90 mM, about 6 mM to about 80 mM, about 6 mM to about 70 mM, about 6 mM to about 60 mM, about 6 mM to about 50 mM, about 6 mM to about 45 mM, about 6 mM to about 40 mM, about 6 mM to about 35 mM, about 6 mM to about 30 mM, about 6 mM to about 25 mM, about 6 mM to about 20 mM, about 6 mM to about 18 mM, about 6 mM to about 16 mM, about 6 mM to about 14 mM, about 6 mM to about 12 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 150 mM, about 8 mM to about 140 mM, about 8 mM to about 130 mM, about 8 mM to about 120 mM, about 8 mM to about 110 mM, about 8 mM to about 100 mM, about 8 mM to about 90 mM, about 8 mM to about 80 mM, about 8 mM to about 70 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 8 mM to about 45 mM, about 8 mM to about 40 mM, about 8 mM to about 35 mM, about 8 mM to about 30 mM, about 8 mM to about 25 mM, about 8 mM to about 20 mM, about 8 mM to about 18 mM, about 8 mM to about 16 mM, about 8 mM to about 14 mM, about 8 mM to about 12 mM, about 8 mM to about 10 mM, about 10 mM to about 150 mM, about 10 mM to about 140 mM, about 10 mM to about 130 mM, about 10 mM to about 120 mM, about 10 mM to about 110 mM, about 10 mM to about 100 mM, about 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 150 mM, about 12 mM to about 140 mM, about 12 mM to about 130 mM, about 12 mM to about 120 mM, about 12 mM to about 110 mM, about 12 mM to about 100 mM, about 12 mM to about 90 mM, about 12 mM to about 80 mM, about 12 mM to about 70 mM, about 12 mM to about 60 mM, about 12 mM to about 50 mM, about 12 mM to about 45 mM, about 12 mM to about 40 mM, about 12 mM to about 35 mM, about 12 mM to about 30 mM, about 12 mM to about 25 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 150 mM, about 14 mM to about 140 mM, about 14 mM to about 130 mM, about 14 mM to about 120 mM, about 14 mM to about 110 mM, about 14 mM to about 100 mM, about 14 mM to about 90 mM, about 14 mM to about 80 mM, about 14 mM to about 70 mM, about 14 mM to about 60 mM, about 14 mM to about 50 mM, about 14 mM to about 45 mM, about 14 mM to about 40 mM, about 14 mM to about 35 mM, about 14 mM to about 30 mM, about 14 mM to about 25 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 150 mM, about 16 mM to about 140 mM, about 16 mM to about 130 mM, about 16 mM to about 120 mM, about 16 mM to about 110 mM, about 16 mM to about 100 mM, about 16 mM to about 90 mM, about 16 mM to about 80 mM, about 16 mM to about 70 mM, about 16 mM to about 60 mM, about 16 mM to about 50 mM, about 16 mM to about 45 mM, about 16 mM to about 40 mM, about 16 mM to about 35 mM, about 16 mM to about 30 mM, about 16 mM to about 25 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, about 18 mM to about 150 mM, about 18 mM to about 140 mM, about 18 mM to about 130 mM, about 18 mM to about 120 mM, about 18 mM to about 110 mM, about 18 mM to about 100 mM, about 18 mM to about 90 mM, about 18 mM to about 80 mM, about 18 mM to about 70 mM, about 18 mM to about 60 mM, about 18 mM to about 50 mM, about 18 mM to about 45 mM, about 18 mM to about 40 mM, about 18 mM to about 35 mM, about 18 mM to about 30 mM, about 18 mM to about 25 mM, about 18 mM to about 20 mM, about 20 mM to about 150 mM, about 20 mM to about 140 mM, about 20 mM to about 130 mM, about 20 mM to about 120 mM, about 20 mM to about 110 mM, about 20 mM to about 100 mM, about 20 mM to about 90 mM, about 20 mM to about 80 mM, about 20 mM to about 70 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 150 mM, about 25 mM to about 140 mM, about 25 mM to about 130 mM, about 25 mM to about 120 mM, about 25 mM to about 110 mM, about 25 mM to about 100 mM, about 25 mM to about 90 mM, about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 150 mM, about 30 mM to about 140 mM, about 30 mM to about 130 mM, about 30 mM to about 120 mM, about 30 mM to about 110 mM, about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 150 mM, about 35 mM to about 140 mM, about 35 mM to about 130 mM, about 35 mM to about 120 mM, about 35 mM to about 110 mM, about 35 mM to about 100 mM, about 35 mM to about 90 mM, about 35 mM to about 80 mM, about 35 mM to about 70 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 150 mM, about 40 mM to about 140 mM, about 40 mM to about 130 mM, about 40 mM to about 120 mM, about 40 mM to about 110 mM, about 40 mM to about 100 mM, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 mM to about 70 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 150 mM, about 45 mM to about 140 mM, about 45 mM to about 130 mM, about 45 mM to about 120 mM, about 45 mM to about 110 mM, about 45 mM to about 100 mM, about 45 mM to about 90 mM, about 45 mM to about 80 mM, about 45 mM to about 70 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 150 mM, about 50 mM to about 140 mM, about 50 mM to about 130 mM, about 50 mM to about 120 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 60 mM to about 150 mM, about 60 mM to about 140 mM, about 60 mM to about 130 mM, about 60 mM to about 120 mM, about 60 mM to about 110 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 150 mM, about 70 mM to about 140 mM, about 70 mM to about 130 mM, about 70 mM to about 120 mM, about 70 mM to about 110 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 150 mM, about 80 mM to about 140 mM, about 80 mM to about 130 mM, about 80 mM to about 120 mM, about 80 mM to about 110 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, about 90 mM to about 150 mM, about 90 mM to about 140 mM, about 90 mM to about 130 mM, about 90 mM to about 120 mM, about 90 mM to about 110 mM, about 90 mM to about 100 mM, about 100 mM to about 150 mM, about 100 mM to about 140 mM, about 100 mM to about 130 mM, about 100 mM to about 120 mM, about 100 mM to about 110 mM, about 110 mM to about 150 mM, about 110 mM to about 140 mM, about 110 mM to about 130 mM, about 110 mM to about 120 mM, about 120 mM to about 150 mM, about 120 mM to about 140 mM, about 120 mM to about 130 mM, about 130 mM to about 150 mM, about 130 mM to about 140 mM, or about 140 mM to about 150 mM). In still another embodiment, the buffer concentration is about 5 mM to 20 mM.
In yet another embodiment, the buffer concentration is about 10 mM.
[0027] In one embodiment, the stabilizer is a sugar. In another embodiment, the stabilizer is sucrose. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine. In some embodiments of any of the aqueous formulations described herein, a stabilizer (e.g., any of the exemplary stabilizers described herein) can be present at a final concentration of about 0.01 % w/v to about 8.0% w/v (e.g., about 0.01 % w/v to about 7.5% w/v, about 0.01 % w/v to about 7.0% w/v, about 0.01 % w/v to about 6.5% w/v, about 0.01 % w/v to about 6.0% w/v, about 0.01 % w/v to about 5.5% w/v, about 0.01 % w/v to about 5.0% w/v, about 0.01 % w/v to about 4.5% w/v, about 0.01 % w/v to about 4.0% w/v, about 0.01 % w/v to about 3.5% w/v, about 0.01 % w/v to about 3.0% w/v, about 0.01 % w/v to about 2.5% w/v, about 0.01 % w/v to about 2.0% w/v, about 0.01 % w/v to about 1 .5% w/v, about 0.01 % w/v, to about 1.0% w/v, about 0.01 % w/v to about 0.5% w/v, about 0.1 % w/v to about 8.0% w/v, about 0.1 % w/v to about 7.5% w/v, about 0.1 % w/v to about 7.0% w/v, about 0.1 % w/v to about 6.5% w/v, about 0.1 % w/v to about 6.0% w/v, about 0.1 % w/v to about 5.5% w/v, about 0.1 % w/v to about 5.0% w/v, about 0.1 % w/v to about 4.5% w/v, about 0.1 % w/v to about 4.0% w/v, about 0.1 % w/v to about 3.5% w/v, about 0.1 % w/v to about 3.0% w/v, about 0.1 % w/v to about 2.5% w/v, about 0.1 % w/v to about 2.0% w/v, about 0.1 % w/v to about 1.5% w/v, about 0.1 % w/v, to about 1.0% w/v, about 0.1 % w/v to about 0.5% w/v, about 0.5% w/v to about 8.0% w/v, about 0.5% w/v to about 7.5% w/v, about 0.5% w/v to about 7.0% w/v, about 0.5% w/v to about 6.5% w/v, about 0.5% w/v to about 6.0% w/v, about 0.5% w/v to about 5.5% w/v, about 0.5% w/v to about 5.0% w/v, about 0.5% w/v to about 4.5% w/v, about 0.5% w/v to about 4.0% w/v, about 0.5% w/v to about 3.5% w/v, about 0.5% w/v to about 3.0% w/v, about 0.5% w/v to about 2.5% w/v, about 0.5% w/v to about 2.0% w/v, about 0.5% w/v to about 1 .5% w/v, about 0.5% w/v to about 1.0% w/v, about 1 .0% w/v to about 8.0% w/v, about 1.0% w/v to about 7.5% w/v, about 1.0% w/v to about 7.0% w/v, about 1.0% w/v to about 6.5% w/v, about 1.0% w/v to about 6.0% w/v, about 1.0% w/v to about 5.5% w/v, about 1 .0% w/v to about 5.0% w/v, about 1.0% w/v to about 4.5% w/v, about 1.0% w/v to about 4.0% w/v, about 1 .0% w/v to about 3.5% w/v, about 1.0% w/v to about 3.0% w/v, about 1.0% w/v to about 2.5% w/v, about 1 .0% w/v to about 2.0% w/v, about 1 .0% w/v to about 1.5% w/v, about 1.5% w/v to about 8.0% w/v, about 1.5% w/v to about 7.5% w/v, about 1.5% w/v to about 7.0% w/v, about 1.5% w/v to about 6.5% w/v, about 1.5% w/v to about 6.0% w/v, about 1 .5% w/v to about 5.5% w/v, about 1.5% w/v to about 5.0% w/v, about 1.5% w/v to about 4.5% w/v, about 1 .5% w/v to about 4.0% w/v, about 1.5% w/v to about 3.5% w/v, about 1.5% w/v to about 3.0% w/v, about 1 .5% w/v to about 2.5% w/v, about 1 .5% w/v to about 2.0% w/v, about 2.0% w/v to about 8.0% w/v, about 2.0% w/v to about 7.5% w/v, about 2.0% w/v to about 7.0% w/v, about 2.0% w/v to about 6.5% w/v, about 2.0% w/v to about 6.0% w/v, about 2.0% w/v to about 5.5% w/v, about 2.0% w/v to about 5.0% w/v, about 2.0 w/v to about 4.5% w/v, about 2.0% w/v to about 4.0% w/v, about 2.0% w/v to about 3.5% w/v, about 2.0% w/v to about 3.0% w/v, about 2.0% w/v to about 2.5% w/v, about 2.5% w/v to about 8.0% w/v, about 2.5% w/v to about 7.5% w/v, about 2.5% w/v to about 7.0% w/v, about 2.5% w/v to about 6.5% w/v, about 2.5% w/v to about 6.0% w/v, about 2.5% w/v to about 5.5% w/v, about 2.5% w/v to about 5.0% w/v, about 2.5% w/v to about 4.5% w/v, about 2.5% w/v to about 4.0% w/v, about 2.5% w/v to about 3.5% w/v, about 2.5% w/v to about 3.0% w/v, about 3.0% w/v to about 8.0% w/v, about 3.0% w/v to about 7.5% w/v, about 3.0% w/v to about 7.0% w/v, about 3.0% w/v to about 6.5% w/v, about 3.0% w/v to about 6.0% w/v, about 3.0% w/v to about 5.5% w/v, about 3.0% w/v to about 5.0% w/v, about 3.0% w/v to about 4.5% w/v, about 3.0% w/v to about 4.0% w/v, about 3.0% w/v to about 3.5% w/v, about 3.5% w/v to about 8.0% w/v, about 3.5% w/v to about 7.5% w/v, about 3.5% w/v to about 7.0% w/v, about 3.5% w/v to about 6.5% w/v, about 3.5% w/v to about 6.0% w/v, about 3.5% w/v to about 5.5% w/v, about 3.5% w/v to about 5.0% w/v, about 3.5% w/v to about 4.5% w/v, about 3.5% w/v to about 4.0% w/v, about 4.0% w/v to about 8.0% w/v, about 4.0% w/v to about 7.5% w/v, about 4.0% w/v to about 7.0% w/v, about 4.0% w/v to about 6.5% w/v, about 4.0% w/v to about 6.0% w/v, about 4.0% w/v to about 5.5% w/v, about 4.0% w/v to about 5.0% w/v, about 4.0% w/v to about 4.5% w/v, about 4.5% w/v to about 8.0% w/v, about 4.5% w/v to about 7.5% w/v, about 4.5% w/v to about 7.0% w/v, about 4.5% w/v to about 6.5% w/v, about 4.5% w/v to about 6.0% w/v, about 4.5% w/v to about 5.5% w/v, about 4.5% w/v to about 5.0% w/v, about 5.0% w/v to about 8.0% w/v, about 5.0% w/v to about 7.5% w/v, about 5.0% w/v to about 7.0% w/v, about 5.0% w/v to about 6.5% w/v, about 5.0% w/v to about 6.0% w/v, about 5.0% w/v to about 5.5% w/v, about 5.5% w/v to about 8.0% w/v, about 5.5% w/v to about 7.5% w/v, about 5.5% w/v to about 7.0% w/v, about 5.5% w/v to about 6.5% w/v, about 5.5% w/v to about 6.0% w/v, about 6.0% w/v to about 8.0% w/v, about 6.0% w/v to about 7.5% w/v, about 6.0% w/v to about 7.0% w/v, about 6.0% w/v to about 6.5% w/v, about 6.5% w/v to about 8.0% w/v, about 6.5% w/v to about 7.5% w/v, about 6.5% w/v to about 7.0% w/v, about 7.0% w/v to about 8.0% w/v, about 7.0% w/v to about 7.5% w/v, or about 7.5% w/v to about 8.0% w/v).
[0028] In one embodiment, the stabilizer is an amino acid (e.g., an aromatic amino acid, e.g., tyrosine, phenylalanine, and tryptophan). In another embodiment, the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof. The amino acids may be present at a concentration of about 5-300 mM (e.g., about 5 mM to about 280 mM, about 5 mM to about 260 mM, about 5 mM to about 240 mM, about 5 mM to about 220 mM, about 5 mM to about 200 mM, about 5 mM to about 180 mM, about 5 mM to about 160 mM, about 5 mM to about 140 mM, about 5 mM to about 120 mM, about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 60 mM, about 5 mM to about 40 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 300 mM, about 10 mM to about 280 mM, about 10 mM to about 260 mM, about 10 mM to about 240 mM, about 10 mM to about 220 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 40 mM, about 10 mM to about 20 mM, about 20 mM to about 300 mM, about 20 mM to about 280 mM, about 20 mM to about 260 mM, about 20 mM to about 240 mM, about 20 mM to about 220 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 40 mM, about 40 mM to about 300 mM, about 40 mM to about 280 mM, about 40 mM to about 260 mM, about 40 mM to about 240 mM, about 40 mM to about 220 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 40 mM, about 60 mM to about 300 mM, about 60 mM to about 280 mM, about 60 mM to about 260 mM, about 60 mM to about 240 mM, about 60 mM to about 220 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 150 mM, about 150 mM to about 300 mM, about 150 mM to about 250 mM, about 150 mM to about 200 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, or about 250 mM to about 300 mM).
[0029] In another embodiment, the co-solvent-free formulations of the invention further comprise one or more salt(s). In one embodiment, the one or more salt(s) can be selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, and potassium chloride. Each of the one or more salt(s) may be present at a concentration of about 10 mM-100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 65 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 10 mM to about 95 mM, about 10 mM to about 90 mM, about 10 mM to about 85 mM, about 10 mM to about 80 mM, about 10 mM to about 75 mM, about 10 mM to about 70 mM, about 10 mM to about 65 mM, about 10 mM to about 60 mM, about 10 mM to about 55 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 100 mM, about 12 mM to about 95 mM, about 12 mM to about 90 mM, about 12 mM to about 85 mM, about 12 mM to about 80 mM, about 12 mM to about 75 mM, about 12 mM to about 70 mM, about 12 mM to about 65 mM, about 12 mM to about 60 mM, about 12 mM to about 55 mM, about 12 mM to about 50 mM, about 12 mM to about 45 mM, about 12 mM to about 40 mM, about 12 mM to about 35 mM, about 12 mM to about 30 mM, about 12 mM to about 25 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 100 mM, about 14 mM to about 95 mM, about 14 mM to about 90 mM, about 14 mM to about 85 mM, about 14 mM to about 80 mM, about 14 mM to about 75 mM, about 14 mM to about 70 mM, about 14 mM to about 65 mM, about 14 mM to about 60 mM, about 14 mM to about 55 mM, about 14 mM to about 50 mM, about 14 mM to about 45 mM, about 14 mM to about 40 mM, about 14 mM to about 35 mM, about 14 mM to about 30 mM, about 14 mM to about 25 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 100 mM, about 16 mM to about 95 mM, about 16 mM to about 90 mM, about 16 mM to about 85 mM, about 16 mM to about 80 mM, about 16 mM to about 75 mM, about 16 mM to about 70 mM, about 16 mM to about 65 mM, about 16 mM to about 60 mM, about 16 mM to about 55 mM, about 16 mM to about 50 mM, about 16 mM to about 45 mM, about 16 mM to about 40 mM, about 16 mM to about 35 mM, about 16 mM to about 30 mM, about 16 mM to about 25 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, about 18 mM to about 100 mM, about 18 mM to about 95 mM, about 18 mM to about 90 mM, about 18 mM to about 85 mM, about 18 mM to about 80 mM, about 18 mM to about 75 mM, about 18 mM to about 70 mM, about 18 mM to about 65 mM, about 18 mM to about 60 mM, about 18 mM to about 55 mM, about 18 mM to about 50 mM, about 18 mM to about 45 mM, about 18 mM to about 40 mM, about 18 mM to about 35 mM, about 18 mM to about 30 mM, about 18 mM to about 25 mM, about 18 mM to about 20 mM, about 20 mM to about 100 mM, about 20 mM to about 95 mM, about 20 mM to about 90 mM, about 20 mM to about 85 mM, about 20 mM to about 80 mM, about 20 mM to about 75 mM, about 20 mM to about 70 mM, about 20 mM to about 65 mM, about 20 mM to about 60 mM, about 20 mM to about 55 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 100 mM, about 25 mM to about 95 mM, about 25 mM to about 90 mM, about 25 mM to about 85 mM, about 25 mM to about 80 mM, about 25 mM to about 75 mM, about 25 mM to about 70 mM, about 25 mM to about 65 mM, about 25 mM to about 60 mM, about 25 mM to about 55 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 100 mM, about 30 mM to about 95 mM, about 30 mM to about 90 mM, about 30 mM to about 85 mM, about 30 mM to about 80 mM, about 30 mM to about 75 mM, about 30 mM to about 70 mM, about 30 mM to about 65 mM, about 30 mM to about 60 mM, about 30 mM to about 55 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 100 mM, about 35 mM to about 95 mM, about 35 mM to about 90 mM, about 35 mM to about 85 mM, about 35 mM to about 80 mM, about 35 mM to about 75 mM, about 35 mM to about 70 mM, about 35 mM to about 65 mM, about 35 mM to about 60 mM, about 35 mM to about 55 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 100 mM, about 40 mM to about 95 mM, about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 100 mM, about 45 mM to about 95 mM, about 45 mM to about 90 mM, about 45 mM to about 85 mM, about 45 mM to about 80 mM, about 45 mM to about 75 mM, about 45 mM to about 70 mM, about 45 mM to about 65 mM, about 45 mM to about 60 mM, about 45 mM to about 55 mM, about 45 mM to about 50 mM, about 50 mM to about 100 mM, about 50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, about 50 mM to about 55 mM, about 55 mM to about 100 mM, about 55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, about 55 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM, about 60 mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, about 60 mM to about 65 mM, about 65 mM to about 100 mM, about 65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM, about 65 mM to about 80 mM, about 65 mM to about 75 mM, about 65 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM, about 70 mM to about 80 mM, about 70 mM to about 75 mM, about 75 mM to about 100 mM, about 75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM, about 75 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 95 mM, about 80 mM to about 90 mM, about 80 mM to about 85 mM, about 85 mM to about 100 mM, about 85 mM to about 95 mM, about 85 mM to about 90 mM, about 90 mM to about 100 mM, about 90 mM to about 95 mM, or about 95 mM to about 100 mM, or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
[0030] In some embodiments of any of the formulations described herein, the formulation can include one or more of: sodium ion at a final concentration of about 100 mM to about 200 mM (e.g., about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); potassium ion at a final concentration of about 1 mM to about 20 mM (e.g., about 1 mM to about 18 mM, about 1 mM to about 16 mM, about 1 mM to about 14 mM, about 1 mM to about 12 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 20 mM, about 2 mM to about 18 mM, about 2 mM to about 16 mM, about 2 mM to about 14 mM, about 2 mM to about 12 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 20 mM, about 4 mM to about 18 mM, about 4 mM to about 16 mM, about 4 mM to about 14 mM, about 4 mM to about 12 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 20 mM, about 6 mM to about 18 mM, about 6 mM to about 16 mM, about 6 mM to about 14 mM, about 6 mM to about 12 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 20 mM, about 8 mM to about 18 mM, about 8 mM to about 16 mM, about 8 mM to about 14 mM, about 8 mM to about 12 mM, about 8 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, or about 18 mM to about 20 mM); chloride ion at a final concentration of about 10 mM to about 200 mM (e.g., about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 40 mM, about 10 mM to about 20 mM, about 50 mM to about 200 mM, 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); cacium ion at a final concentration of about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM); magnesium ion at a final concentration of about 0.1 mM to about 3 mM (e.g., about 0.1 mM to about 2.5 mM, about 0.1 mM to about 2.0 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 1 .0 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 0.5 mM to about 1 .5 mM, about 0.5 mM to about 1.0 mM, about 1.0 mM to about 3.0 mM, about 1.0 mM to about 2.5 mM, about 1.0 mM to about 2.0 mM, about 1.0 mM to about 1.5 mM, about 1.5 mM to about 3.0 mM, about 1 .5 mM to about 2.5 mM, about 1 .5 mM to about 2.0 mM, about 2.0 mM to about 3.0 mM, about 2.0 mM to about 2.5 mM, or about 2.5 mM to about 3.0 mM); sugar (e.g., glucose) at a final concentration of about 0.5 mM to about 10 mM (e.g., about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); lactate at a final concentration of about 1 mM to about 10 mM (e.g., about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about
2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); beta-hyrdoxybutyrate at a final concentration of about 0.01 mM to about 0.3 mM (e.g., about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about
3 mM, about 0.1 mM to about 0.2 mM, or about 0.2 mM to about 0.3 mM); copper ion at a concentration of about 0.1 mM to about 1.0 mM (e.g., about 0.1 mM to about 0.8 mM, about 0.1 mM to about 0.6 mM, about 0.1 mM to about 0.4 mM, or about 0.1 mM to about 0.2 mM); zinc ion at a final concentration of about 0.1 mM to about 10 mM (e.g., about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, or about 0.1 mM to about 2 mM); selenium ion at a final concentration of about 0.01 mM to about 0.2 mM (e.g., about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.10 mM, or about 0.01 mM to about 0.5 mM); iron ion at a final concentration of about 0.01 mM to about 8 mM (e.g., about 0.01 mM to about 6 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 0.05 mM); ferritin at a final concentration of about 0.1 mM to about 50 mM (e.g., 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 1 mM); and transferrin at final concentration of about 0.01 mM to about 0.2 mM (e.g., about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.10 mM, about 0.01 mM to about 0.05 mM).
[0031] In one embodiment, the formulation is iso-osmolar relative to the vitreous.
Formulations Free of Buffer and Organic Co-Solvent [0032] In another embodiment, the invention provides an aqueous formulation comprising aflibercept and a stabilizer, wherein the formulation is free of both an organic co-solvent and a buffer.
[0033] In one embodiment, the formulation has a pH of about 5.5 to about 7.0 (e.g., or any of the subranges of this range described herein, e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0). In another embodiment, the pH is about 6.0 to about 6.5. In yet another embodiment, the pH is about 6.2.
[0034] In one embodiment, the stabilizer is selected from the group consisting of amino acids, sugars (e.g., sucrose), polyols, polymers, block copolymers, alkyl saccharides (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000, or any combination thereof.
[0035] In one embodiment, the stabilizer is a block copolymer. In another embodiment, the block copolymer is poloxamer (a water-soluble nonionic A-B-A and B-A-B triblock copolymers, where A is polyethylene oxide) (PEO) and B is polypropylene oxide) (PPO)). In a futher embodiment, the poloxamer is one described in Bodratti AM, Alexandridis P. Formulation of Poloxamers for Drug Delivery. J Fund Biomater. 2018;9(1 ):11.
[0036] In one embodiment, the stabilizer is a sugar. In another embodiment, the stabilizer is sucrose.
[0037] In one embodiment, the stabilizer is an amino acid (e.g., an aromatic amino acid, e.g., tyrosine, phenylalanine, and tryptophan). In another embodiment, the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof. The amino acids may be present at a concentration of about 5-300 mM (e.g., about 25 mM to about 280 mM, about 25 mM to about 260 mM, about 25 mM to about 240 mM, about 25 mM to about 220 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 95 mM, about 25 mM to about 90 mM, about 25 mM to about 85 mM, about 25 mM to about 80 mM, about 25 mM to about 75 mM, about 25 mM to about 70 mM, about 25 mM to about 65 mM, about 25 mM to about 60 mM, about 25 mM to about 55 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 300 mM, about 30 mM to about 280 mM, about 30 mM to about 260 mM, about 30 mM to about 240 mM, about 30 mM to about 220 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 95 mM, about 30 mM to about 90 mM, about 30 mM to about 85 mM, about 30 mM to about 80 mM, about 30 mM to about 75 mM, about 30 mM to about 70 mM, about 30 mM to about 65 mM, about 30 mM to about 60 mM, about 30 mM to about 55 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 300 mM, about 35 mM to about 280 mM, about 35 mM to about 260 mM, about 35 mM to about 240 mM, about 35 mM to about 220 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 95 mM, about 35 mM to about 90 mM, about 35 mM to about 85 mM, about 35 mM to about 80 mM, about 35 mM to about 75 mM, about 35 mM to about 70 mM, about 35 mM to about 65 mM, about 35 mM to about 60 mM, about 35 mM to about 55 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 300 mM, about 40 mM to about 280 mM, about 40 mM to about 260 mM, about 40 mM to about 240 mM, about 40 mM to about 220 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 95 mM, about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 300 mM, about 45 mM to about 280 mM, about 45 mM to about 260 mM, about 45 mM to about 240 mM, about 45 mM to about 220 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 95 mM, about 45 mM to about 90 mM, about 45 mM to about 85 mM, about 45 mM to about 80 mM, about 45 mM to about 75 mM, about 45 mM to about 70 mM, about 45 mM to about 65 mM, about 45 mM to about 60 mM, about 45 mM to about 55 mM, about 45 mM to about 50 mM, about 50 mM to about 300 mM, about 50 mM to about 280 mM, about 50 mM to about 260 mM, about 50 mM to about 240 mM, about 50 mM to about 220 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, about 50 mM to about 55 mM, about 55 mM to about 300 mM, about 55 mM to about 280 mM, about 55 mM to about 260 mM, about 55 mM to about 240 mM, about 55 mM to about 220 mM, about 55 mM to about 200 mM, about 55 mM to about 180 mM, about 55 mM to about 160 mM, about 55 mM to about 140 mM, about 55 mM to about 120 mM, about 55 mM to about 100 mM, about 55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, about 55 mM to about 60 mM, about 60 mM to about 300 mM, about 60 mM to about 280 mM, about 60 mM to about 260 mM, about 60 mM to about 240 mM, about 60 mM to about 220 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM, about 60 mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, about 60 mM to about 65 mM, about 65 mM to about 300 mM, about 65 mM to about 280 mM, about 65 mM to about 260 mM, about 65 mM to about 240 mM, about 65 mM to about 220 mM, about 65 mM to about 200 mM, about 65 mM to about 180 mM, about 65 mM to about 160 mM, about 65 mM to about 140 mM, about 65 mM to about 120 mM, about 65 mM to about 100 mM, about 65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM, about 65 mM to about 80 mM, about 65 mM to about 75 mM, about 65 mM to about 70 mM, about 70 mM to about 300 mM, about 70 mM to about 280 mM, about 70 mM to about 260 mM, about 70 mM to about 240 mM, about 70 mM to about 220 mM, about 70 mM to about 200 mM, about 70 mM to about 180 mM, about 70 mM to about 160 mM, about 70 mM to about 140 mM, about 70 mM to about 120 mM, about 70 mM to about 100 mM, about 70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM, about 70 mM to about 80 mM, about 70 mM to about 75 mM, about 75 mM to about 300 mM, about 75 mM to about 280 mM, about 75 mM to about 260 mM, about 75 mM to about 240 mM, about 75 mM to about 220 mM, about 75 mM to about 200 mM, about 75 mM to about 180 mM, about 75 mM to about 160 mM, about 75 mM to about 140 mM, about 75 mM to about 120 mM, about 75 mM to about 100 mM, about 75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM, about 75 mM to about 80 mM, about 80 mM to about 300 mM, about 80 mM to about 280 mM, about 80 mM to about 260 mM, about 80 mM to about 240 mM, about 80 mM to about 220 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 80 mM to about 95 mM, about 80 mM to about 90 mM, about 80 mM to about 85 mM, about 85 mM to about 300 mM, about 85 mM to about 280 mM, about 85 mM to about 260 mM, about 85 mM to about 240 mM, about 85 mM to about 220 mM, about 85 mM to about 200 mM, about 85 mM to about 180 mM, about 85 mM to about 160 mM, about 85 mM to about 140 mM, about 85 mM to about 120 mM, about 85 mM to about 100 mM, about 85 mM to about 95 mM, about 85 mM to about 90 mM, about 90 mM to about 300 mM, about 90 mM to about 280 mM, about 90 mM to about 260 mM, about 90 mM to about 240 mM, about 90 mM to about 220 mM, about 90 mM to about 200 mM, about 90 mM to about 180 mM, about 90 mM to about 160 mM, about 90 mM to about 140 mM, about 90 mM to about 120 mM, about 90 mM to about 100 mM, about 90 mM to about 95 mM, about 95 mM to about 300 mM, about 95 mM to about 280 mM, about 95 mM to about 260 mM, about 95 mM to about 240 mM, about 95 mM to about 220 mM, about 95 mM to about 200 mM, about 95 mM to about 180 mM, about 95 mM to about 160 mM, about 95 mM to about 140 mM, about 95 mM to about 120 mM, about 95 mM to about 100 mM, about 100 mM to about 300 mM, about 100 mM to about 280 mM, about 100 mM to about 260 mM, about 100 mM to about 240 mM, about 100 mM to about 220 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 300 mM, about 120 mM to about 280 mM, about 120 mM to about 260 mM, about 120 mM to about 240 mM, about 120 mM to about 220 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 300 mM, about 140 mM to about 280 mM, about 140 mM to about 260 mM, about 140 mM to about 240 mM, about 140 mM to about 220 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 300 mM, about 160 mM to about 280 mM, about 160 mM to about 260 mM, about 160 mM to about 240 mM, about 160 mM to about 220 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 300 mM, about 180 mM to about 280 mM, about 180 mM to about 260 mM, about 180 mM to about 240 mM, about 180 mM to about 220 mM, about 180 mM to about 200 mM, about 200 mM to about 300 mM, about 200 mM to about 280 mM, about 200 mM to about 260 mM, about 200 mM to about 240 mM, about 200 mM to about 220 mM, about 220 mM to about 300 mM, about 220 mM to about 280 mM, about 220 mM to about 260 mM, about 220 mM to about 240 mM, about 240 mM to about 300 mM, about 240 mM to about 280 mM, about 240 mM to about 260 mM, about 260 mM to about 300 mM, about 260 mM to about 280 mM, or about 280 mM to about 300 mM, or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about 300 mM).ln another embodiment, the co-solvent-free and buffer-free formulations of the invention further comprise a salt. In one embodiment, the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride. The salts may be present at a concentration of about 10 mM-100 mM (e.g., or any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
[0038] In another embodiment, the co-solvent-free formulations of the invention further comprise one or more salt(s). In one embodiment, the one or more salt(s) can be selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, and potassium chloride. Each of the one or more salt(s) may be present at a concentration of about 10 mM-100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 65 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 10 mM to about 95 mM, about 10 mM to about 90 mM, about 10 mM to about 85 mM, about 10 mM to about 80 mM, about 10 mM to about 75 mM, about 10 mM to about 70 mM, about 10 mM to about 65 mM, about 10 mM to about 60 mM, about 10 mM to about 55 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 100 mM, about 12 mM to about 95 mM, about 12 mM to about 90 mM, about 12 mM to about 85 mM, about 12 mM to about 80 mM, about 12 mM to about 75 mM, about 12 mM to about 70 mM, about 12 mM to about 65 mM, about 12 mM to about 60 mM, about 12 mM to about 55 mM, about 12 mM to about 50 mM, about 12 mM to about 45 mM, about 12 mM to about 40 mM, about 12 mM to about 35 mM, about 12 mM to about 30 mM, about 12 mM to about 25 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 100 mM, about 14 mM to about 95 mM, about 14 mM to about 90 mM, about 14 mM to about 85 mM, about 14 mM to about 80 mM, about 14 mM to about 75 mM, about 14 mM to about 70 mM, about 14 mM to about 65 mM, about 14 mM to about 60 mM, about 14 mM to about 55 mM, about 14 mM to about 50 mM, about 14 mM to about 45 mM, about 14 mM to about 40 mM, about 14 mM to about 35 mM, about 14 mM to about 30 mM, about 14 mM to about 25 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 100 mM, about 16 mM to about 95 mM, about 16 mM to about 90 mM, about 16 mM to about 85 mM, about 16 mM to about 80 mM, about 16 mM to about 75 mM, about 16 mM to about 70 mM, about 16 mM to about 65 mM, about 16 mM to about 60 mM, about 16 mM to about 55 mM, about 16 mM to about 50 mM, about 16 mM to about 45 mM, about 16 mM to about 40 mM, about 16 mM to about 35 mM, about 16 mM to about 30 mM, about 16 mM to about 25 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, about 18 mM to about 100 mM, about 18 mM to about 95 mM, about 18 mM to about 90 mM, about 18 mM to about 85 mM, about 18 mM to about 80 mM, about 18 mM to about 75 mM, about 18 mM to about 70 mM, about 18 mM to about 65 mM, about 18 mM to about 60 mM, about 18 mM to about 55 mM, about 18 mM to about 50 mM, about 18 mM to about 45 mM, about 18 mM to about 40 mM, about 18 mM to about 35 mM, about 18 mM to about 30 mM, about 18 mM to about 25 mM, about 18 mM to about 20 mM, about 20 mM to about 100 mM, about 20 mM to about 95 mM, about 20 mM to about 90 mM, about 20 mM to about 85 mM, about 20 mM to about 80 mM, about 20 mM to about 75 mM, about 20 mM to about 70 mM, about 20 mM to about 65 mM, about 20 mM to about 60 mM, about 20 mM to about 55 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 100 mM, about 25 mM to about 95 mM, about 25 mM to about 90 mM, about 25 mM to about 85 mM, about 25 mM to about 80 mM, about 25 mM to about 75 mM, about 25 mM to about 70 mM, about 25 mM to about 65 mM, about 25 mM to about 60 mM, about 25 mM to about 55 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 100 mM, about 30 mM to about 95 mM, about 30 mM to about 90 mM, about 30 mM to about 85 mM, about 30 mM to about 80 mM, about 30 mM to about 75 mM, about 30 mM to about 70 mM, about 30 mM to about 65 mM, about 30 mM to about 60 mM, about 30 mM to about 55 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 100 mM, about 35 mM to about 95 mM, about 35 mM to about 90 mM, about 35 mM to about 85 mM, about 35 mM to about 80 mM, about 35 mM to about 75 mM, about 35 mM to about 70 mM, about 35 mM to about 65 mM, about 35 mM to about 60 mM, about 35 mM to about 55 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 100 mM, about 40 mM to about 95 mM, about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 100 mM, about 45 mM to about 95 mM, about 45 mM to about 90 mM, about 45 mM to about 85 mM, about 45 mM to about 80 mM, about 45 mM to about 75 mM, about 45 mM to about 70 mM, about 45 mM to about 65 mM, about 45 mM to about 60 mM, about 45 mM to about 55 mM, about 45 mM to about 50 mM, about 50 mM to about 100 mM, about 50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, about 50 mM to about 55 mM, about 55 mM to about 100 mM, about 55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, about 55 mM to about 60 mM, about 60 mM to about 100 mM, about 60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM, about 60 mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, about 60 mM to about 65 mM, about 65 mM to about 100 mM, about 65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM, about 65 mM to about 80 mM, about 65 mM to about 75 mM, about 65 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM, about 70 mM to about 80 mM, about 70 mM to about 75 mM, about 75 mM to about 100 mM, about 75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM, about 75 mM to about 80 mM, about 80 mM to about 100 mM, about 80 mM to about 95 mM, about 80 mM to about 90 mM, about 80 mM to about 85 mM, about 85 mM to about 100 mM, about 85 mM to about 95 mM, about 85 mM to about 90 mM, about 90 mM to about 100 mM, about 90 mM to about 95 mM, or about 95 mM to about 100 mM, or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
[0039] In some embodiments of any of the formulations described herein, the formulation can include one or more of: sodium ion at a final concentration of about 100 mM to about 200 mM (e.g., about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); potassium ion at a final concentration of about 1 mM to about 20 mM (e.g., about 1 mM to about 18 mM, about 1 mM to about 16 mM, about 1 mM to about 14 mM, about 1 mM to about 12 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 20 mM, about 2 mM to about 18 mM, about 2 mM to about 16 mM, about 2 mM to about 14 mM, about 2 mM to about 12 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 20 mM, about 4 mM to about 18 mM, about 4 mM to about 16 mM, about 4 mM to about 14 mM, about 4 mM to about 12 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 20 mM, about 6 mM to about 18 mM, about 6 mM to about 16 mM, about 6 mM to about 14 mM, about 6 mM to about 12 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 20 mM, about 8 mM to about 18 mM, about 8 mM to about 16 mM, about 8 mM to about 14 mM, about 8 mM to about 12 mM, about 8 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 18 mM, about 10 mM to about 16 mM, about 10 mM to about 14 mM, about 10 mM to about 12 mM, about 12 mM to about 20 mM, about 12 mM to about 18 mM, about 12 mM to about 16 mM, about 12 mM to about 14 mM, about 14 mM to about 20 mM, about 14 mM to about 18 mM, about 14 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 18 mM, or about 18 mM to about 20 mM); chloride ion at a final concentration of about 10 mM to about 200 mM (e.g., about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 40 mM, about 10 mM to about 20 mM, about 50 mM to about 200 mM, 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, or about 180 mM to about 200 mM); cacium ion at a final concentration of about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM); magnesium ion at a final concentration of about 0.1 mM to about 3 mM (e.g., about 0.1 mM to about 2.5 mM, about 0.1 mM to about 2.0 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 1 .0 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 0.5 mM to about 1 .5 mM, about 0.5 mM to about 1.0 mM, about 1.0 mM to about 3.0 mM, about 1.0 mM to about 2.5 mM, about 1.0 mM to about 2.0 mM, about 1.0 mM to about 1.5 mM, about 1.5 mM to about 3.0 mM, about 1 .5 mM to about 2.5 mM, about 1 .5 mM to about 2.0 mM, about 2.0 mM to about 3.0 mM, about 2.0 mM to about 2.5 mM, or about 2.5 mM to about 3.0 mM); sugar (e.g., glucose) at a final concentration of about 0.5 mM to about 10 mM (e.g., about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); lactate at a final concentration of about 1 mM to about 10 mM (e.g., about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about
2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); beta-hyrdoxybutyrate at a final concentration of about 0.01 mM to about 0.3 mM (e.g., about 0.01 mM to about 0.2 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.05 mM, about 0.05 mM to about 3 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.1 mM to about
3 mM, about 0.1 mM to about 0.2 mM, or about 0.2 mM to about 0.3 mM); copper ion at a concentration of about 0.1 mM to about 1.0 mM (e.g., about 0.1 mM to about 0.8 mM, about 0.1 mM to about 0.6 mM, about 0.1 mM to about 0.4 mM, or about 0.1 mM to about 0.2 mM); zinc ion at a final concentration of about 0.1 mM to about 10 mM (e.g., about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, or about 0.1 mM to about 2 mM); selenium ion at a final concentration of about 0.01 mM to about 0.2 mM (e.g., about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.10 mM, or about 0.01 mM to about 0.5 mM); iron ion at a final concentration of about 0.01 mM to about 8 mM (e.g., about 0.01 mM to about 6 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 0.05 mM); ferritin at a final concentration of about 0.1 mM to about 50 mM (e.g., 0.1 mM to about 40 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 1 mM); and transferrin at final concentration of about 0.01 mM to about 0.2 mM (e.g., about 0.01 mM to about 0.15 mM, about 0.01 mM to about 0.10 mM, about 0.01 mM to about 0.05 mM).
[0040] In one embodiment, the formulation is iso-osmolar relative to the vitreous.
[0041] These and other aspects will become apparent from the following description of the various embodiments, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
[0042] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
DESCRIPTION OF DRAWINGS
[0043] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0044] FIG. 1 is a graphic showing the effect of pH and acetate according to a PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0045] FIG. 2 is a graphic showing the effect of pH and acetate in the presence of 100 mM NaCI according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0046] FIG. 3 is a graphic showing the effect of pH and His according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0047] FIG. 4 is a graphic showing the effect of sucrose and NaCI according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at
5.5, and His at 20 mM.
[0048] FIG. 5 is a graphic showing the effect of PS20 and PS80 according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at
5.5, and His at 10 mM.
[0049] FIG. 6 is a graphic showing the effect of glycerol and PEG 3350 according to the PLS1 model using monomer content after one week at 40 ° as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at
5.5, and His at 0 mM.
[0050] FIG. 7 is a graphic showing the effect of Gly and Pro according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at 5.5, and His at 0 mM.
[0051] FIG. 8 is a graphic showing the effect of trehalose and calcium chloride according to the PLS1 model using monomer content after one week at 40 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at 5.5, and His at 0 mM.
[0052] FIG. 9 is a graphic showing the effect of pH and His according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0053] FIG. 10 is a graph showing the effect of pH and His in the presence of 100 mM NaCI according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0054] FIG. 11 is a graph showing the effect of pH and acetate according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0055] FIG. 12 is a graph showing the effect of NaCI and sucrose according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at 6.0, and the His at 20 mM.
[0056] FIG. 13 is a graphic showing the effect of PS20 and PS80 according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH 6.0, and His at 20 mM.
[0057] FIG. 14 is a graphic showing the effect of ArgHCI and PEG 3350 according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at 6.0, and His at 20 mM.
[0058] FIG. 15 is a graphic showing the effect of calcium chloride and glycine according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at 6.0, and His at 20 mM.
[0059] FIG. 16 is a graphic showing the effect of magnesium chloride and glycerol according to the PLS1 model using monomer content after two weeks at 25 °C as the endpoint. The protein concentration was fixed at 40 mg/mL, the pH at 6.0, and the His at 20 mM.
[0060] FIG. 17 is a graphic showing the effect of pH and His according to the PSL1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0061] FIG. 18 is a graphic showing the effect of pH (down to pH 7.0) and His according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0062] FIG. 19 is a graphic showing the effect of pH and acetate according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0063] FIG. 20 is a graphic showing the effect of pH and Tris according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL.
[0064] FIG. 21 is a graphic showing the effect of NaCI and sucrose according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL and pH at 6.0.
[0065] FIG. 22 is a graphic showing the effect of PS20 and PS80 according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL and pH at 6.0.
[0066] FIG. 23 is a graphic showing the effect of PEG 3360 and Gly according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL and pH 6.0.
[0067] FIG. 24 is a graphic showing the effect of ArgHCI and proline according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL and pH at 6.0.
[0068] FIG. 25 is a graphic showing the effect of magnesium chloride and calcium chloride according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL and pH at 6.0.
[0069] FIG. 26 is a graphic showing the effect of propylene glycol (PG) and glycerol according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint. The protein concentration was fixed at 40 mg/mL and pH at 6.0.
[0070] FIG. 27 is a graphic showing the effect of pH and protein concentration according to the PLS1 model using monomer content after four weeks at 5 °C as the endpoint.
[0071] FIG. 28 is a graphic showing the effect of pH and His according to the PLS2 model using the total particle concentration (counts per ml_) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL.
[0072] FIG. 29 is a graphic showing the effect of pH and acetate according to the PLS2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL.
[0073] FIG. 30 is a graphic showing the effect of pH and phosphate according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL.
[0074] FIG. 31 is a graphic showing the effect of sucrose and NaCI according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL.
[0075] FIG. 32 is a graphic showing the effect of PS20 and PS80 according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
[0076] FIG. 33 is a graphic showing the effect of glycine and ArgHCI according to the PSL2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
[0077] FIG. 34 is a graphic showing the effect of proline and PEG 3350 according to the PLS2 model using the total particle concentration (counts per ml_) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
[0078] FIG. 35 is a graphic showing the effect of Pluronic F-68 and magnesium chloride according to the PLS2 model using the total particle concentration (counts per ml_) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
[0079] FIG. 36 is a graphic showing the effect of trehalose and glycerol according to the PLS2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL and the pH set at pH 6.0.
[0080] FIG. 37 is a graphic showing the effect of propylene glycol (PG) and calcium chloride according to the PLS2 model using the total particle concentration (counts per mL) at t1 w/ 40 °C, t2 w/ 25 °C, and t4 w/ 5 °C as the endpoints. The protein concentration was fixed at 40 mg/mL and the pH set at 6.0.
[0081] FIG. 38 is a graphic showing the effect of pH and acetate according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL.
[0082] FIG. 39 is a graphic showing the e Effect of pH and His according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL.
[0083] FIG. 40 is a graphic showing the effect of pH and citrate according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL.
[0084] FIG. 41 is a graphic showing the effect of sucrose and NaCI according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
[0085] FIG. 42 is a graphic showing the effect of PS 20 and PS 80 according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
[0086] FIG. 43 is a graphic showing the effect of PEG 3350 and MgCh according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
[0087] FIG. 44 is a graphic showing the effect of Gly and Pro according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
[0088] FIG. 45 is a graphic showing the effect of glycerol and CaCh according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
[0089] FIG. 46 is a graphic showing the effect of ArgHCI and trehalose according to the PLS1 model using the difference in average pi value at t4w/5° C (compared to tO) as the endpoint. The protein concentration is fixed at 40 mg/mL and the pH is set to 5.6.
DETAILED DESCRIPTION
[0090] The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.
[0091] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present document, including definitions will control. [0092] Singleton, et a!., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991 ) provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.
[0093] The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
Definitions
[0094] The term “aflibercept” is intended to be synonymous with the active pharmaceutical ingredient in Eylea® (Regeneron Pharmaceuticals,
Inc.) and in Zaltrap® (Sanofi and Regeneron Pharmaceuticals, Inc.). Aflibercept is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF)-binding portions from the extracellular domains of human VEGF Receptors 1 and 2 fused to the Fc portion of the human lgG1 . Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation, constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa. Amino acid and nucleic acid sequences of aflibercept are known in the art. See, US 7,070,959 sequences 15 and 16 (figure 24).
[0095] The term “aflibercept” encompasses the active pharmaceutical ingredient in products intended to be, or approved as, biosimilar, interchangeable, or bio-better product of commercially available Eylea® and Zaltrap® (also referred to ziv-aflibercept). According the FDA, A biosimilar is a biological product that is highly similar to and has no clinically meaningful differences from an existing FDA-approved reference product. Therefore, a biosimilar for Eylea® or Zaltrap® may have essentially the same pharmacological effects as commercially available Eylea® or Zaltrap® even though the active pharmaceutical product in the biosimilar exhibits certain physical properties, such as glycosylation profile, that may be similar but not identical to Eylea® or Zaltrap®.
[0096] For the purposes of the present application, the term "aflibercept" also encompasses aflibercept with minor modifications in the amino acid structure (including deletions, additions, and/or substitutions of amino acids) or in the glycosylation properties, which do not significantly affect the function of the polypeptide.
[0097] The term "antibody", as used herein, refers to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order:
FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
[0098] The term “sugar” refers to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, but are not limited to, sucrose, glucose, dextrose, trehalose, lactose, and maltose.
[0099] The term “polyol” refers to an alcohol containing multiple hydroxyl groups. Examples of polyols include, but are not limited to, mannitol, sorbitol, glycerol, xylitol and inositol.
[00100] The term “metal ion” refers to a metal atom with a net positive or negative electric charge. For the purposes of the present application, the term “metal ion” also includes sources of metal ions, including but not limited to metal salts.
[00101] The term "stable" is understood to mean that aflibercept contained in the formulation or pharmaceutical compositions does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the formulation or composition at the beginning of storage. The term also should be understood to mean that the aflibercept formulations or compositions are at least comparable to, or better than commercially available aflibercept compositions, in terms of their stability and/or ability to resist formation of particulates during storage. In another embodiment, term is understood to mean the aflibercept formulations or compositions is stable when agitated, mixed, or handled.
[00102] Stability is also intended to denote the ability of the pharmaceutical aflibercept compositions disclosed herein to resist particulates formation such that the compositions, under storage conditions typical of protein therapeutics, exhibits levels and types of particulates that meet standard guidelines. A reduced tendency to form particulates in formulations disclosed herein results in aflibercept formulations having reduced immunogenicity, and therefore reduced potential to cause harm to patients resulting from such immunogenicity.
[00103] The term "long-term storage” in connection with “formulation” or “pharmaceutical composition” is understood to mean that the formulation or pharmaceutical composition can be stored for three months or more, for six months or more, or for one year or more. In one embodiment of long-term storage, the composition is stored at about 2-8°C. In another embodiment of long-term storage, the composition is stored at about 0°C. In another embodiment of long-term storage, the composition is stored at about -20°C. In yet another embodiment of long-term storage, the composition is stored at about -40°C. In another embodiment of long-term storage, the composition is stored at about -70°C. In still another embodiment of long-term storage, the composition is stored at about -80°C. Generally speaking, the terms "long term storage" and "long term stability" further include stable storage durations that are at least comparable to or better than the stability of currently available commercial formulations of aflibercept, without losses in stability that would render the formulation unsuitable for its intended pharmaceutical application.
It is also contemplated that the composition can be frozen and thawed more than once.
[00104] Stability of a protein in an aqueous formulation may also be defined as the percentage of monomer, aggregate, or fragment, or combinations thereof, of the protein in the formulation. A protein “retains its physical stability” in a formulation if it shows substantially no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography. In one aspect of the invention, a stable aqueous formulation is a formulation having less than about 10%, or less than about 5% of the protein being present as aggregate in the formulation.
[00105] A protein in an aqueous formulation can be stable at frozen storage temperatures, liquid storage temperatures such as refrigeration (e.g. 2-8°C) and room temperature (about 25°C), and under accelerated liquid stability testing temperatures (e.g. 40°C).
[00106] Various analytical techniques for measuring protein stability, including techniques for measuring the type and degree of particulates that may be present in protein formulations, are available in the art and are reviewed in PEPTIDE AND PROTEIN DRUG DELIVERY, 247-301 (Vincent Lee ed., New York, N.Y., 1991) and Jones, 1993 Adv. Drug Delivery Rev. 10: 29-90, for examples. Stability can be measured at a selected temperature for a selected time period, e.g., as described in the examples below.
[00107] The term “mammal” includes, but is not limited to, a human.
[00108] The term “ophthalmically suitable” or “suitable for ophthalmic administration” means a composition as a whole, or the individual components (e.g. excipients) of a composition are safe for administration to a subject in, on, or around the subject’s eye. Examples of such excipients are found in products already approved by the FDA for administration to a person’s eye and are known to person of skill in the art. In one embodiment, a composition suitable for ophthalmic administration has a non-stinging formulation. In one embodiment, a composition suitable for ophthalmic administration is iso-osmolar relative to the vitreous. In one embodiment, a composition suitable for ophthalmic administration has an osmolality is between about 200 and about 350 mOsM. In another embodiment, a composition suitable for ophthalmic administration can be administered intravitreously. In one embodiment, a composition suitable for ophthalmic administration is administed by intravitreal injection. In one embodiment, a composition suitable for ophthalmic administration is administed topically. In one embodiment, a composition suitable for ophthalmic administration is administed via a port. In one embodiment, a composition suitable for ophthalmic administration has a volume of about 0.1 ml_ or less. In one embodiment, a composition suitable for ophthalmic administration has a volume of about 0.05 ml_ or less. In one embodiment, a composition suitable for ophthalmic administration has a volume of about 0.025 ml_ or less.
[00109] The term “pharmaceutically acceptable carrier” refers to a non toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
[00110] The term “pharmaceutical composition” or “formulation” as used herein refers to a mixture of a protein, such as a fusion protein, e.g., aflibercept, together with one or more additional components. In some embodiments, the additional component is water or a buffer. In other embodiments, the additional components may include, e.g., one or more excipients, such as a stabilizer, tonicity modifier, surfactant, and the like, e.g., a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration to a subject for therapeutic, diagnostic, or prophylactic purposes. In one embodiment, the additional component is suitable for ophthalmic administration. For example, pharmaceutical compositions / formulations according to the present invention may be aqueous formulation suitable for intravitreal administration. [00111 ] The term "substantially free" of a particular substance means that either the substance is not present or only minimal, trace amounts of the substance are present which do not have any substantial impact on the properties of the composition. If reference is made to no amount of a substance (or that the substance is not present), it should be understood as "no detectable amount."
[00112] By “iso-osmolar relative to the vitreous” is meant that the formulation of interest has essentially the same osmotic pressure as vitreous.
[00113] As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In one example, a buffer consists of a weak acid and a conjugate salt or a weak base and a conjugate salt (e.g sodium acetate). Buffers suitable for use in connection with this invention may have a pH in the range from about 4.0 to about 9.0; from about pH 4.0 to about 7.0; or from about pH 4.5 to about 6.5.
A pH of any point in between the above ranges is also contemplated.
[00114] As used herein, the terms “buffer-free” or “free of buffer” are synonymous and intend to exclude components traditionally used to introduce buffer capacity in the formulation in addition to any buffering capacity present in the formulation absent the buffer component. For example, proteins are known to provide buffering capacity to a formulation. Hence, these terms do not mean a formulation cannot have any buffering capacity. In one embodiment, a buffer-free protein formulation is self-buffering.
[00115] As used herein, “purified” means that a molecule is present in a sample at a concentration of at least 95% by weight, or at least 98% by weight of the sample in which it is contained.
[00116] The term “organic co-solvent” is used herein in the same sense as it is used in U.S. 9,580,489. See, for example, column 2, lines 25-38. U.S. 9,580,489 states “organic co-solvent may be polysorbate, for example, polysorbate 20 or polysorbate 80, polyethylene glycol (PEG), for example, PEG 3350, or propylene glycol, or a combination thereof.” An organic co solvent is an organic (i.e. , carbon-based) substance that is added to composition primarily to keep the ingredients of the composition in solution (i.e. dissolved). This includes preventing precipitation, aggregation, and adsorption of composition ingredients.
[00117] Substances defined or recited separately from “organic co solvent” in U.S. 9,580,489 are excluded from term “organic co-solvent” in the present application. For example, U.S. 9,580,489 lists types of substances, and gives examples thereof, as distinct from an “organic co-solvent.” See, column 2, lines 25-38. In particular, U.S. 9,580,489 provides the following examples of substances that are not organic co-solvents: a tonicity agent for example sodium chloride or potassium chloride; a stabilizing agent for example sucrose, sorbitol, glycerol, trehalose, or mannitol; and a buffering agent for example phosphate buffer. Examples of compounds that are not an “organic co-solvent,” as used herein, include salt (e.g. sodium chloride, magnesium chloride), sugar (e.g. dextran or other polysaccharides), polyol (e.g. sorbitol, glycerol, mannitol), and amino acid (e.g. arginine, glycine, proline). Additional examples of compounds that are not an organic co solvent include block copolymers, alkyl saccharides (e.g., a ProTek alkyl saccharides, octyl glucoside, N-decyl-pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000, or any combination thereof.
[00118] When pharmaceutical compositions containing aflibercept are stored, the activity of aflibercept can be lost or decreased due to aggregation and/or degradation. Thus, the present invention provides aqueous formulations of aflibercept that allow stable storage of aflibercept, so that aflibercept is stable over the course of storage either in liquid or frozen states. The provided formulations do not require any extra steps such as rehydrating.
[00119] Numerous embodiments of the present invention are explained in a greater detail below.
A. Aflibercept
[00120] Aflibercept suitable for storage in one of the present pharmaceutical compositions or formulations can be produced by standard methods known in the art. For example, U.S. Pat. No. 9,580,480 and WO 2006/104852 describe various methods that a skilled artisan could use to prepare aflibercept protein for use in the formulations of the present invention. These methods are incorporated by reference herein.
[00121] Aflibercept can also be produced by purifying and/or diluting from commercially-available Eylea® and/or Zaltrap® preparations using standard methods
[00122] Purification of the expressed aflibercept can be performed by standard methods known in the art. When aflibercept is produced intracellularly by host cells, the host cells are lysed and particulate debris is removed, for example, by centrifugation or ultrafiltration. When aflibercept is secreted into the medium by host cells, supernatants from such expression systems can be first concentrated using standard polypeptide concentration filters. Protease inhibitors can also be added to inhibit proteolysis and antibiotics can be included to prevent the growth of microorganisms.
[00123] For example, aflibercept can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography and affinity chromatography, and any combination of known or yet to be discovered purification techniques, including but not limited to Protein A chromatography, fractionation on an ion-exchange column, hydrophobic interaction chromatography (HIC), ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.
B. pH/Buffer Selection for Aflibercept Formulations [00124] The formulations of the invention may include buffers, tonicity modifiers, excipients, pharmaceutically acceptable carriers and other commonly used inactive ingredients of the pharmaceutical compositions. However, formulations that indicate that an ingredient is excluded (e.g. buffer- free formulations) do not include such ingredient. [00125] Buffers maintain pH in a desired range, e.g., between pH 4 and pH 9. Buffers may also serve to stabilize aflibercept by a variety of other mechanisms, meaning they may be used outside of the nominal buffer capacity range indicated by their respective pKa values. Examples of suitable buffers include acetate (e.g., at pH 4 to 5.5), citrate (e.g., at pH 5 to 6.5), histidine (e.g., at pH 5 to 7), phosphate (e.g., at about pH 6 and 8), Tris (e.g., at pH 7 to 8), and glycine (e.g., at pH 8 to 9). Specific embodiments include, without limitation, sodium or potassium phosphate, sodium or potassium citrate, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), various forms of acetate and diethanolamine. Other suitable buffers include succinate, histidine, tartrate, bicarbonate, borate, imidazole, and maleate. In one embodiment, the concentration of the buffer in the formulation is between about 1 mM to about 1 M (e.g., about 1 mM to about 950 mM, about 1 mM to about 900 mM, about 1 mM to about 850 mM, about 1 mM to about 800 mM, about 1 mM to about 750 mM, about 1 mM to about 700 mM, about 1 mM to about 650 mM, about 1 mM to about 600 mM, about 1 mM to about 550 mM, about 1 mM to about 500 mM, about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 90 mM, about 1 mM to about 80 mM, about 1 mM to about 70 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, about 5 mM to about 1 M, about 5 mM to about 950 mM, about 5 mM to about 900 mM, about 5 mM to about 850 mM, about 5 mM to about 800 mM, about 5 mM to about 750 mM, about 5 mM to about 700 mM, about 5 mM to about 650 mM, about 5 mM to about 600 mM, about 5 mM to about 550 mM, about 5 mM to about 500 mM, about 5 mM to about 450 mM, about 5 mM to about 400 mM, about 5 mM to about 350 mM, about 5 mM to about 300 mM, about 5 mM to about 250 mM, about 5 mM to about 200 mM, about 5 mM to about 180 mM, about 5 mM to about 160 mM, about 5 mM to about 140 mM, about 5 mM to about 120 mM, about 5 mM to about 100 mM, about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 10 mM, about 10 mM to about 1 M, about 10 mM to about 950 mM, about 10 mM to about 900 mM, about 10 mM to about 850 mM, about 10 mM to about 800 mM, about 10 mM to about 750 mM, about 10 mM to about 700 mM, about 10 mM to about 650 mM, about 10 mM to about 600 mM, about 10 mM to about 550 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 70 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 1 M, about 15 mM to about 950 mM, about 15 mM to about 900 mM, about 15 mM to about 850 mM, about 15 mM to about 800 mM, about 15 mM to about 750 mM, about 15 mM to about 700 mM, about 15 mM to about 650 mM, about 15 mM to about 600 mM, about 15 mM to about 550 mM, about 15 mM to about 500 mM, about 15 mM to about 450 mM, about 15 mM to about 400 mM, about 15 mM to about 350 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, about 15 mM to about 180 mM, about 15 mM to about 160 mM, about 15 mM to about 140 mM, about 15 mM to about 120 mM, about 15 mM to about 100 mM, about 15 mM to about 90 mM, about 15 mM to about 80 mM, about 15 mM to about 70 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 1 M, about 20 mM to about 950 mM, about 20 mM to about 900 mM, about 20 mM to about 850 mM, about 20 mM to about 800 mM, about 20 mM to about 750 mM, about 20 mM to about 700 mM, about 20 mM to about 650 mM, about 20 mM to about 600 mM, about 20 mM to about 550 mM, about 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM, about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 90 mM, about 20 mM to about 80 mM, about 20 mM to about 70 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 1 M, about 25 mM to about 950 mM, about 25 mM to about 900 mM, about 25 mM to about 850 mM, about 25 mM to about 800 mM, about 25 mM to about 750 mM, about 25 mM to about 700 mM, about 25 mM to about 650 mM, about 25 mM to about 600 mM, about 25 mM to about 550 mM, about 25 mM to about 500 mM, about 25 mM to about 450 mM, about 25 mM to about 400 mM, about 25 mM to about 350 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 90 mM, about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 1 M, about 30 mM to about 950 mM, about 30 mM to about 900 mM, about 30 mM to about 850 mM, about 30 mM to about 800 mM, about 30 mM to about 750 mM, about 30 mM to about 700 mM, about 30 mM to about 650 mM, about 30 mM to about 600 mM, about 30 mM to about 550 mM, about 30 mM to about 500 mM, about 30 mM to about 450 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 1 M, about 35 mM to about 950 mM, about 35 mM to about 900 mM, about 35 mM to about 850 mM, about 35 mM to about 800 mM, about 35 mM to about 750 mM, about 35 mM to about 700 mM, about 35 mM to about 650 mM, about 35 mM to about 600 mM, about 35 mM to about 550 mM, about 35 mM to about 500 mM, about 35 mM to about 450 mM, about 35 mM to about 400 mM, about 35 mM to about 350 mM, about 35 mM to about 300 mM, about 35 mM to about 250 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 90 mM, about 35 mM to about 80 mM, about 35 mM to about 70 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 1 M, about 40 mM to about 950 mM, about 40 mM to about 900 mM, about 40 mM to about 850 mM, about 40 mM to about 800 mM, about 40 mM to about 750 mM, about 40 mM to about 700 mM, about 40 mM to about 650 mM, about 40 mM to about 600 mM, about 40 mM to about 550 mM, about 40 mM to about 500 mM, about 40 mM to about 450 mM, about 40 mM to about 400 mM, about 40 mM to about 350 mM, about 40 mM to about 300 mM, about 40 mM to about 250 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 mM to about 70 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 1 M, about 45 mM to about 950 mM, about 45 mM to about 900 mM, about 45 mM to about 850 mM, about 45 mM to about 800 mM, about 45 mM to about 750 mM, about 45 mM to about 700 mM, about 45 mM to about 650 mM, about 45 mM to about 600 mM, about 45 mM to about 550 mM, about 45 mM to about 500 mM, about 45 mM to about 450 mM, about 45 mM to about 400 mM, about 45 mM to about 350 mM, about 45 mM to about 300 mM, about 45 mM to about 250 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 90 mM, about 45 mM to about 80 mM, about 45 mM to about 70 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 1 M, about 50 mM to about 950 mM, about 50 mM to about 900 mM, about 50 mM to about 850 mM, about 50 mM to about 800 mM, about 50 mM to about 750 mM, about 50 mM to about 700 mM, about 50 mM to about 650 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 50 mM to about 60 mM, about 60 mM to about 1 M, about 60 mM to about 950 mM, about 60 mM to about 900 mM, about 60 mM to about 850 mM, about 60 mM to about 800 mM, about 60 mM to about 750 mM, about 60 mM to about 700 mM, about 60 mM to about 650 mM, about 60 mM to about 600 mM, about 60 mM to about 550 mM, about 60 mM to about 500 mM, about 60 mM to about 450 mM, about 60 mM to about 400 mM, about 60 mM to about 350 mM, about 60 mM to about 300 mM, about 60 mM to about 250 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 1 M, about 70 mM to about 950 mM, about 70 mM to about 900 mM, about 70 mM to about 850 mM, about 70 mM to about 800 mM, about 70 mM to about 750 mM, about 70 mM to about 700 mM, about 70 mM to about 650 mM, about 70 mM to about 600 mM, about 70 mM to about 550 mM, about 70 mM to about 500 mM, about 70 mM to about 450 mM, about 70 mM to about 400 mM, about 70 mM to about 350 mM, about 70 mM to about 300 mM, about 70 mM to about 250 mM, about 70 mM to about 200 mM, about 70 mM to about 180 mM, about 70 mM to about 160 mM, about 70 mM to about 140 mM, about 70 mM to about 120 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 1 M, about 80 mM to about 950 mM, about 80 mM to about 900 mM, about 80 mM to about 850 mM, about 80 mM to about 800 mM, about 80 mM to about 750 mM, about 80 mM to about 700 mM, about 80 mM to about 650 mM, about 80 mM to about 600 mM, about 80 mM to about 550 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, about 90 mM to about 1 M, about 90 mM to about 950 mM, about 90 mM to about 900 mM, about 90 mM to about 850 mM, about 90 mM to about 800 mM, about 90 mM to about 750 mM, about 90 mM to about 700 mM, about 90 mM to about 650 mM, about 90 mM to about 600 mM, about 90 mM to about 550 mM, about 90 mM to about 500 mM, about 90 mM to about 450 mM, about 90 mM to about 400 mM, about 90 mM to about 350 mM, about 90 mM to about 300 mM, about 90 mM to about 250 mM, about 90 mM to about 200 mM, about 90 mM to about 180 mM, about 90 mM to about 160 mM, about 90 mM to about 140 mM, about 90 mM to about 120 mM, about 90 mM to about 100 mM, about 100 mM to about 1 M, about 100 mM to about 950 mM, about 100 mM to about 900 mM, about 100 mM to about 850 mM, about 100 mM to about 800 mM, about 100 mM to about 750 mM, about 100 mM to about 700 mM, about 100 mM to about 650 mM, about 100 mM to about 600 mM, about 100 mM to about 550 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 1 M, about 120 mM to about 950 mM, about 120 mM to about 900 mM, about 120 mM to about 850 mM, about 120 mM to about 800 mM, about 120 mM to about 750 mM, about 120 mM to about 700 mM, about 120 mM to about 650 mM, about 120 mM to about 600 mM, about 120 mM to about 550 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 1 M, about 140 mM to about 950 mM, about 140 mM to about 900 mM, about 140 mM to about 850 mM, about 140 mM to about 800 mM, about 140 mM to about 750 mM, about 140 mM to about 700 mM, about 140 mM to about 650 mM, about 140 mM to about 600 mM, about 140 mM to about 550 mM, about 140 mM to about 500 mM, about 140 mM to about 450 mM, about 140 mM to about 400 mM, about 140 mM to about 350 mM, about 140 mM to about 300 mM, about 140 mM to about 250 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 1 M, about 160 mM to about 950 mM, about 160 mM to about 900 mM, about 160 mM to about 850 mM, about 160 mM to about 800 mM, about 160 mM to about 750 mM, about 160 mM to about 700 mM, about 160 mM to about 650 mM, about 160 mM to about 600 mM, about 160 mM to about 550 mM, about 160 mM to about 500 mM, about 160 mM to about 450 mM, about 160 mM to about 400 mM, about 160 mM to about 350 mM, about 160 mM to about 300 mM, about 160 mM to about 250 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 1 M, about 180 mM to about 950 mM, about 180 mM to about 900 mM, about 180 mM to about 850 mM, about 180 mM to about 800 mM, about 180 mM to about 750 mM, about 180 mM to about 700 mM, about 180 mM to about 650 mM, about 180 mM to about 600 mM, about 180 mM to about 550 mM, about 180 mM to about 500 mM, about 180 mM to about 450 mM, about 180 mM to about 400 mM, about 180 mM to about 350 mM, about 180 mM to about 300 mM, about 180 mM to about 250 mM, about 180 mM to about 200 mM, about 200 mM to about 1 M, about 200 mM to about 950 mM, about 200 mM to about 900 mM, about 200 mM to about 850 mM, about 200 mM to about 800 mM, about 200 mM to about 750 mM, about 200 mM to about 700 mM, about 200 mM to about 650 mM, about 200 mM to about 600 mM, about 200 mM to about 550 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 1 M, about 250 mM to about 950 mM, about 250 mM to about 900 mM, about 250 mM to about 850 mM, about 250 mM to about 800 mM, about 250 mM to about 750 mM, about 250 mM to about 700 mM, about 250 mM to about 650 mM, about 250 mM to about 600 mM, about 250 mM to about 550 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 1 M, about 300 mM to about 950 mM, about 300 mM to about 900 mM, about 300 mM to about 850 mM, about 300 mM to about 800 mM, about 300 mM to about 750 mM, about 300 mM to about 700 mM, about 300 mM to about 650 mM, about 300 mM to about 600 mM, about 300 mM to about 550 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 1 M, about 350 mM to about 950 mM, about 350 mM to about 900 mM, about 350 mM to about 850 mM, about 350 mM to about 800 mM, about 350 mM to about 750 mM, about 350 mM to about 700 mM, about 350 mM to about 650 mM, about 350 mM to about 600 mM, about 350 mM to about 550 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 1 M, about 400 mM to about 950 mM, about 400 mM to about 900 mM, about 400 mM to about 850 mM, about 400 mM to about 800 mM, about 400 mM to about 750 mM, about 400 mM to about 700 mM, about 400 mM to about 650 mM, about 400 mM to about 600 mM, about 400 mM to about 550 mM, about 400 mM to about 500 mM, about 400 mM to about 450 mM, about 450 mM to about 1 M, about 450 mM to about 950 mM, about 450 mM to about 900 mM, about 450 mM to about 850 mM, about 450 mM to about 800 mM, about 450 mM to about 750 mM, about 450 mM to about 700 mM, about 450 mM to about 650 mM, about 450 mM to about 600 mM, about 450 mM to about 550 mM, about 450 mM to about 500 mM, about 500 mM to about 1 M, about 500 mM to about 950 mM, about 500 mM to about 900 mM, about 500 mM to about 850 mM, about 500 mM to about 800 mM, about 500 mM to about 750 mM, about 500 mM to about 700 mM, about 500 mM to about 650 mM, about 500 mM to about 600 mM, about 500 mM to about 550 mM, about 550 mM to about 1 M, about 550 mM to about 950 mM, about 550 mM to about 900 mM, about 550 mM to about 850 mM, about 550 mM to about 800 mM, about 550 mM to about 750 mM, about 550 mM to about 700 mM, about 550 mM to about 650 mM, about 550 mM to about 600 mM, about 600 mM to about 1 M, about 600 mM to about 950 mM, about 600 mM to about 900 mM, about 600 mM to about 850 mM, about 600 mM to about 800 mM, about 600 mM to about 750 mM, about 600 mM to about 700 mM, about 600 mM to about 650 mM, about 650 mM to about 1 M, about 650 mM to about 950 mM, about 650 mM to about 900 mM, about 650 mM to about 850 mM, about 650 mM to about 800 mM, about 650 mM to about 750 mM, about 650 mM to about 700 mM, about 700 mM to about 1 M, about 700 mM to about 950 mM, about 700 mM to about 900 mM, about 700 mM to about 850 mM, about 700 mM to about 800 mM, about 700 mM to about 750 mM, about 750 mM to about 1 M, about 750 mM to about 950 mM, about 750 mM to about 900 mM, about 750 mM to about 850 mM, about 750 mM to about 800 mM, about 800 mM to about 1 M, about 800 mM to about 950 mM, about 800 mM to about 900 mM, about 800 mM to about 850 mM, about 850 mM to about 1 M, about 850 mM to about 950 mM, about 850 mM to about 900 mM, about 900 mM to about 1 M, about 900 mM to about 950 mM, or 950 mM to 1 M). In one embodiment, the concentration of the buffer is between 1 mM to about 150 mM. In one embodiment, the concentration of the buffer is about 5 mM to about 20 mM. In one embodiment, the concentration of the buffer is about 10 mM. Buffers are well known in the art and are manufactured by known methods and available from commercial suppliers. The selection of an appropriate buffer may be informed by data on any interactions of specific buffers with other formulation components. For example, US Patent 8,349,321 discloses that degradation of polysorbate 80 (thought to be due to a metal catalyzed oxidation of polysorbate) is significantly impeded by replacing histidine buffer with phosphate buffer in a tested placebo formulation.
[00126] The pH of the pharmaceutical compositions of the invention is generally between 5 and 7 (e.g., about 5.0 to about 6.9, about 5.0 to about 6.8, about 5.0 to about 6.7, about 5.0 to about 6.6, about 5.0 to about 6.5, about 5.0 to about 6.4, about 5.0 to about 6.3, about 5.0 to about 6.2, about 5.0 to about 6.1 , about 5.0 to about 6.0, about 5.0 to about 5.9, about 5.0 to about 5.8, about 5.0 to about 5.7, about 5.0 to about 5.6, about 5.0 to about
5.5, about 5.0 to about 5.4, about 5.0 to about 5.3, about 5.0 to about 5.2, about 5.1 to about 7.0, about 5.1 to about 6.9, about 5.1 to about 6.8, about
5.1 to about 6.7, about 5.1 to about 6.6, about 5.1 to about 6.5, about 5.1 to about 6.4, about 5.1 to about 6.3, about 5.1 to about 6.2, about 5.1 to about
6.1 , about 5.1 to about 6.0, about 5.1 to about 5.9, about 5.1 to about 5.8, about 5.1 to about 5.7, about 5.1 to about 5.6, about 5.1 to about 5.5, about
5.1 to about 5.4, about 5.1 to about 5.3, about 5.2 to about 7.0, about 5.2 to about 6.9, about 5.2 to about 6.8, about 5.2 to about 6.7, about 5.2 to about
6.6, about 5.2 to about 6.5, about 5.2 to about 6.4, about 5.2 to about 6.3, about 5.2 to about 6.2, about 5.2 to about 6.1 , about 5.2 to about 6.0, about
5.2 to about 5.9, about 5.2 to about 5.8, about 5.2 to about 5.7, about 5.2 to about 5.6, about 5.2 to about 5.5, about 5.2 to about 5.4, about 5.3 to about 7.0, about 5.3 to about 6.9, about 5.3 to about 6.8, about 5.3 to about 6.7, about 5.3 to about 6.6, about 5.3 to about 6.5, about 5.3 to about 6.4, about
5.3 to about 6.3, about 5.3 to about 6.2, about 5.3 to about 6.1 , about 5.3 to about 6.0, about 5.3 to about 5.9, about 5.3 to about 5.8, about 5.3 to about
5.7, about 5.3 to about 5.6, about 5.3 to about 5.5, about 5.4 to about 7.0, about 5.4 to about 6.9, about 5.4 to about 6.8, about 5.4 to about 6.7, about
5.4 to about 6.6, about 5.4 to about 6.5, about 5.4 to about 6.4, about 5.4 to about 6.3, about 5.4 to about 6.2, about 5.4 to about 6.1 , about 5.4 to about 6.0, about 5.4 to about 5.9, about 5.4 to about 5.8, about 5.4 to about 5.7, about 5.4 to about 5.6, about 5.5 to about 7.0, about 5.5 to about 6.9, about
5.5 to about 6.8, about 5.5 to about 6.7, about 5.5 to about 6.6, about 5.5 to about 6.5, about 5.5 to about 6.4, about 5.5 to about 6.3, about 5.5 to about
6.2, about 5.5 to about 6.1 , about 5.5 to about 6.0, about 5.5 to about 5.9, about 5.5 to about 5.8, about 5.5 to about 5.7, about 5.6 to about 7.0, about
5.6 to about 6.9, about 5.6 to about 6.8, about 5.6 to about 6.7, about 5.6 to about 6.6, about 5.6 to about 6.5, about 5.6 to about 6.4, about 5.6 to about 6.3, about 5.6 to about 6.2, about 5.6 to about 6.1 , about 5.6 to about 6.0, about 5.6 to about 5.9, about 5.6 to about 5.8, about 5.7 to about 7.0, about
5.7 to about 6.9, about 5.7 to about 6.8, about 5.7 to about 6.7, about 5.7 to about 6.6, about 5.7 to about 6.5, about 5.7 to about 6.4, about 5.7 to about 6.3, about 5.7 to about 6.2, about 5.7 to about 6.1 , about 5.7 to about 6.0, about 5.7 to about 5.9, about 5.8 to about 7.0, about 5.8 to about 6.9, about
5.8 to about 6.8, about 5.8 to about 6.7, about 5.8 to about 6.6, about 5.8 to about 6.5, about 5.8 to about 6.4, about 5.8 to about 6.3, about 5.8 to about 6.2, about 5.8 to about 6.1 , about 5.8 to about 6.0, about 5.9 to about 7.0, about 5.9 to about 6.9, about 5.9 to about 6.8, about 5.9 to about 6.7, about
5.9 to about 6.6, about 5.9 to about 6.5, about 5.9 to about 6.4, about 5.9 to about 6.3, about 5.9 to about 6.2, about 5.9 to about 6.1 , about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5, about 6.0 to about 6.4, about 6.0 to about 6.3, about 6.0 to about 6.2, about 6.1 to about 7.0, about 6.1 to about 6.9, about 6.1 to about 6.8, about 6.1 to about 6.7, about 6.1 to about 6.6, about 6.1 to about 6.5, about 6.1 to about 6.4, about 6.1 to about 6.3, about 6.2 to about 7.0, about 6.2 to about 6.9, about 6.2 to about 6.8, about 6.2 to about 6.7, about 6.2 to about 6.6, about 6.2 to about 6.5, about 6.2 to about 6.4, about 6.3 to about 7.0, about 6.3 to about 6.9, about 6.3 to about 6.8, about 6.3 to about 6.7, about 6.3 to about 6.6, about 6.3 to about 6.5, about 6.4 to about 7.0, about 6.4 to about 6.9, about 6.4 to about 6.8, about 6.4 to about 6.7, about 6.4 to about 6.6, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.0, about 6.6 to about 6.9, about 6.6 to about 6.8, about 6.7 to about 7.0, about 6.7 to about 6.9, or about 6.8 to about 7.0, or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0). In one embodiment, the pH is between 5.5 and 7.0. In another embodiment, the pH is between 6.0 and 6.5. In another embodiment, the pH is about 6.2.
[00127] A person of ordinary skill in the art will understand that the pH can be adjusted as necessary to maximize stability and solubility of aflibercept in a particular formulation. For example, a strong acid (e.g. HCI) or a strong base (NaOH) can be added to a formulation to adjust the pH down or up.
Such adjustment to a target pH can be made in buffer-free formulations and such formulations are still considered buffer-free
C. Excipients Suitable for use in Aflibercept Formulations [00128] Excipients include components of a pharmaceutical formulation other than the active ingredient and are typically added during formulation development for a specific purpose, such as to stabilize the polypeptide while in solution, change viscosity of the formulation, adjust tonicity, and confer other desired properties to the formulation. Excipients are well known in the art and are manufactured by known methods and available from commercial suppliers. Excipients may include, for example, tonicity modifiers (e.g., any of the exemplary tonicity modifiers described herein), stabilizers (e.g., any of the exemplary stabilizers described herein), salts (e.g., any of the exemplary salts described herein), chelating agents (e.g., any of the exemplary chelating agents described herein), sacrificial additives and surfactants (e.g., any of the exemplary additives or surfactants described herein), and miscellaneous excipients such as ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta- mannoglycerate, and the like. In one embodiment, the concentration of an excipient in a formulation of the invention is between about 0.001 to 30 weight percent (e.g., about 0.001 to about 28 weight percent, about 0.001 to about 26 weight percent, about 0.001 to about 24 weight percent, about 0.001 to about 22 weight percent, about 0.001 to about 20 weight percent, about 0.001 to about 18 weight percent, about 0.001 to about 16 weight percent, about 0.001 to about 14 weight percent, about 0.001 to about 12 weight percent, about 0.001 to about 10 weight percent, about 0.001 to about 9 weight percent, about 0.001 to about 8 weight percent, about 0.001 to about 7 weight percent, about 0.001 to about 6 weight percent, about 0.001 to about about 5 weight percent, about 0.001 to about 4.5 weight percent, about 0.001 to about 4.0 weight percent, about 0.001 to about 3.5 weight percent, about 0.001 to about 3.0 weight percent, about 0.001 to about 2.5 weight percent, about 0.001 to about 2.0 weight percent, about 0.001 to about 1.5 weight percent, about 0.001 to about 1 .0 weight percent, about 0.001 to about 0.5 weight percent, about 0.001 to about 0.1 weight percent, about 0.001 to about 0.01 weight percent, about 0.01 to about 30 weight percent, about 0.01 to about 28 weight percent, about 0.01 to about 26 weight percent, about 0.01 to about 24 weight percent, about 0.01 to about 22 weight percent, about 0.01 to about 20 weight percent, about 0.01 to about 18 weight percent, about 0.01 to about 16 weight percent, about 0.01 to about 14 weight percent, about 0.01 to about 12 weight percent, about 0.01 to about 10 weight percent, about 0.01 to about 9 weight percent, about 0.01 to about 8 weight percent, about 0.01 to about 7 weight percent, about 0.01 to about 6 weight percent, about 0.01 to about about 5 weight percent, about 0.01 to about 4.5 weight percent, about 0.01 to about 4.0 weight percent, about 0.01 to about 3.5 weight percent, about 0.01 to about 3.0 weight percent, about 0.01 to about 2.5 weight percent, about 0.01 to about 2.0 weight percent, about 0.01 to about 1.5 weight percent, about 0.01 to about 1.0 weight percent, about 0.01 to about 0.5 weight percent, about 0.01 to about 0.1 weight percent, about 0.1 to about 30 weight percent, about 0.1 to about 28 weight percent, about 0.1 to about 26 weight percent, about 0.1 to about 24 weight percent, about 0.1 to about 22 weight percent, about 0.1 to about 20 weight percent, about 0.1 to about 18 weight percent, about 0.1 to about 16 weight percent, about 0.1 to about 14 weight percent, about 0.1 to about 12 weight percent, about 0.1 to about 10 weight percent, about 0.1 to about 9 weight percent, about 0.1 to about 8 weight percent, about 0.1 to about 7 weight percent, about 0.1 to about 6 weight percent, about 0.1 to about about 5 weight percent, about 0.1 to about 4.5 weight percent, about 0.1 to about 4.0 weight percent, about 0.1 to about 3.5 weight percent, about 0.1 to about 3.0 weight percent, about 0.1 to about 2.5 weight percent, about 0.1 to about 2.0 weight percent, about 0.1 to about 1.5 weight percent, about 0.1 to about 1.0 weight percent, about 0.1 to about 0.5 weight percent, about 0.5 to about 30 weight percent, about 0.5 to about 28 weight percent, about 0.5 to about 26 weight percent, about 0.5 to about 24 weight percent, about 0.5 to about 22 weight percent, about 0.5 to about 20 weight percent, about 0.5 to about 18 weight percent, about 0.5 to about 16 weight percent, about 0.5 to about 14 weight percent, about 0.5 to about 12 weight percent, about 0.5 to about 10 weight percent, about 0.5 to about 9 weight percent, about 0.5 to about 8 weight percent, about 0.5 to about 7 weight percent, about 0.5 to about 6 weight percent, about 0.5 to about about 5 weight percent, about 0.5 to about 4.5 weight percent, about 0.5 to about 4.0 weight percent, about 0.5 to about 3.5 weight percent, about 0.5 to about 3.0 weight percent, about 0.5 to about 2.5 weight percent, about 0.5 to about 2.0 weight percent, about 0.5 to about 1.5 weight percent, about 0.5 to about 1 .0 weight percent, about 1 .0 to about 30 weight percent, about 1 .0 to about 28 weight percent, about 1.0 to about 26 weight percent, about 1.0 to about
24 weight percent, about 1.0 to about 22 weight percent, about 1.0 to about
20 weight percent, about 1.0 to about 18 weight percent, about 1.0 to about
16 weight percent, about 1.0 to about 14 weight percent, about 1.0 to about
12 weight percent, about 1.0 to about 10 weight percent, about 1.0 to about 9 weight percent, about 1.0 to about 8 weight percent, about 1.0 to about 7 weight percent, about 1.0 to about 6 weight percent, about 1.0 to about about 5 weight percent, about 1.0 to about 4.5 weight percent, about 1.0 to about 4.0 weight percent, about 1 .0 to about 3.5 weight percent, about 1.0 to about 3.0 weight percent, about 1 .0 to about 2.5 weight percent, about 1.0 to about 2.0 weight percent, about 1 .0 to about 1.5 weight percent, about 1.5 to about 30 weight percent, about 1.5 to about 28 weight percent, about 1.5 to about
26 weight percent, about 1.5 to about 24 weight percent, about 1.5 to about
22 weight percent, about 1.5 to about 20 weight percent, about 1.5 to about
18 weight percent, about 1.5 to about 16 weight percent, about 1.5 to about
14 weight percent, about 1.5 to about 12 weight percent, about 1.5 to about
10 weight percent, about 1.5 to about 9 weight percent, about 1 .5 to about 8 weight percent, about 1.5 to about 7 weight percent, about 1.5 to about 6 weight percent, about 1.5 to about about 5 weight percent, about 1.5 to about 4.5 weight percent, about 1 .5 to about 4.0 weight percent, about 1.5 to about
3.5 weight percent, about 1 .5 to about 3.0 weight percent, about 1.5 to about
2.5 weight percent, about 1 .5 to about 2.0 weight percent, about 2.0 to about
30 weight percent, about 2.0 to about 28 weight percent, about 2.0 to about
26 weight percent, about 2.0 to about 24 weight percent, about 2.0 to about
22 weight percent, about 2.0 to about 20 weight percent, about 2.0 to about
18 weight percent, about 2.0 to about 16 weight percent, about 2.0 to about
14 weight percent, about 2.0 to about 12 weight percent, about 2.0 to about
10 weight percent, about 2.0 to about 9 weight percent, about 2.0 to about 8 weight percent, about 2.0 to about 7 weight percent, about 2.0 to about 6 weight percent, about 2.0 to about about 5 weight percent, about 2.0 to about
4.5 weight percent, about 2.0 to about 4.0 weight percent, about 2.0 to about
3.5 weight percent, about 2.0 to about 3.0 weight percent, about 2.0 to about
2.5 weight percent, about 2.5 to about 30 weight percent, about 2.5 to about
28 weight percent, about 2.5 to about 26 weight percent, about 2.5 to about
24 weight percent, about 2.5 to about 22 weight percent, about 2.5 to about
20 weight percent, about 2.5 to about 18 weight percent, about 2.5 to about
16 weight percent, about 2.5 to about 14 weight percent, about 2.5 to about
12 weight percent, about 2.5 to about 10 weight percent, about 2.5 to about 9 weight percent, about 2.5 to about 8 weight percent, about 2.5 to about 7 weight percent, about 2.5 to about 6 weight percent, about 2.5 to about about 5 weight percent, about 2.5 to about 4.5 weight percent, about 2.5 to about 4.0 weight percent, about 2.5 to about 3.5 weight percent, about 2.5 to about 3.0 weight percent, about 3.0 to about 30 weight percent, about 3.0 to about 28 weight percent, about 3.0 to about 26 weight percent, about 3.0 to about
24 weight percent, about 3.0 to about 22 weight percent, about 3.0 to about
20 weight percent, about 3.0 to about 18 weight percent, about 3.0 to about
16 weight percent, about 3.0 to about 14 weight percent, about 3.0 to about
12 weight percent, about 3.0 to about 10 weight percent, about 3.0 to about 9 weight percent, about 3.0 to about 8 weight percent, about 3.0 to about 7 weight percent, about 3.0 to about 6 weight percent, about 3.0 to about about 5 weight percent, about 3.0 to about 4.5 weight percent, about 3.0 to about 4.0 weight percent, about 3.0 to about 3.5 weight percent, about 3.5 to about 30 weight percent, about 3.5 to about 28 weight percent, about 3.5 to about
26 weight percent, about 3.5 to about 24 weight percent, about 3.5 to about
22 weight percent, about 3.5 to about 20 weight percent, about 3.5 to about
18 weight percent, about 3.5 to about 16 weight percent, about 3.5 to about
14 weight percent, about 3.5 to about 12 weight percent, about 3.5 to about
10 weight percent, about 3.5 to about 9 weight percent, about 3.5 to about 8 weight percent, about 3.5 to about 7 weight percent, about 3.5 to about 6 weight percent, about 3.5 to about about 5 weight percent, about 3.5 to about 4.5 weight percent, about 3.5 to about 4.0 weight percent, about 4.0 to about 30 weight percent, about 4.0 to about 28 weight percent, about 4.0 to about
26 weight percent, about 4.0 to about 24 weight percent, about 4.0 to about
22 weight percent, about 4.0 to about 20 weight percent, about 4.0 to about
18 weight percent, about 4.0 to about 16 weight percent, about 4.0 to about
14 weight percent, about 4.0 to about 12 weight percent, about 4.0 to about
10 weight percent, about 4.0 to about 9 weight percent, about 4.0 to about 8 weight percent, about 4.0 to about 7 weight percent, about 4.0 to about 6 weight percent, about 4.0 to about about 5 weight percent, about 4.0 to about
4.5 weight percent, about 4.5 to about 30 weight percent, about 4.5 to about 28 weight percent, about 4.5 to about 26 weight percent, about 4.5 to about
24 weight percent, about 4.5 to about 22 weight percent, about 4.5 to about
20 weight percent, about 4.5 to about 18 weight percent, about 4.5 to about
16 weight percent, about 4.5 to about 14 weight percent, about 4.5 to about
12 weight percent, about 4.5 to about 10 weight percent, about 4.5 to about 9 weight percent, about 4.5 to about 8 weight percent, about 4.5 to about 7 weight percent, about 4.5 to about 6 weight percent, about 4.5 to about about
5 weight percent, about 5 to about 30 weight percent, about 5 to about 28 weight percent, about 5 to about 26 weight percent, about 5 to about 24 weight percent, about 5 to about 22 weight percent, about 5 to about 20 weight percent, about 5 to about 18 weight percent, about 5 to about 16 weight percent, about 5 to about 14 weight percent, about 5 to about 12 weight percent, about 5 to about 10 weight percent, about 5 to about 9 weight percent, about 5 to about 8 weight percent, about 5 to about 7 weight percent, about 5 to about 6 weight percent, about 6 to about 30 weight percent, about 6 to about 28 weight percent, about 6 to about 26 weight percent, about 6 to about 24 weight percent, about 6 to about 22 weight percent, about 6 to about 20 weight percent, about 6 to about 18 weight percent, about 6 to about 16 weight percent, about 6 to about 14 weight percent, about 6 to about 12 weight percent, about 6 to about 10 weight percent, about 6 to about 9 weight percent, about 6 to about 8 weight percent, about 6 to about 7 weight percent, about 7 to about 30 weight percent, about 7 to about 28 weight percent, about
7 to about 26 weight percent, about 7 to about 24 weight percent, about 7 to about 22 weight percent, about 7 to about 20 weight percent, about 7 to about 18 weight percent, about 7 to about 16 weight percent, about 7 to about 14 weight percent, about 7 to about 12 weight percent, about 7 to about 10 weight percent, about 7 to about 9 weight percent, about 7 to about 8 weight percent, about 8 to about 30 weight percent, about 8 to about 28 weight percent, about 8 to about 26 weight percent, about 8 to about 24 weight percent, about 8 to about 22 weight percent, about 8 to about 20 weight percent, about 8 to about 18 weight percent, about 8 to about 16 weight percent, about 8 to about 14 weight percent, about 8 to about 12 weight percent, about 8 to about 10 weight percent, about 8 to about 9 weight percent, about 9 to about 30 weight percent, about 9 to about 28 weight percent, about 9 to about 26 weight percent, about 9 to about 24 weight percent, about 9 to about 22 weight percent, about 9 to about 20 weight percent, about 9 to about 18 weight percent, about 9 to about 16 weight percent, about 9 to about 14 weight percent, about 9 to about 12 weight percent, about 9 to about 10 weight percent, about 10 to about 30 weight percent, about 10 to about 28 weight percent, about 10 to about 26 weight percent, about 10 to about 24 weight percent, about 10 to about 22 weight percent, about 10 to about 20 weight percent, about 10 to about 18 weight percent, about 10 to about 16 weight percent, about 10 to about 14 weight percent, about 10 to about 12 weight percent, about 12 to about 30 weight percent, about 12 to about 28 weight percent, about 12 to about 26 weight percent, about 12 to about 24 weight percent, about 12 to about 22 weight percent, about 12 to about 20 weight percent, about 12 to about 18 weight percent, about 12 to about 16 weight percent, about 12 to about 14 weight percent, about 14 to about 30 weight percent, about 14 to about 28 weight percent, about 14 to about 26 weight percent, about 14 to about 24 weight percent, about 14 to about 22 weight percent, about 14 to about 20 weight percent, about 14 to about 18 weight percent, about 14 to about 16 weight percent, about 16 to about 30 weight percent, about 16 to about 28 weight percent, about 16 to about 26 weight percent, about 16 to about 24 weight percent, about 16 to about 22 weight percent, about 16 to about 20 weight percent, about 16 to about 18 weight percent, about 18 to about 30 weight percent, about 18 to about 28 weight percent, about 18 to about 26 weight percent, about 18 to about 24 weight percent, about 18 to about 22 weight percent, about 18 to about 20 weight percent, about 20 to about 30 weight percent, about 20 to about 28 weight percent, about 20 to about 26 weight percent, about 20 to about 24 weight percent, about 20 to about 22 weight percent, about 22 to about 30 weight percent, about 22 to about 28 weight percent, about 22 to about 26 weight percent, about 22 to about 24 weight percent, about 24 to about 30 weight percent, about 24 to about 28 weight percent, about 24 to about 26 weight percent, about 26 to about 30 weight percent, about 26 to about 28 weight percent, or about 28 to about 30 weight percent).
[00129] In another embodiment, the concentration of the excipient is about 0.01 to 10 weight percent.
[00130] In one embodiment, the concentration of an excipient in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 500 mM, about 2 mM to about 450 mM, about 2 mM to about 400 mM, about 2 mM to about 350 mM, about 2 mM to about 300 mM, about 2 mM to about 250 mM, about 2 mM to about 200 mM, about 2 mM to about 180 mM, about 2 mM to about 160 mM, about 2 mM to about 140 mM, about 2 mM to about 120 mM, about 2 mM to about 100 mM, about 2 mM to about 80 mM, about 2 mM to about 60 mM, about 2 mM to about 50 mM, about 2 mM to about 45 mM, about 2 mM to about 40 mM, about 2 mM to about 35 mM, about 2 mM to about 30 mM, about 2 mM to about 25 mM, about 2 mM to about 20 mM, about 2 mM to about 15 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 500 mM, about 4 mM to about 450 mM, about 4 mM to about 400 mM, about 4 mM to about 350 mM, about 4 mM to about 300 mM, about 4 mM to about 250 mM, about 4 mM to about 200 mM, about 4 mM to about 180 mM, about 4 mM to about 160 mM, about 4 mM to about 140 mM, about 4 mM to about 120 mM, about 4 mM to about 100 mM, about 4 mM to about 80 mM, about 4 mM to about 60 mM, about 4 mM to about 50 mM, about 4 mM to about 45 mM, about 4 mM to about 40 mM, about 4 mM to about 35 mM, about 4 mM to about 30 mM, about 4 mM to about 25 mM, about 4 mM to about 20 mM, about 4 mM to about 15 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 500 mM, about 6 mM to about 450 mM, about 6 mM to about 400 mM, about 6 mM to about 350 mM, about 6 mM to about 300 mM, about 6 mM to about 250 mM, about 6 mM to about 200 mM, about 6 mM to about 180 mM, about 6 mM to about 160 mM, about 6 mM to about 140 mM, about 6 mM to about 120 mM, about 6 mM to about 100 mM, about 6 mM to about 80 mM, about 6 mM to about 60 mM, about 6 mM to about 50 mM, about 6 mM to about 45 mM, about 6 mM to about 40 mM, about 6 mM to about 35 mM, about 6 mM to about 30 mM, about 6 mM to about 25 mM, about 6 mM to about 20 mM, about 6 mM to about 15 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 500 mM, about 8 mM to about 450 mM, about 8 mM to about 400 mM, about 8 mM to about 350 mM, about 8 mM to about 300 mM, about 8 mM to about 250 mM, about 8 mM to about 200 mM, about 8 mM to about 180 mM, about 8 mM to about 160 mM, about 8 mM to about 140 mM, about 8 mM to about 120 mM, about 8 mM to about 100 mM, about 8 mM to about 80 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 8 mM to about 45 mM, about 8 mM to about 40 mM, about 8 mM to about 35 mM, about 8 mM to about 30 mM, about 8 mM to about 25 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 10 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 500 mM, about 15 mM to about 450 mM, about 15 mM to about 400 mM, about 15 mM to about 350 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, about 15 mM to about 180 mM, about 15 mM to about 160 mM, about 15 mM to about 140 mM, about 15 mM to about 120 mM, about 15 mM to about 100 mM, about 15 mM to about 80 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM, about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 500 mM, about 25 mM to about 450 mM, about 25 mM to about 400 mM, about 25 mM to about 350 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 80 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 500 mM, about 30 mM to about 450 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 80 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 500 mM, about 35 mM to about 450 mM, about 35 mM to about 400 mM, about 35 mM to about 350 mM, about 35 mM to about 300 mM, about 35 mM to about 250 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 80 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 500 mM, about 40 mM to about 450 mM, about 40 mM to about 400 mM, about 40 mM to about 350 mM, about 40 mM to about 300 mM, about 40 mM to about 250 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 500 mM, about 45 mM to about 450 mM, about 45 mM to about 400 mM, about 45 mM to about 350 mM, about 45 mM to about 300 mM, about 45 mM to about 250 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 80 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 60 mM to about 500 mM, about 60 mM to about 450 mM, about 60 mM to about 400 mM, about 60 mM to about 350 mM, about 60 mM to about 300 mM, about 60 mM to about 250 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 500 mM, about 140 mM to about 450 mM, about 140 mM to about 400 mM, about 140 mM to about 350 mM, about 140 mM to about 300 mM, about 140 mM to about 250 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 500 mM, about 160 mM to about 450 mM, about 160 mM to about 400 mM, about 160 mM to about 350 mM, about 160 mM to about 300 mM, about 160 mM to about 250 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 500 mM, about 180 mM to about 450 mM, about 180 mM to about 400 mM, about 180 mM to about 350 mM, about 180 mM to about 300 mM, about 180 mM to about 250 mM, about 180 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM, about 400 mM to about 450 mM, or about 450 mM to about 500 mM).
I. Tonicity Modifiers
[00131] A tonicity modifier is an ingredient that contributes to the osmolality of a solution. A tonicity modifier may also provide some degree of conformational or colloidal stabilization as well. The osmolality of a pharmaceutical composition is adjusted to minimize discomfort to the patient upon administration. Additionally, osmolality of a composition may impact the active ingredient's stability and can be adjusted to improve the stability. It is generally preferred that a pharmaceutical composition for intravitreal administration to a patient be isotonic with vitreous, i.e. , have the same or similar osmolality, which is achieved by addition of a tonicity modifier. However, hypertonic formulations which would then be diluted in an isotonic vehicle are also within the scope of this invention.
[00132] In one embodiment, the osmolality of the provided formulations is from about 180 to about 500mOsM (e.g., about 180 to about 450 mOsM, about 180 to about 400 mOsM, about 180 to about 350 mOsM, about 180 to about 300 mOsM, about 180 to about 250 mOsM, about 180 to about 200 mOsM, about 200 to about 500 mOsM, about 200 to about 450 mOsM, about 200 to about 400 mOsM, about 200 to about 350 mOsM, about 200 to about 300 mOsM, about 200 to about 250 mOsM, about 250 to about 500 mOsM, about 250 to about 450 mOsM, about 250 to about 400 mOsM, about 250 to about 350 mOsM, about 250 to about 300 mOsM, about 300 to about 500 mOsM, about 300 to about 450 mOsM, about 300 to about 400 mOsM, about 300 to about 350 mOsM, about 350 to about 500 mOsM, about 350 to about 450 mOsM, about 350 to about 400 mOsM, about 400 to about 500 mOsM, about 400 to about 450 mOsM, or about 450 to about 500 mOsM).
[00133] In one embodiment, the osmolality is between about 200 and about 300 mOsM. However, it is to be understood that the osmolality can be either higher or lower as specific conditions require.
[00134] Examples of tonicity modifiers suitable for modifying osmolality include, but are not limited to amino acids (e.g., cysteine, arginine, histidine and glycine), salts (e.g., sodium chloride, potassium chloride and sodium citrate) and/or nonelectrolytes (e.g., sugars or polyols, such as, for example, sucrose, glucose and mannitol).
[00135] In one embodiment, the concentration of the tonicity modifier in the formulation is between about 1 mM to about 1 M (e.g., any of the subranges of this range described herein). In another embodiment, the concentration is about 50 mM to about 500 mM.
[00136] In one embodiment, the concentration of the tonicity modifier in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 500 mM, about 2 mM to about 450 mM, about 2 mM to about 400 mM, about 2 mM to about 350 mM, about 2 mM to about 300 mM, about 2 mM to about 250 mM, about 2 mM to about 200 mM, about 2 mM to about 180 mM, about 2 mM to about 160 mM, about 2 mM to about 140 mM, about 2 mM to about 120 mM, about 2 mM to about 100 mM, about 2 mM to about 80 mM, about 2 mM to about 60 mM, about 2 mM to about 50 mM, about 2 mM to about 45 mM, about 2 mM to about 40 mM, about 2 mM to about 35 mM, about 2 mM to about 30 mM, about 2 mM to about 25 mM, about 2 mM to about 20 mM, about 2 mM to about 15 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 500 mM, about 4 mM to about 450 mM, about 4 mM to about 400 mM, about 4 mM to about 350 mM, about 4 mM to about 300 mM, about 4 mM to about 250 mM, about 4 mM to about 200 mM, about 4 mM to about 180 mM, about 4 mM to about 160 mM, about 4 mM to about 140 mM, about 4 mM to about 120 mM, about 4 mM to about 100 mM, about 4 mM to about 80 mM, about 4 mM to about 60 mM, about 4 mM to about 50 mM, about 4 mM to about 45 mM, about 4 mM to about 40 mM, about 4 mM to about 35 mM, about 4 mM to about 30 mM, about 4 mM to about 25 mM, about 4 mM to about 20 mM, about 4 mM to about 15 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 500 mM, about 6 mM to about 450 mM, about 6 mM to about 400 mM, about 6 mM to about 350 mM, about 6 mM to about 300 mM, about 6 mM to about 250 mM, about 6 mM to about 200 mM, about 6 mM to about 180 mM, about 6 mM to about 160 mM, about 6 mM to about 140 mM, about 6 mM to about 120 mM, about 6 mM to about 100 mM, about 6 mM to about 80 mM, about 6 mM to about 60 mM, about 6 mM to about 50 mM, about 6 mM to about 45 mM, about 6 mM to about 40 mM, about 6 mM to about 35 mM, about 6 mM to about 30 mM, about 6 mM to about 25 mM, about 6 mM to about 20 mM, about 6 mM to about 15 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 500 mM, about 8 mM to about 450 mM, about 8 mM to about 400 mM, about 8 mM to about 350 mM, about 8 mM to about 300 mM, about 8 mM to about 250 mM, about 8 mM to about 200 mM, about 8 mM to about 180 mM, about 8 mM to about 160 mM, about 8 mM to about 140 mM, about 8 mM to about 120 mM, about 8 mM to about 100 mM, about 8 mM to about 80 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 8 mM to about 45 mM, about 8 mM to about 40 mM, about 8 mM to about 35 mM, about 8 mM to about 30 mM, about 8 mM to about 25 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 10 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 500 mM, about 15 mM to about 450 mM, about 15 mM to about 400 mM, about 15 mM to about 350 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, about 15 mM to about 180 mM, about 15 mM to about 160 mM, about 15 mM to about 140 mM, about 15 mM to about 120 mM, about 15 mM to about 100 mM, about 15 mM to about 80 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM, about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 500 mM, about 25 mM to about 450 mM, about 25 mM to about 400 mM, about 25 mM to about 350 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 80 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 500 mM, about 30 mM to about 450 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 80 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 500 mM, about 35 mM to about 450 mM, about 35 mM to about 400 mM, about 35 mM to about 350 mM, about 35 mM to about 300 mM, about 35 mM to about 250 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 80 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 500 mM, about 40 mM to about 450 mM, about 40 mM to about 400 mM, about 40 mM to about 350 mM, about 40 mM to about 300 mM, about 40 mM to about 250 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 500 mM, about 45 mM to about 450 mM, about 45 mM to about 400 mM, about 45 mM to about 350 mM, about 45 mM to about 300 mM, about 45 mM to about 250 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 80 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 60 mM to about 500 mM, about 60 mM to about 450 mM, about 60 mM to about 400 mM, about 60 mM to about 350 mM, about 60 mM to about 300 mM, about 60 mM to about 250 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 500 mM, about 140 mM to about 450 mM, about 140 mM to about 400 mM, about 140 mM to about 350 mM, about 140 mM to about 300 mM, about 140 mM to about 250 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 500 mM, about 160 mM to about 450 mM, about 160 mM to about 400 mM, about 160 mM to about 350 mM, about 160 mM to about 300 mM, about 160 mM to about 250 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 500 mM, about 180 mM to about 450 mM, about 180 mM to about 400 mM, about 180 mM to about 350 mM, about 180 mM to about 300 mM, about 180 mM to about 250 mM, about 180 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM, about 400 mM to about 450 mM, or about 450 mM to about 500 mM).
[00137] Tonicity modifiers are well known in the art and are manufactured by known methods and available from commercial suppliers.
II. Stabilizers
[00138] Stabilizers are a class of excipients that may stabilize a composition, and the active pharmaceutical ingredient, in various way including providing physical stability, conformational stability, and chemical stability. Examples of types of excipients that are used as a stabilizer include sugars, polymers, polyols, amino acids, a block copolymer, an alkyl saccharide (e.g., a ProTek alkyl saccharide, octyl glucoside, N-decyl- pyranoside, N-octyl-glucopyranoside, Intravail®, and those described in U.S. Patent Nos. 9,446,134; 8,846,044, 8,226,949; 7,998,927; 8,084,022; 7,425,542; and 10,046,025), and FM1000. In some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is human serum albumin or Recombumin®. In some embodiments, the stabilizer is betaine.
[00139] Specific examples of sugars include sucrose, dextrose, maltose, lactose, raffinose, and trehalose. Examples of polyols include sorbitol, maltitol, xylitol, and mannitol. Examples of amino acids include glycine, arginine, glutamate, and proline (either as the free base form or as a salt form). The invention further includes specific combinations of stabilizers that have particularly desirable properties. Examples include combinations of amino acids, e.g., Arg and Glu, as well as combinations of one or more amino acids with one or more polyols (e.g., Gly together with sorbitol or mannitol).
[00140] Improvements in formulation stability, due to such stabilizers, may be observed over a period of hours, days, months, or may last for extended periods, e.g., 6 months, 9 months, a year, or up to two years or more. Stabilizers may improve stability of a formulation overfree-thaw cycles. a. Aflibercept Formulations Stabilized with Sugars
[00141] In one embodiment, the invention provides a stable aqueous formulation comprising aflibercept and a sugar. Optionally, the formulation may further comprise a polyol and/or an amino acid. Examples of sugars include sucrose, lactose, maltose, trehalose, raffinose, and glucose. The composition may be a buffer-free and/or free of an organic co-solvent.
[00142] Inclusion of a sugar in the formulation improves the physical stability and other properties of specific aflibercept formulations (e.g., reduce tendency of aflibercept to associate in an undesirable conformation, and therefore reduce aggregation in aflibercept formulations). Thus, a sugar may stabilize aqueous pharmaceutical compositions containing aflibercept.
[00143] In one embodiment, the concentration of a sugar (e.g., sucrose) in the provided formulations is between about 0.01% (w/v) to 40% (e.g., about 0.01 % to about 35% w/v, about 0.01 % to about 30% w/v, about 0.01 % to about 25% w/v, about 0.01 % to about 20% w/v, about 0.01 % to about 18% w/v, about 0.01% to about 16% w/v, about 0.01% to about 14% w/v, about 0.01 % to about 12% w/v, about 0.01 % to about 10% w/v, about 0.01 % to about 9% w/v, about 0.01 % to about 8% w/v, about 0.01 % to about 7% w/v, about 0.01 % to about 6% w/v, about 0.01 % to about 5% w/v, about 0.01 % to about 4% w/v, about 0.01 % to about 3% w/v, about 0.01 % to about 2% w/v, about 0.01 % to about 1 % w/v, about 0.01 % to about 0.5% w/v, about 0.01 % to about 0.1 % w/v, about 0.1 % to about 40% w/v, about 0.1 % to about 35% w/v, about 0.1 % to about 30% w/v, about 0.1 % to about 25% w/v, about 0.1 % to about 20% w/v, about 0.1% to about 18% w/v, about 0.1% to about 16% w/v, about 0.1 % to about 14% w/v, about 0.1 % to about 12% w/v, about 0.1 % to about 10% w/v, about 0.1 % to about 9% w/v, about 0.1 % to about 8% w/v, about 0.1 % to about 7% w/v, about 0.1 % to about 6% w/v, about 0.1 % to about 5% w/v, about 0.1 % to about 4% w/v, about 0.1 % to about 3% w/v, about 0.1 % to about 2% w/v, about 0.1 % to about 1 % w/v, about 0.1 % to about 0.5% w/v, about 0.5% to about 40% w/v, about 0.5% to about 35% w/v, about 0.5% to about 30% w/v, about 0.5% to about 25% w/v, about 0.5% to about 20% w/v, about 0.5% to about 18% w/v, about 0.5% to about 16% w/v, about 0.5% to about 14% w/v, about 0.5% to about 12% w/v, about 0.5% to about 10% w/v, about 0.5% to about 9% w/v, about 0.5% to about 8% w/v, about 0.5% to about 7% w/v, about 0.5% to about 6% w/v, about 0.5% to about 5% w/v, about 0.5% to about 4% w/v, about 0.5% to about 3% w/v, about 0.5% to about 2% w/v, about 0.5% to about 1 % w/v, about 1 % to about 40% w/v, about 1 % to about 35% w/v, about 1 % to about 30% w/v, about 1 % to about 25% w/v, about 1 % to about 20% w/v, about 1 % to about 18% w/v, about 1% to about 16% w/v, about 1% to about 14% w/v, about 1% to about 12% w/v, about 1 % to about 10% w/v, about 1 % to about 9% w/v, about 1 % to about 8% w/v, about 1 % to about 7% w/v, about 1 % to about 6% w/v, about 1 % to about 5% w/v, about 1 % to about 4% w/v, about 1 % to about 3% w/v, about 1 % to about 2% w/v, about 2% to about 40% w/v, about 2% to about 35% w/v, about 2% to about 30% w/v, about 2% to about 25% w/v, about 2% to about 20% w/v, about 2% to about 18% w/v, about 2% to about 16% w/v, about 2% to about 14% w/v, about 2% to about 12% w/v, about 2% to about 10% w/v, about 2% to about 9% w/v, about 2% to about 8% w/v, about 2% to about 7% w/v, about 2% to about 6% w/v, about 2% to about 5% w/v, about 2% to about 4% w/v, about 2% to about 3% w/v, about 3% to about 40% w/v, about 3% to about 35% w/v, about 3% to about 30% w/v, about 3% to about 25% w/v, about 3% to about 20% w/v, about 3% to about 18% w/v, about 3% to about 16% w/v, about 3% to about 14% w/v, about 3% to about 12% w/v, about 3% to about 10% w/v, about 3% to about 9% w/v, about 3% to about 8% w/v, about 3% to about 7% w/v, about 3% to about 6% w/v, about 3% to about 5% w/v, about 3% to about 4% w/v, about 4% to about 40% w/v, about 4% to about 35% w/v, about 4% to about 30% w/v, about 4% to about 25% w/v, about 4% to about 20% w/v, about 4% to about 18% w/v, about 4% to about 16% w/v, about 4% to about 14% w/v, about 4% to about 12% w/v, about 4% to about 10% w/v, about 4% to about 9% w/v, about 4% to about 8% w/v, about 4% to about 7% w/v, about 4% to about 6% w/v, about 4% to about 5% w/v, about 5% to about 40% w/v, about 5% to about 35% w/v, about 5% to about 30% w/v, about 5% to about 25% w/v, about 5% to about 20% w/v, about 5% to about 18% w/v, about 5% to about 16% w/v, about 5% to about 14% w/v, about 5% to about 12% w/v, about 5% to about 10% w/v, about 5% to about 9% w/v, about 5% to about 8% w/v, about 5% to about 7% w/v, about 5% to about 6% w/v, about 6% to about 40% w/v, about 6% to about 35% w/v, about 6% to about 30% w/v, about 6% to about 25% w/v, about 6% to about 20% w/v, about 6% to about 18% w/v, about 6% to about 16% w/v, about 6% to about 14% w/v, about 6% to about 12% w/v, about 6% to about 10% w/v, about 6% to about 9% w/v, about 6% to about 8% w/v, about 6% to about 7% w/v, about 7% to about 40% w/v, about 7% to about 35% w/v, about 7% to about 30% w/v, about 7% to about 25% w/v, about 7% to about 20% w/v, about 7% to about 18% w/v, about 7% to about 16% w/v, about 7% to about 14% w/v, about 7% to about 12% w/v, about 7% to about 10% w/v, about 7% to about 9% w/v, about 7% to about 8% w/v, about 8% to about 40% w/v, about 8% to about 35% w/v, about 8% to about 30% w/v, about 8% to about 25% w/v, about 8% to about 20% w/v, about 8% to about 18% w/v, about 8% to about 16% w/v, about 8% to about 14% w/v, about 8% to about 12% w/v, about 8% to about 10% w/v, about 8% to about 9% w/v, about 9% to about 40% w/v, about 9% to about 35% w/v, about 9% to about 30% w/v, about 9% to about 25% w/v, about 9% to about 20% w/v, about 9% to about 18% w/v, about 9% to about 16% w/v, about 9% to about 14% w/v, about 9% to about 12% w/v, about 9% to about 10% w/v, about 10% to about 40% w/v, about 10% to about 35% w/v, about 10% to about 30% w/v, about 10% to about 25% w/v, about 10% to about 20% w/v, about 10% to about 18% w/v, about 10% to about 16% w/v, about 10% to about 14% w/v, about 10% to about 12% w/v, about 12% to about 40% w/v, about 12% to about 35% w/v, about 12% to about 30% w/v, about 12% to about 25% w/v, about 12% to about 20% w/v, about 12% to about 18% w/v, about 12% to about 16% w/v, about 12% to about 14% w/v, about 14% to about 40% w/v, about 14% to about 35% w/v, about 14% to about 30% w/v, about 14% to about 25% w/v, about 14% to about 20% w/v, about 14% to about 18% w/v, about 14% to about 16% w/v, about 16% to about 40% w/v, about 16% to about 35% w/v, about 16% to about 30% w/v, about 16% to about 25% w/v, about 16% to about 20% w/v, about 16% to about 18% w/v, about 18% to about 40% w/v, about 18% to about 35% w/v, about 18% to about 30% w/v, about 18% to about 25% w/v, about 18% to about 20% w/v, about 20% to about 40% w/v, about 20% to about 35% w/v, about 20% to about 30% w/v, about 20% to about 25% w/v, about 25% to about 40% w/v, about 25% to about 35% w/v, about 25% to about 30% w/v, about 30% to about 40% w/v, about 30% to about 35% w/v, or about 35% to about 40% w/v), for example about 1 % to about 20%, about 2 % to about 10%, or about 5% to 9%.
[00144] In one embodiment, the concentration of the sugar in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 500 mM, about 2 mM to about 450 mM, about 2 mM to about 400 mM, about 2 mM to about 350 mM, about 2 mM to about 300 mM, about 2 mM to about 250 mM, about 2 mM to about 200 mM, about 2 mM to about 180 mM, about 2 mM to about 160 mM, about 2 mM to about 140 mM, about 2 mM to about 120 mM, about 2 mM to about 100 mM, about 2 mM to about 80 mM, about 2 mM to about 60 mM, about 2 mM to about 50 mM, about 2 mM to about 45 mM, about 2 mM to about 40 mM, about 2 mM to about 35 mM, about 2 mM to about 30 mM, about 2 mM to about 25 mM, about 2 mM to about 20 mM, about 2 mM to about 15 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 500 mM, about 4 mM to about 450 mM, about 4 mM to about 400 mM, about 4 mM to about 350 mM, about 4 mM to about 300 mM, about 4 mM to about 250 mM, about 4 mM to about 200 mM, about 4 mM to about 180 mM, about 4 mM to about 160 mM, about 4 mM to about 140 mM, about 4 mM to about 120 mM, about 4 mM to about 100 mM, about 4 mM to about 80 mM, about 4 mM to about 60 mM, about 4 mM to about 50 mM, about 4 mM to about 45 mM, about 4 mM to about 40 mM, about 4 mM to about 35 mM, about 4 mM to about 30 mM, about 4 mM to about 25 mM, about 4 mM to about 20 mM, about 4 mM to about 15 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 500 mM, about 6 mM to about 450 mM, about 6 mM to about 400 mM, about 6 mM to about 350 mM, about 6 mM to about 300 mM, about 6 mM to about 250 mM, about 6 mM to about 200 mM, about 6 mM to about 180 mM, about 6 mM to about 160 mM, about 6 mM to about 140 mM, about 6 mM to about 120 mM, about 6 mM to about 100 mM, about 6 mM to about 80 mM, about 6 mM to about 60 mM, about 6 mM to about 50 mM, about 6 mM to about 45 mM, about 6 mM to about 40 mM, about 6 mM to about 35 mM, about 6 mM to about 30 mM, about 6 mM to about 25 mM, about 6 mM to about 20 mM, about 6 mM to about 15 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 500 mM, about 8 mM to about 450 mM, about 8 mM to about 400 mM, about 8 mM to about 350 mM, about 8 mM to about 300 mM, about 8 mM to about 250 mM, about 8 mM to about 200 mM, about 8 mM to about 180 mM, about 8 mM to about 160 mM, about 8 mM to about 140 mM, about 8 mM to about 120 mM, about 8 mM to about 100 mM, about 8 mM to about 80 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 8 mM to about 45 mM, about 8 mM to about 40 mM, about 8 mM to about 35 mM, about 8 mM to about 30 mM, about 8 mM to about 25 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 10 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 500 mM, about 15 mM to about 450 mM, about 15 mM to about 400 mM, about 15 mM to about 350 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, about 15 mM to about 180 mM, about 15 mM to about 160 mM, about 15 mM to about 140 mM, about 15 mM to about 120 mM, about 15 mM to about 100 mM, about 15 mM to about 80 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM, about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 500 mM, about 25 mM to about 450 mM, about 25 mM to about 400 mM, about 25 mM to about 350 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 80 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 500 mM, about 30 mM to about 450 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 80 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 500 mM, about 35 mM to about 450 mM, about 35 mM to about 400 mM, about 35 mM to about 350 mM, about 35 mM to about 300 mM, about 35 mM to about 250 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 80 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 500 mM, about 40 mM to about 450 mM, about 40 mM to about 400 mM, about 40 mM to about 350 mM, about 40 mM to about 300 mM, about 40 mM to about 250 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 500 mM, about 45 mM to about 450 mM, about 45 mM to about 400 mM, about 45 mM to about 350 mM, about 45 mM to about 300 mM, about 45 mM to about 250 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 80 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 60 mM to about 500 mM, about 60 mM to about 450 mM, about 60 mM to about 400 mM, about 60 mM to about 350 mM, about 60 mM to about 300 mM, about 60 mM to about 250 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 500 mM, about 140 mM to about 450 mM, about 140 mM to about 400 mM, about 140 mM to about 350 mM, about 140 mM to about 300 mM, about 140 mM to about 250 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 500 mM, about 160 mM to about 450 mM, about 160 mM to about 400 mM, about 160 mM to about 350 mM, about 160 mM to about 300 mM, about 160 mM to about 250 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 500 mM, about 180 mM to about 450 mM, about 180 mM to about 400 mM, about 180 mM to about 350 mM, about 180 mM to about 300 mM, about 180 mM to about 250 mM, about 180 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM, about 400 mM to about 450 mM, or about 450 mM to about 500 mM).
[00145] In one embodiment, a formulation of the invention comprises about 10 to about 200 mg/mL (e.g., any of the subranges of this range described herein) of aflibercept; about 10 mM to about 350 mM (e.g., any of the subranges of this range described herein) sucrose; about 0.01 mM to about 100 mM (e.g., any of the subranges of this range described herein) sodium chloride; about 0.0001 % to about 0.1% (e.g., about 0.0001 % to about 0.05%, about 0.0001 % to about 0.01 %, about 0.0001 % to about 0.0005%, about 0.0005% to about 0.1 %, about 0.0005% to about 0.05%, about 0.0005% to about 0.01%, about 0.01% to about 0.1 %, about 0.01 % to about 0.05%, or about 0.05% to about 0.1%) surfactant; and about 0.1 to about 5% (e.g., about 0.1 % to about 4.5%, about 0.1% to about 4.0%, about 0.1% to about 3.5%, about 0.1% to about 3.0%, about 0.1 % to about 2.5%, about 0.1 % to about 2.0%, about 0.1% to about 1 .5%, about 0.1% to about 1 .0%, about 0.1% to about 0.5%, about 0.5% to about 5.0%, about 0.5% to about 4.5%, about 0.5% to about 4.0%, about 0.5% to about 3.5%, about 0.5% to about 3.0%, about 0.5% to about 2.5%, about 0.5% to about 2.0%, about 0.5% to about 1.5%, about 0.5% to about 1 .0%, about 1.0% to about 5.0%, about 1.0% to about 4.5%, about 1.0% to about 4.0%, about 1.0% to about 3.5%, about 1.0% to about 3.0%, about 1.0% to about 2.5%, about 1 .0% to about 2.0%, about 1.0% to about 1.5%, about 1 .5% to about 5.0%, about 1.5% to about 4.5%, about 1.5% to about 4.0%, about 1.5% to about 3.5%, about 1.5% to about 3.0%, about 1.5% to about 2.5%, about 1.5% to about 2.0%, about 2.0% to about 5.0%, about 2.0% to about 4.5%, about 2.0% to about 4.0%, about 2.0% to about 3.5%, about 2.0% to about 3.0%, about 2.0% to about 2.5%, about 2.5% to about 5.0%, about 2.5% to about 4.5%, about 2.5% to about 4.0%, about 2.5% to about 3.5%, about 2.5% to about 3.0%, about 3.0% to about 5.0%, about 3.0% to about 4.5%, about 3.0% to about 4.0%, about 3.0% to about 3.5%, about 3.5% to about 5.0%, about 3.5% to about 4.5%, about 3.5% to about 4.0%, about 4.0% to about 5.0%, about 4.0% to about 4.5%, or about 4.0% to about 5.0%) polyethylene glycol, at about pH 5 to about pH 7 (e.g., any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
[00146] In another embodiment, sucrose can be replaced with another sugar such as trehalose (at about 10 mM to about 350 mM, or any of the subranges of this range described herein) in the formulation. In yet another embodiment, sodium chloride can be replaced with another salt such as magnesium chloride (at about 0.01 mM to about 100 mM, e.g., any of the subranges of this range described herein or about 0.01 mM, about 0.1 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM)) in the formulation.
[00147] In another embodiment, a formulation of the invention comprises about 10 to about 200 mg/mL (e.g., any of the subranges of this range described herein) of aflibercept; about 10 mM to about 350 mM (e.g., any of the subranges of this range described herein) sucrose; about 0.01 mM to about 100 mM (e.g., any of the subranges of this range described herein) sodium chloride; about 0.01 mM to about 10 mM buffer (e.g., about 0.01 mM to about 8 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.1 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 10 mM, about 3 mM to about 8 mM, about 3 mM to about 6 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 4 mM to about 5 mM, about 5 mM to about 10 mM, about 5 mM to about 8 mM, about 5 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM); at about pH 5 to about pH 7 (e.g., any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
[00148] The formulations of the invention may also include buffering agents, tonicity modifiers, excipients, and other commonly used inactive ingredients of the pharmaceutical compositions. b. Aflibercept Formulations Stabilized with Polyol
[00149] In one embodiment, the invention provides a stable aqueous formulation comprising aflibercept and a polyol. Optionally, the formulation may further comprise a sugar and/or an amino acid. Examples of polyols include glycerol, sorbitol, mannitol, xylitol, and maltitol. The composition may be a buffer-free and/or free of an organic co-solvent.
[00150] In one embodiment, the concentration of a polyol in the provided formulations is between about 0.1% to 30% (e.g., about 0.1% to about 28%, about 0.1% to about 26%, about 0.1% to about 24%, about 0.1 % to about 22%, about 0.1% to about 20%, about 0.1% to about 18%, about 0.1 % to about 16%, about 0.1% to about 14%, about 0.1% to about 12%, about 0.1% to about 10%, about 0.1% to about 8%, about 0.1% to about 6%, about 0.1% to about 4%, about 0.1% to about 2%, about 0.1% to about 1 %, about 0.1% to about 0.5%, about 0.5% to about 30%, about 0.5% to about 28%, about 0.5% to about 26%, about 0.5% to about 24%, about 0.5% to about 22%, about 0.5% to about 20%, about 0.5% to about 18%, about 0.5% to about 16%, about 0.5% to about 14%, about 0.5% to about 12%, about 0.5% to about 10%, about 0.5% to about 8%, about 0.5% to about 6%, about 0.5% to about 4%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 30%, about 1 % to about 28%, about 1 % to about 26%, about 1 % to about 24%, about 1 % to about 22%, about 1 % to about 20%, about 1 % to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1 % to about 8%, about 1% to about 6%, about 1 % to about 4%, about 1 % to about 2%, about 2% to about 30%, about 2% to about 28%, about 2% to about 26%, about 2% to about 24%, about 2% to about 22%, about 2% to about 20%, about 2% to about 18%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 2% to about 4%, about 4% to about 30%, about 4% to about 28%, about 4% to about 26%, about 4% to about 24%, about 4% to about 22%, about 4% to about 20%, about 4% to about 18%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 4% to about 6%, about 6% to about 30%, about 6% to about 28%, about 6% to about 26%, about 6% to about 24%, about 6% to about 22%, about 6% to about 20%, about 6% to about 18%, about 6% to about 16%, about 6% to about 14%, about 6% to about 12%, about 6% to about 10%, about 6% to about 8%, about 8% to about 30%, about 8% to about 28%, about 8% to about 26%, about 8% to about 24%, about 8% to about 22%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8% to about 14%, about 8% to about 12%, about 8% to about 10%, about 10% to about 30%, about 10% to about 28%, about 10% to about 26%, about 10% to about 24%, about 10% to about 22%, about 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 30%, about 12% to about 28%, about 12% to about 26%, about 12% to about 24%, about 12% to about 22%, about 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 30%, about 14% to about 28%, about 14% to about 26%, about 14% to about 24%, about 14% to about 22%, about 14% to about 20%, about 14% to about 18%, about 14% to about 16%, about 16% to about 30%, about 16% to about 28%, about 16% to about 26%, about 16% to about 24%, about 16% to about 22%, about 16% to about 20%, about 16% to about 18%, about 18% to about 30%, about 18% to about 28%, about 18% to about 26%, about 18% to about 24%, about 18% to about 22%, about 18% to about 20%, about 20% to about 30%, about 20% to about 28%, about 20% to about 26%, about 20% to about 24%, about 20% to about 22%, about 22% to about 30%, about 22% to about 28%, about 22% to about 26%, about 22% to about 24%, about 24% to about 30%, about 24% to about 28%, about 24% to about 26%, about 26% to about 30%, about 26% to about 28%, or about 28% to about 30%), for example about 1 % to about 10%, or about 2% to about 5%.
[00151] In one embodiment, the concentration of the polyol in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 500 mM, about 2 mM to about 450 mM, about 2 mM to about 400 mM, about 2 mM to about 350 mM, about 2 mM to about 300 mM, about 2 mM to about 250 mM, about 2 mM to about 200 mM, about 2 mM to about 180 mM, about 2 mM to about 160 mM, about 2 mM to about 140 mM, about 2 mM to about 120 mM, about 2 mM to about 100 mM, about 2 mM to about 80 mM, about 2 mM to about 60 mM, about 2 mM to about 50 mM, about 2 mM to about 45 mM, about 2 mM to about 40 mM, about 2 mM to about 35 mM, about 2 mM to about 30 mM, about 2 mM to about 25 mM, about 2 mM to about 20 mM, about 2 mM to about 15 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 500 mM, about 4 mM to about 450 mM, about 4 mM to about 400 mM, about 4 mM to about 350 mM, about 4 mM to about 300 mM, about 4 mM to about 250 mM, about 4 mM to about 200 mM, about 4 mM to about 180 mM, about 4 mM to about 160 mM, about 4 mM to about 140 mM, about 4 mM to about 120 mM, about 4 mM to about 100 mM, about 4 mM to about 80 mM, about 4 mM to about 60 mM, about 4 mM to about 50 mM, about 4 mM to about 45 mM, about 4 mM to about 40 mM, about 4 mM to about 35 mM, about 4 mM to about 30 mM, about 4 mM to about 25 mM, about 4 mM to about 20 mM, about 4 mM to about 15 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 500 mM, about 6 mM to about 450 mM, about 6 mM to about 400 mM, about 6 mM to about 350 mM, about 6 mM to about 300 mM, about 6 mM to about 250 mM, about 6 mM to about 200 mM, about 6 mM to about 180 mM, about 6 mM to about 160 mM, about 6 mM to about 140 mM, about 6 mM to about 120 mM, about 6 mM to about 100 mM, about 6 mM to about 80 mM, about 6 mM to about 60 mM, about 6 mM to about 50 mM, about 6 mM to about 45 mM, about 6 mM to about 40 mM, about 6 mM to about 35 mM, about 6 mM to about 30 mM, about 6 mM to about 25 mM, about 6 mM to about 20 mM, about 6 mM to about 15 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 500 mM, about 8 mM to about 450 mM, about 8 mM to about 400 mM, about 8 mM to about 350 mM, about 8 mM to about 300 mM, about 8 mM to about 250 mM, about 8 mM to about 200 mM, about 8 mM to about 180 mM, about 8 mM to about 160 mM, about 8 mM to about 140 mM, about 8 mM to about 120 mM, about 8 mM to about 100 mM, about 8 mM to about 80 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 8 mM to about 45 mM, about 8 mM to about 40 mM, about 8 mM to about 35 mM, about 8 mM to about 30 mM, about 8 mM to about 25 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 10 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 500 mM, about 15 mM to about 450 mM, about 15 mM to about 400 mM, about 15 mM to about 350 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, about 15 mM to about 180 mM, about 15 mM to about 160 mM, about 15 mM to about 140 mM, about 15 mM to about 120 mM, about 15 mM to about 100 mM, about 15 mM to about 80 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM, about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 500 mM, about 25 mM to about 450 mM, about 25 mM to about 400 mM, about 25 mM to about 350 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 80 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 500 mM, about 30 mM to about 450 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 80 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 500 mM, about 35 mM to about 450 mM, about 35 mM to about 400 mM, about 35 mM to about 350 mM, about 35 mM to about 300 mM, about 35 mM to about 250 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 80 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 500 mM, about 40 mM to about 450 mM, about 40 mM to about 400 mM, about 40 mM to about 350 mM, about 40 mM to about 300 mM, about 40 mM to about 250 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 500 mM, about 45 mM to about 450 mM, about 45 mM to about 400 mM, about 45 mM to about 350 mM, about 45 mM to about 300 mM, about 45 mM to about 250 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 80 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 60 mM to about 500 mM, about 60 mM to about 450 mM, about 60 mM to about 400 mM, about 60 mM to about 350 mM, about 60 mM to about 300 mM, about 60 mM to about 250 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 500 mM, about 140 mM to about 450 mM, about 140 mM to about 400 mM, about 140 mM to about 350 mM, about 140 mM to about 300 mM, about 140 mM to about 250 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 500 mM, about 160 mM to about 450 mM, about 160 mM to about 400 mM, about 160 mM to about 350 mM, about 160 mM to about 300 mM, about 160 mM to about 250 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 500 mM, about 180 mM to about 450 mM, about 180 mM to about 400 mM, about 180 mM to about 350 mM, about 180 mM to about 300 mM, about 180 mM to about 250 mM, about 180 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM, about 400 mM to about 450 mM, or about 450 mM to about 500 mM). c. Aflibercept Stabilized with Amino Acids [00152] Amino acids, e.g., proline, serine, sodium glutamic acid, glutamate, alanine, histidine, tryptophan, tyrosine, arginine, glycine, lysine, methionine, threonine, glutamic acid, aspartic acid, sarcosine, glycine betaine, and mixtures of the foregoing may be employed as stabilizers in certain aflibercept formulations.
[00153] These compositions may use the free base form of the amino acid or any conjugate acid form such as a hydrochloride salt. Such amino acids are readily available from commercial suppliers. For example, in one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept and one or more amino acids, wherein the amino acid(s) is selected from the group consisting of serine, proline, glycine, alanine, glutamate, arginine and combinations thereof. The composition may further optionally include a sugar and/or polyol. The composition may be a buffer-free and/or free of an organic co-solvent.
[00154] Such additives may improve the properties of the aflibercept formulations, e.g., by reducing aggregation or tendency for aflibercept to associate in undesired ternary or quaternary complexes. Without wishing to be bound to a particular theory, it is believed that amino acids such as serine, proline and glutamate are able to stabilize aqueous pharmaceutical compositions containing aflibercept because they are excluded from the surface of the protein, resulting in net conformation stabilization. [00155] In one embodiment, the concentration of the amino acids in the provided formulations is between about 1 mM and about 500 mM (e.g., about 1 mM to about 450 mM, about 1 mM to about 400 mM, about 1 mM to about 350 mM, about 1 mM to about 300 mM, about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 180 mM, about 1 mM to about 160 mM, about 1 mM to about 140 mM, about 1 mM to about 120 mM, about 1 mM to about 100 mM, about 1 mM to about 80 mM, about 1 mM to about 60 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 35 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about
1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 500 mM, about 2 mM to about 450 mM, about 2 mM to about 400 mM, about 2 mM to about 350 mM, about 2 mM to about 300 mM, about 2 mM to about 250 mM, about 2 mM to about 200 mM, about 2 mM to about 180 mM, about 2 mM to about 160 mM, about 2 mM to about 140 mM, about 2 mM to about 120 mM, about 2 mM to about 100 mM, about 2 mM to about 80 mM, about 2 mM to about 60 mM, about 2 mM to about 50 mM, about 2 mM to about 45 mM, about 2 mM to about 40 mM, about 2 mM to about 35 mM, about 2 mM to about 30 mM, about 2 mM to about 25 mM, about 2 mM to about 20 mM, about 2 mM to about 15 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about
2 mM to about 4 mM, about 4 mM to about 500 mM, about 4 mM to about 450 mM, about 4 mM to about 400 mM, about 4 mM to about 350 mM, about 4 mM to about 300 mM, about 4 mM to about 250 mM, about 4 mM to about 200 mM, about 4 mM to about 180 mM, about 4 mM to about 160 mM, about 4 mM to about 140 mM, about 4 mM to about 120 mM, about 4 mM to about
100 mM, about 4 mM to about 80 mM, about 4 mM to about 60 mM, about 4 mM to about 50 mM, about 4 mM to about 45 mM, about 4 mM to about 40 mM, about 4 mM to about 35 mM, about 4 mM to about 30 mM, about 4 mM to about 25 mM, about 4 mM to about 20 mM, about 4 mM to about 15 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 500 mM, about 6 mM to about 450 mM, about 6 mM to about 400 mM, about 6 mM to about 350 mM, about 6 mM to about 300 mM, about 6 mM to about 250 mM, about 6 mM to about 200 mM, about 6 mM to about 180 mM, about 6 mM to about 160 mM, about 6 mM to about 140 mM, about 6 mM to about 120 mM, about 6 mM to about 100 mM, about 6 mM to about 80 mM, about 6 mM to about 60 mM, about 6 mM to about 50 mM, about 6 mM to about 45 mM, about 6 mM to about 40 mM, about 6 mM to about 35 mM, about 6 mM to about 30 mM, about 6 mM to about 25 mM, about 6 mM to about 20 mM, about 6 mM to about 15 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, about 8 mM to about 500 mM, about 8 mM to about 450 mM, about 8 mM to about 400 mM, about 8 mM to about 350 mM, about 8 mM to about 300 mM, about 8 mM to about 250 mM, about 8 mM to about 200 mM, about 8 mM to about 180 mM, about 8 mM to about 160 mM, about 8 mM to about 140 mM, about 8 mM to about 120 mM, about 8 mM to about 100 mM, about 8 mM to about 80 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 8 mM to about 45 mM, about 8 mM to about 40 mM, about 8 mM to about 35 mM, about 8 mM to about 30 mM, about 8 mM to about 25 mM, about 8 mM to about 20 mM, about 8 mM to about 15 mM, about 8 mM to about 10 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 180 mM, about 10 mM to about 160 mM, about 10 mM to about 140 mM, about 10 mM to about 120 mM, about 10 mM to about 100 mM, about 10 mM to about 80 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 500 mM, about 15 mM to about 450 mM, about 15 mM to about 400 mM, about 15 mM to about 350 mM, about 15 mM to about 300 mM, about 15 mM to about 250 mM, about 15 mM to about 200 mM, about 15 mM to about 180 mM, about 15 mM to about 160 mM, about 15 mM to about 140 mM, about 15 mM to about 120 mM, about 15 mM to about 100 mM, about 15 mM to about 80 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 500 mM, about 20 mM to about 450 mM, about 20 mM to about 400 mM, about 20 mM to about 350 mM, about 20 mM to about 300 mM, about 20 mM to about 250 mM, about 20 mM to about 200 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 140 mM, about 20 mM to about 120 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 500 mM, about 25 mM to about 450 mM, about 25 mM to about 400 mM, about 25 mM to about 350 mM, about 25 mM to about 300 mM, about 25 mM to about 250 mM, about 25 mM to about 200 mM, about 25 mM to about 180 mM, about 25 mM to about 160 mM, about 25 mM to about 140 mM, about 25 mM to about 120 mM, about 25 mM to about 100 mM, about 25 mM to about 80 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 500 mM, about 30 mM to about 450 mM, about 30 mM to about 400 mM, about 30 mM to about 350 mM, about 30 mM to about 300 mM, about 30 mM to about 250 mM, about 30 mM to about 200 mM, about 30 mM to about 180 mM, about 30 mM to about 160 mM, about 30 mM to about 140 mM, about 30 mM to about 120 mM, about 30 mM to about 100 mM, about 30 mM to about 80 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 500 mM, about 35 mM to about 450 mM, about 35 mM to about 400 mM, about 35 mM to about 350 mM, about 35 mM to about 300 mM, about 35 mM to about 250 mM, about 35 mM to about 200 mM, about 35 mM to about 180 mM, about 35 mM to about 160 mM, about 35 mM to about 140 mM, about 35 mM to about 120 mM, about 35 mM to about 100 mM, about 35 mM to about 80 mM, about 35 mM to about 60 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 500 mM, about 40 mM to about 450 mM, about 40 mM to about 400 mM, about 40 mM to about 350 mM, about 40 mM to about 300 mM, about 40 mM to about 250 mM, about 40 mM to about 200 mM, about 40 mM to about 180 mM, about 40 mM to about 160 mM, about 40 mM to about 140 mM, about 40 mM to about 120 mM, about 40 mM to about 100 mM, about 40 mM to about 80 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 500 mM, about 45 mM to about 450 mM, about 45 mM to about 400 mM, about 45 mM to about 350 mM, about 45 mM to about 300 mM, about 45 mM to about 250 mM, about 45 mM to about 200 mM, about 45 mM to about 180 mM, about 45 mM to about 160 mM, about 45 mM to about 140 mM, about 45 mM to about 120 mM, about 45 mM to about 100 mM, about 45 mM to about 80 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, about 50 mM to about 200 mM, about 50 mM to about 180 mM, about 50 mM to about 160 mM, about 50 mM to about 140 mM, about 50 mM to about 120 mM, about 50 mM to about 100 mM, about 50 mM to about 80 mM, about 50 mM to about 60 mM, about 60 mM to about 500 mM, about 60 mM to about 450 mM, about 60 mM to about 400 mM, about 60 mM to about 350 mM, about 60 mM to about 300 mM, about 60 mM to about 250 mM, about 60 mM to about 200 mM, about 60 mM to about 180 mM, about 60 mM to about 160 mM, about 60 mM to about 140 mM, about 60 mM to about 120 mM, about 60 mM to about 100 mM, about 60 mM to about 80 mM, about 80 mM to about 500 mM, about 80 mM to about 450 mM, about 80 mM to about 400 mM, about 80 mM to about 350 mM, about 80 mM to about 300 mM, about 80 mM to about 250 mM, about 80 mM to about 200 mM, about 80 mM to about 180 mM, about 80 mM to about 160 mM, about 80 mM to about 140 mM, about 80 mM to about 120 mM, about 80 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, about 100 mM to about 350 mM, about 100 mM to about 300 mM, about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 100 mM to about 180 mM, about 100 mM to about 160 mM, about 100 mM to about 140 mM, about 100 mM to about 120 mM, about 120 mM to about 500 mM, about 120 mM to about 450 mM, about 120 mM to about 400 mM, about 120 mM to about 350 mM, about 120 mM to about 300 mM, about 120 mM to about 250 mM, about 120 mM to about 200 mM, about 120 mM to about 180 mM, about 120 mM to about 160 mM, about 120 mM to about 140 mM, about 140 mM to about 500 mM, about 140 mM to about 450 mM, about 140 mM to about 400 mM, about 140 mM to about 350 mM, about 140 mM to about 300 mM, about 140 mM to about 250 mM, about 140 mM to about 200 mM, about 140 mM to about 180 mM, about 140 mM to about 160 mM, about 160 mM to about 500 mM, about 160 mM to about 450 mM, about 160 mM to about 400 mM, about 160 mM to about 350 mM, about 160 mM to about 300 mM, about 160 mM to about 250 mM, about 160 mM to about 200 mM, about 160 mM to about 180 mM, about 180 mM to about 500 mM, about 180 mM to about 450 mM, about 180 mM to about 400 mM, about 180 mM to about 350 mM, about 180 mM to about 300 mM, about 180 mM to about 250 mM, about 180 mM to about 200 mM, about 200 mM to about 500 mM, about 200 mM to about 450 mM, about 200 mM to about 400 mM, about 200 mM to about 350 mM, about 200 mM to about 300 mM, about 200 mM to about 250 mM, about 250 mM to about 500 mM, about 250 mM to about 450 mM, about 250 mM to about 400 mM, about 250 mM to about 350 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, about 300 mM to about 450 mM, about 300 mM to about 400 mM, about 300 mM to about 350 mM, about 350 mM to about 500 mM, about 350 mM to about 450 mM, about 350 mM to about 400 mM, about 400 mM to about 500 mM, about 400 mM to about 450 mM, or about 450 mM to about 500 mM). In another embodiment, the concentration of the amino acid(s)is between about 10 mM and about 350 mM; in related embodiments, the concentration of the amino acid(s) is about 50 mM, 100 mM, 150 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, 300 mM, 320 mM and 340 mM, for example, 50-100 mM, 100-150 mM, 150-200 mM, 200- 300 mM, 200-250 mM, 250-300 mM, and 300-350 mM. Aflibercept Stabilized with Combinations of Stabilizers
[00156] In some embodiments, combinations of stabilizers may act in concert to stabiliz aflibercept. In one embodiment, an aflibercept formulation comprises a sugar and a polyol. In another example, amino acids, such as proline, serine, or glutamate maybe used together with a sugar to achieve a stability profile better than either excipient could provide on its own. In one embodiment, the ratio of a sugar to a polyol (or amino acid) in the formulation is between 5:1 and 1 :5 (e.g., about 5:1 , about 4:1 , about 3:1 , about 2:1 , about 1 :1 , about 1 :2, about 1 :3, about 1 :4, or about 1 :5). In another embodiment, a formulation with a combination of stabilizers has better stability than a formulation with just one of the stabilizers. In another embodiment, a formulation with a combination of stabilizers has better stability than a formulation with just one of the stabilizers where the formulation with the combination of stabilizers has a lower total amount of stabilizers than the formulation with the single stabilizer (e.g. a formulation with 100 mM of sucrose and 50 mM arginine has better stability than a formulation with 200 mM sucrose).
III. Salts
[00157] Salts, e.g., NaCI, are often part of protein (e.g., antibody) formulations. For instance, salts such as NaCI, Na2SC>4, CaCh, MgCh, and KCI, together with specific buffer combinations as described above, can confer particularly advantageous stability properties. Such salts may be used with as well as without a sugar added (e.g., sucrose and NaCI) or a polyol added (e.g., mannitol and NaCI). For example, concentrations as low as 20 to 50 mM can provide decreased levels of proteolysis (such as hinge region hydrolysis), oxidation, deamidation, or other chemical instabilities.
Furthermore, low levels of salt (< 50 mM) may lead to improved colloidal stability. Likewise, it is known that high concentrations of certain salts (up to 400 mM or more) can increase conformational stability. The specific effects of NaCI and other salts on aflibercept is dependent on pH, buffer composition, and stress condition. [00158] In specific embodiments, sodium chloride may be replaced with Na2SC>4, KCI, MgCh, CaCh, MgSC , ZnCh, or other physiologically- acceptable salts.
IV. Surfactants
[00159] Surfactants can confer protection against agitation and freeze/thaw damage, as well as stabilizing a formulation during storage. Surfactants which may be employed with aflibercept formulations of the present invention include Tween®-80 (polysorbate 80, PS 80), Tween®-20 (polysorbate 20, PS 20), SDS, polysorbate, polyoxyethylene copolymer, Brij 35, Triton X-10, poloxamer 188 (Pluronic F-68), Pluronic F127, and Maltosides, e.g., n-Decyl- -D-maltopyranoside (DM), n-Dodecyl- -D- maltopyranoside (DDM), and 6-Cyclohexyl-1-hexyl- -D-maltopyranoside (Cymal-6). In certain embodiments, surfactants that may be particularly advantageous include PS 20, PS 40, PS 60, and PS 80 at different concentrations, as well as DMM and poloxamer 188 (Pluronic F-68). Other zwitterionic or nonionic surfactants may be used as well.
[00160] In the embodiments of the invention where organic co-solvents are excluded, surfactants cannot be present.
[00161] As above, optimal formulation employing such surfactants may also employ specific buffers at specific pH ranges, and may optionally include other tonicity modifier such as NaCI, polyol, or amino acid(s).
V. Polymers
[00162] Polymers, such as dextrans, starches (e.g., hydroxyl ethyl starch (HETA)), polyethylene glycols (PEGs), e.g., PEG-3350 or PEG-4000, may also provide stabilization to aflibercept, presumably by being excluded from the surface of the protein due to steric effects arising from their higher molecular weight. In particular, hydrophilic polymers, such as polyethylene glycols (PEGs), polysaccharides, and inert proteins, and may be used to stabilize proteins and enhance protein assembly. Examples include dextran, hydroxyl ethyl starch (HETA), PEG-4000, and gelatin. Additionally, non-polar moieties on certain polymers such as PEGs and Pluronics can decrease water surface tension rendering them as surfactants that suppress surface adsorption induced aggregation. In one embodiment, the concentration of polymer is between 0.01% and 40% (e.g., about 0.01% to about 35%, about 0.01 % to about 30%, about 0.01 % to about 25%, about 0.01 % to about 20%, about 0.01% to about 18%, about 0.01% to about 16%, about 0.01% to about 14%, about 0.01% to about 12%, about 0.01% to about 10%, about 0.01% to about 8%, about 0.01 % to about 6%, about 0.01 % to about 5%, about 0.01 % to about 4.5%, about 0.01% to about 4.0%, about 0.01% to about 3.5%, about 0.01 % to about 3.0%, about 0.01 % to about 2.5%, about 0.01 % to about 2.0%, about 0.01 % to about 1.5%, about 0.01 % to about 1 .0%, about 0.01 % to about 0.5%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, about 0.05% to about 40%, about 0.05% to about 35%, about 0.05% to about 30%, about 0.05% to about 25%, about 0.05% to about 20%, about 0.05% to about 18%, about 0.05% to about 16%, about 0.05% to about 14%, about 0.05% to about 12%, about 0.05% to about 10%, about 0.05% to about 8%, about 0.05% to about 6%, about 0.05% to about 5%, about 0.05% to about 4.5%, about 0.05% to about 4.0%, about 0.05% to about 3.5%, about 0.05% to about 3.0%, about 0.05% to about 2.5%, about 0.05% to about 2.0%, about 0.05% to about 1 .5%, about 0.05% to about 1.0%, about 0.05% to about 0.5%, about 0.05% to about 0.1%, about 0.1% to about 40%, about 0.1 % to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1 % to about 20%, about 0.1% to about 18%, about 0.1% to about 16%, about 0.1 % to about 14%, about 0.1% to about 12%, about 0.1% to about 10%, about 0.1% to about 8%, about 0.1% to about 6%, about 0.1 % to about 5%, about 0.1% to about 4.5%, about 0.1% to about 4.0%, about 0.1% to about 3.5%, about 0.1 % to about 3.0%, about 0.1 % to about 2.5%, about 0.1% to about 2.0%, about 0.1% to about 1.5%, about 0.1 % to about 1.0%, about 0.1 % to about 0.5%, about 0.5% to about 40%, about 0.5% to about 35%, about 0.5% to about 30%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 18%, about 0.5% to about 16%, about 0.5% to about 14%, about 0.5% to about 12%, about 0.5% to about 10%, about 0.5% to about 8%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4.5%, about 0.5% to about 4.0%, about 0.5% to about 3.5%, about 0.5% to about 3.0%, about 0.5% to about 2.5%, about 0.5% to about 2.0%, about 0.5% to about 1.5%, about 0.5% to about 1 .0%, about 1.0% to about 40%, about 1.0% to about 35%, about 1.0% to about 30%, about 1.0% to about 25%, about 1 .0% to about 20%, about 1 .0% to about 18%, about 1.0% to about 16%, about 1 .0% to about 14%, about 1.0% to about 12%, about 1.0% to about 10%, about 1.0% to about 8%, about 1.0% to about 6%, about 1.0% to about 5%, about 1.0% to about 4.5%, about 1 .0% to about 4.0%, about 1.0% to about 3.5%, about 1.0% to about 3.0%, about 1.0% to about 2.5%, about 1.0% to about 2.0%, about 1.0% to about 1.5%, about 1 .5% to about 40%, about 1 .5% to about 35%, about 1 .5% to about 30%, about 1.5% to about 25%, about 1 .5% to about 20%, about 1.5% to about 18%, about 1.5% to about 16%, about 1 .5% to about 14%, about 1 .5% to about 12%, about 1 .5% to about 10%, about 1.5% to about 8%, about 1.5% to about 6%, about 1.5% to about 5%, about 1.5% to about 4.5%, about 1.5% to about 4.0%, about 1.5% to about 3.5%, about 1.5% to about 3.0%, about 1 .5% to about 2.5%, about 1.5% to about 2.0%, about 2.0% to about 40%, about 2.0% to about 35%, about 2.0% to about 30%, about 2.0% to about 25%, about 2.0% to about 20%, about 2.0% to about 18%, about 2.0% to about 16%, about 2.0% to about 14%, about 2.0% to about 12%, about 2.0% to about 10%, about 2.0% to about 8%, about 2.0% to about 6%, about 2.0% to about 5%, about 2.0% to about 4.5%, about 2.0% to about 4.0%, about 2.0% to about 3.5%, about 2.0% to about 3.0%, about 2.0% to about 2.5%, about 2.5% to about 40%, about 2.5% to about 35%, about 2.5% to about 30%, about 2.5% to about 25%, about 2.5% to about 20%, about 2.5% to about 18%, about 2.5% to about 16%, about 2.5% to about 14%, about 2.5% to about 12%, about 2.5% to about 10%, about 2.5% to about 8%, about 2.5% to about 6%, about 2.5% to about 5%, about 2.5% to about 4.5%, about 2.5% to about 4.0%, about 2.5% to about 3.5%, about 2.5% to about 3.0%, about 3.0% to about 40%, about 3.0% to about 35%, about 3.0% to about 30%, about 3.0% to about 25%, about 3.0% to about 20%, about 3.0% to about 18%, about 3.0% to about 16%, about 3.0% to about 14%, about 3.0% to about 12%, about 3.0% to about 10%, about 3.0% to about 8%, about 3.0% to about 6%, about 3.0% to about 5%, about 3.0% to about 4.5%, about 3.0% to about 4.0%, about 3.0% to about 3.5%, about 3.5% to about 40%, about 3.5% to about 35%, about 3.5% to about 30%, about 3.5% to about 25%, about 3.5% to about 20%, about 3.5% to about 18%, about 3.5% to about 16%, about 3.5% to about 14%, about 3.5% to about 12%, about 3.5% to about 10%, about 3.5% to about 8%, about 3.5% to about 6%, about 3.5% to about 5%, about 3.5% to about 4.5%, about 3.5% to about 4.0%, about 4.0% to about 40%, about 4.0% to about 35%, about 4.0% to about 30%, about 4.0% to about 25%, about 4.0% to about 20%, about 4.0% to about 18%, about 4.0% to about 16%, about 4.0% to about 14%, about 4.0% to about 12%, about 4.0% to about 10%, about 4.0% to about 8%, about 4.0% to about 6%, about 4.0% to about 5%, about 4.0% to about 4.5%, about 4.5% to about 40%, about 4.5% to about 35%, about 4.5% to about 30%, about 4.5% to about 25%, about 4.5% to about 20%, about 4.5% to about 18%, about 4.5% to about 16%, about 4.5% to about 14%, about 4.5% to about 12%, about 4.5% to about 10%, about 4.5% to about 8%, about 4.5% to about 6%, about 4.5% to about 5%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 18%, about 5% to about 16%, about 5% to about 14%, about 5% to about 12%, about 5% to about 10%, about 5% to about 8%, about 5% to about 6%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 6% to about 18%, about 6% to about 16%, about 6% to about 14%, about 6% to about 12%, about 6% to about 10%, about 6% to about 8%, about 8% to about 40%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 8% to about 18%, about 8% to about 16%, about 8% to about 14%, about 8% to about 12%, about 8% to about 10%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 18%, about 10% to about 16%, about 10% to about 14%, about 10% to about 12%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 12% to about 25%, about 12% to about 20%, about 12% to about 18%, about 12% to about 16%, about 12% to about 14%, about 14% to about 40%, about 14% to about 35%, about 14% to about 30%, about 14% to about 25%, about 14% to about 20%, about 14% to about 18%, about 14% to about 16%, about 16% to about 40%, about 16% to about 35%, about 16% to about 30%, about 16% to about 25%, about 16% to about 20%, about 16% to about 18%, about 18% to about 40%, about 18% to about 35%, about 18% to about 30%, about 18% to about 25%, about 18% to about 20%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 40%, about 30% to about 35%, or about 35% to about 40%). In another embodiment, the concentration of polymer is between 1 and 15%. The formulations of the invention may include combination of polymers with sugars, polyols, or amino acids in any combination.
[00163] In the embodiments of the invention where organic co-solvents are excluded, polymers cannot be present if they are organic co-solvents (for example, PEGs).
[00164] Under certain conditions, aflibercept may be stable even in the absence of surfactants, and/or may be stabilized with surfactants other than PS80 and at lower surfactant concentrations. For example, certain polymers, e.g., PEG, can exhibit surfactant-like properties and may be employed to stabilize aflibercept formulations in the absence of surfactants according to the present invention. Additional polymers which may be employed in specific aflibercept formulations include serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextrans (e.g., cyclodextrin, e.g., Captisol®), poly(vinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), and 2-Hydroxypropyl-beta-cyclodextrin (HP-beta-CD).
VI. Chelating agents
[00165] Chelating agents such as EDTA, DPTA, etc., and/or sacrificial additives (e.g., ascorbate, Met), may be employed at specific pH values and with and without buffers (that may also acts as chelating agents, e.g., citrate, phosphate) to enhance the formulation properties, especially in cases where there may be some level of oxidative damage (under certain conditions, certain metals can catalyze the degradation of antibodies, especially at the hinge region). The addition of a chelating agent, such EDTA and DPTA, may be beneficial at improving the storage stability of aflibercept. Such approaches may be employed to stabilize aflibercept formulations according to the present invention.
[00166] Certain buffers, such as citrate, may also function as chelating agents and can serve multiple purposes in stabilization of aflibercept.
[00167] Sacrificial additives are well known to diminish certain oxidation events, such as oxidation of methionine residues. Addition of the free amino acid, methionine, or some derivative, can lead to decreased oxidation of aflibercept. Ascorbate and various thiol derivatives can serve the same purpose. Likewise, Trp and its derivatives can also serve as a sacrificial additive, even in the case of photolytic oxidation.
VII. Preparing Formulations
[00168] In on embodiment, the formulations of the invention can be prepared by isolating or purifying aflibercept or ziv-aflibercept from commercially available Eylea® or Zaltrap® or by modifying commercial aflibercept (Eylea®) or ziv-aflibercept (Zaltrap®) formulations to arrive at the formulations of the invention. In another embodiment, the formulation is prepared with aflibercept purified from a host cell that produces aflibercept. In another embodiment, the formulation of the invention is a biosimilar of Eylea.
[00169] The pharmaceutical compositions of the invention may be prepared by combining aflibercept and an excipient such as a stabilizer (e.g. sugar, amino acid). Further, a buffer (except in buffer-free formulations), an organic co-solvent, such as a surfactant (except in formulations free of organic co-solvents), a tonicity modifier and an additional excipient may be added as needed. A person of ordinary skill in the art will understand that the combining of the various components to be included in the composition can be done in any appropriate order. For example, the buffer can be added first, middle or last, and the tonicity modifier can also be added first, middle or last.
[00170] A person of ordinary skill in the art will also understand that some of these chemicals can be incompatible in certain combinations, and accordingly, must be substituted with different chemicals that have similar properties but are compatible in the relevant mixture. While a person of skill in the art may look to the art for clues on how to create new formulations for aflibercept, due to the complexity of proteins, and the unpredictability of effects of chemicals on proteins (including differing effects a chemical may have on one protein versus another protein), care must be taken when creating new formulations as the differing effects of excipients may not produce stable formulations.
VIII. Formulations Free of Excipients
[00171] In accordance with the FDA approval to market Eylea, Eylea is a stable formulation of aflibercept. Eylea has a pH of 6.2 and contains 40 mg/mL aflibercept, 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose. Since this formulation is stable, it is surprising if an aflibercept formulation that is free of at least one of the specific excipients in Eylea, or a class of excipients that one of the Eylea excipients belongs to, is stable.
[00172] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a buffer. In a further embodiment, the buffer is a phosphate buffer.
[00173] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a tonicity agent. In a further embodiment, the tonicty agent is a salt. In a futher embodiment, the salt is sodium chloride.
[00174] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent. In a further embodiment, the organic co-solvent is a surfactant. In a futher embodiment, the surfactant is polysorbate 20.
[00175] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a stabilizer. In a further embodiment, the stabilizer is a sugar. In a futher embodiment, the sugar is sucrose.
[00176] In another embodiment of the invention, if a stable aflibercept formulation contains an excipient, it is free of other functionally similar excipients. For instance, in one embodiment, a stable aflibercept formulation containing trehalose is free of sucrose. In another embodiment, a stable aflibercept formulation containing acetate buffer is free of phosphate buffer. Likewise, in another embodiment, a stable aflibercept formulation containing acetate buffer is free of histidine buffer. Thus, the formulations of the present invention can be free of one or more excipients.
D. Stabiltiy Characteristics
[00177] In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition comprises at least 90% monomer aflibercept. In another embodiment, the amount of monomer aflibercept is at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%. In another embodiment, the the amount of monomer aflibercept is determined by SEC.
[00178] In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the pH of the composition is stable. In one embodiment the pH does not change more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% during storage.
[00179] In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more total sub-visible particles than Eylea. In another embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more sub-visible particles > 10 pm in diameter than Eylea. In another embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more sub-visible particles > 25 pm in diameter than Eylea. In another embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more sub-visible particles > 50 pm in diameter than Eylea.
[00180] In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition meets the requirments of USP <789>, which is incorporated herein by reference in its enterity. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 50 particles per ml_ of particles that are > 10 pm in diameter, wherein the particles are counted by a light obscuration test. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 5 particles per ml_ of particles that are > 25 pm in diameter, wherein the particles are counted by a light obscuration test. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 50 particles per mL of particles that are > 10 pm in diameter, wherein the particles are counted by a miscroscopic method. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 5 particles per mL of particles that are > 25 pm in diameter, wherein the particles are counted by a miscroscopic method. In one embodiment, the invention provides stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition has no more than 2 particles per mL of particles that are > 50 pm in diameter, wherein the particles are counted by a miscroscopic method.
E. Aflibercept Formulations
I. Formulations of Aflibercept which Exclude Buffer (Buffer-Free Formulations)
[00181] In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a buffer. The term "free of buffer" should be understood to allow inclusion of the inherent buffering effect of the protein itself. Such “self-buffering” or “buffer-free” protein formulations comprise a protein, e.g., a pharmaceutical protein, and are buffered by the protein itself, i.e., the formulations do not require additional buffering agents to maintain a desired pH. The protein is substantially the only buffering agent in such formulations (i.e., other ingredients, if any, do not act substantially as buffering agents in the formulation).
[00182] In another embodiment, the buffer-free aflibercept composition further comprises a stabilizer, a tonicity agent, an organic co-solvent, or combinations thereof.
[00183] In one embodiment, the buffer-free formulation has a pH of about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0). In another embodiment, the pH is about 5.0 to about 5.8. In another embodiment, the PH is about 5.5.
[00184] In one embodiment, the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
[00185] In one embodiment, the organic co-solvent is selected from the group consisting of a surfactant, polyethylene glycol (PEG), propylene glycol, and combinations thereof.
[00186] In one embodiment, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, SDS, DDM and poloxamer 188 (Pluronic F-68).
[00187] In one embodiment, the buffer-free formulations of the invention may comprise a salt, which may be selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride. In one embodiment, the salt is present at a concentration of about 10 mM-100 mM (e.g., any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
[00188] In one embodiment, the stabilizer is sucrose.
[00189] In one embodiment, the stabilizer is an amino acid, which may be selected from the group consisting of glycine, arginine, and proline or combinations thereof. The amino acids may be present at a concentration of about 25-300 mM (e.g., any of the subranges of this range described herein or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about
300 mM).
[00190] In some embodiments, the stabilizer is sucrose, the organic co solvent is polysorbate 20 and the pH of the formulation is about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0). In another embodiment, the pH is about 5.0 to about 5.8. In another embodiment, the PH is about 5.5.
[00191] In one embodiment, the formulation is iso-osmolar relative to the vitreous.
[00192] In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of a buffer and the composition has an osmolality of about 200 to 400 mOsm. In another embodiment, the composition is suitable for administration to a subject as a single dose. In still another embodiment, the single dose is about 2mg of aflibercept. In another embodiment, the aflibercept is present at about 40 mg/mL.
[00193] In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is substantially free of a buffer the composition has an osmolality of about 200 to 400 mOsm, the composition is suitable for administration to a subject as a single dose, and the single dose is about 2mg of aflibercept. In another embodiment the single dose of aflibercept has a volume of about 0.05 ml_.
II. Formulations of Aflibercept which Exclude Organic Co-Solvent [00194] In another embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent. In another embodiment, the co-solvent-free aflibercept composition further comprises a buffer, a stabilizer, a tonicity agent, or combinations thereof.
[00195] In one embodiment, the formulation has a pH of about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
In another embodiment, the pH is about 5.5 to about 6.3. In another embodiment, the PH is about 6.2.
[00196] In one embodiment, the buffer is selected from the group consisting of acetate, histidine, phosphate, citrate, succinate, tartrate and maleate. In one embodiment, the buffer is present at a concentration of about 1 mM to 150 mM (e.g., any of the subranges of this range described herein).
In another embodiment, the buffer is present at a concentration of about 5 mM to 20 mM. In another embodiment, the buffer is present at a concentration at about 10 mM.
[00197] In one embodiment, the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof. [00198] In some organic co-solvent-free formulations, the stabilizer is sucrose.
[00199] In other organic co-solvent-free formulations, the stabilizer is an amino acid, which may be selected from the group consisting of glycine, arginine, and proline or combinations thereof. The amino acid may be present at a concentration of about 25-300 mM (e.g., any of the subranges of this range described herein or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about 300 mM).
[00200] In one embodiment, the formulation may comprise a salt, which may be selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride. In one embodiment, the salt is present at a concentration of about 10 mM-100 mM (e.g., any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
[00201] In one embodiment, the formulation is iso-osmolar relative to the vitreous.
[00202] In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent and the composition has an osmolality of about 200 to 400 mOsm. In another embodiment, the composition is suitable for administration to a subject as a single dose. In still another embodiment, the single dose is about 2mg of aflibercept. In another embodiment, the aflibercept is present at about 40 mg/mL.
In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of an organic co-solvent, the composition has an osmolality of about 200 to 400 mOsm, the composition is suitable for administration to a subject as a single dose, and the single dose is about 2mg of aflibercept. In another embodiment the single dose of aflibercept has a volume of about 0.05 ml_.
III. Formulations of Aflibercept which Exclude Both a Buffer and an Organic Co-Solvent
[00203] In a further embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is substantially free of both an organic co-solvent and a buffer. In another embodiment, the buffer-free and co-solvent-free aflibercept composition further comprises a stabilizer, a tonicity agent, or combinations thereof.
[00204] In one embodiment, the formulation has a pH of about 5.0 to about 7.0 (e.g., or any of the subranges of this range described herein or about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 , about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).
In another embodiment, the pH is about 5.0 to about 6.2. In another embodiment, the pH is about 5.5.
[00205] In one embodiment, the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
[00206] In one embodiment, the stabilizer is sucrose.
[00207] In one embodiment, the stabilizer is an amino acid, which may be selected from the group consisting of glycine, arginine, and proline or combinations thereof. The amino acids may be present at a concentration of about 25-300 mM (e.g., any of the subranges of this range described herein or about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, or about
300 mM).
[00208] In one embodiment, the formulation may comprise a salt, which may be selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride. In one embodiment, the salt is present at a concentration of about 10 mM-100 mM (e.g., any of the subranges of this range described herein or about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM about 95 mM, or about 100 mM).
[00209] In one embodiment, the formulation is iso-osmolar relative to the vitreous.
[00210] In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of both a buffer and an organic co-solvent and the composition has an osmolality of about 200 to 400 mOsm. In another embodiment, the composition is suitable for administration to a subject as a single dose. In still another embodiment, the single dose is about 2mg of aflibercept. In another embodiment, the aflibercept is present at about 40 mg/mL. In another embodiment the single dose of aflibercept has a volume of about 0.05 ml_. [00211] In a one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, wherein the composition is free or substantially free of both a buffer and an organic co-solvent, the composition has an osmolality of about 200 to 400 mOsm, the composition is suitable for administration to a subject as a single dose, and the single dose is about 2mg of aflibercept.
IV. Exemplary Aflibercept Formulations
[00212] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, a stabilizer, and an organic co-solvent, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer. In one embodiment, the stabilizer is a sugar, polyol, amino acid, salt, or a combination thereof. In another embodiment, the sugar is sucrose. In another embodiment, the salt is NaCI or CaCI2. In another embodiment, the amino acid is glycine. In another embodiment, the organic so-solvent is a surfactant. In a further embodiment, the surfactant is a polysorbate. In a further embodiment, the polysorbate is polysorbate 20 or polysorbate 80.
[00213] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, sucrose, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer. In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, sucrose, a salt selected from the group consisting of NaCI and CaCI2, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer. In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, an amino acid, a salt selected from the group consisting of NaCI and CaCI2, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer. In a further embodiment the amino acid is glycine. In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, sucrose, CaCI2, and a polysorbate, wherein the composition has a pH of about 5.0 to about 5.8 and is free or substantially free of a buffer. In a further embodiment, the composition is free of NaCI.
[00214] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, a buffer, and a stabilizer, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent. In one embodiment, the buffer is histidine or acetate. In one embodiment, the stabilizer is a sugar, polyol, amino acid, or a combination thereof. In another embodiment, the sugar is sucrose. In another embodiment, the amino acid is glycine.
[00215] In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, a buffer, and sucrose, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent. In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, a buffer, and an amino acid, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co solvent. In a further embodiment, the amino acid is glycine. In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, histidine, and sucrose, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent. In one embodiment, the invention provides a stable aqueous pharmaceutical composition comprising aflibercept, histidine, and glycine, wherein the composition has a pH of about 5.5 to about 6.3 and is free or substantially free of an organic co-solvent. In another embodiment, a co-solvent-free composition further comprises a salt. In one embodiment, the salt is selected from the group consisting of NaCI and CaCI2.
[00216] In a further embodiment, exemplary aflibercept formulations are listed in Table 1.1 below. Formulations in Table 1.1 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co- solvent-free. All formulations Table 1.1 contain 40 mg/mL aflibercept.
Table 1.1
Figure imgf000138_0001
[00217] In some embodiments, the formulation can include aflibercept
(e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about
6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, or about 50 mM to about 60 mM) of a salt (e.g., NaCI); about 0.01 % w/v to about 0.1 % w/v (e.g., about 0.01 % w/v to about 0.05% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., any of the polysorbates described herein); having a pH of about 5.5 to about 6.2 (e.g., about 6.0), and is free of buffer.
[00218] In some embodiments, the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 0.01 % w/v to about 0.1 % w/v (e.g., about 0.01 % w/v to about 0.05% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., any of the polysorbates described herein); having a pH of about 5.5 to about 6.2 (e.g., about 6.0), and is free of buffer.
[00219] In some embodiments, the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 0.1 mM to about 10 mM (e.g., about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM) about a buffer (e.g., any of the buffers described herein, e.g., histidine or imidazole buffer); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, or about 50 mM to about 60 mM) of a salt (e.g., NaCI); about 0.01% w/v to about 0.1 % w/v (e.g., about 0.01 % w/v to about 0.05% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., any of the polysorbates described herein); having a pH of about 5.5 to about 6.2 (e.g., about 6.0).
[00220] In some embodiments, the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); and is free of both a buffer and an organic co-solvent.
[00221] In some embodiments, the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, or about 50 mM to about 60 mM) of a salt (e.g., NaCI); about 0.01 % w/v to about 0.2% w/v (e.g., about 0.01 % w/v to about 0.2% w/v, about 0.05% to about 0.2% w/v, about 0.1 % w/v to about 0.2% w/v, or about 0.15% w/v to about 0.2% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., human serum albumin); about 0.5 mM to about 10 mM (e.g., about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM) potassium ion (e.g., provided as potassium chloride); about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM) calcium ion (e.g., provided as calcium chloride); about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM) magnesium ion (e.g., provided as magnesium chloride); having a pH of about 5.5 to about 6.2 (e.g., about 6.0), and is free of a buffer and an organic co-solvent.
[00222] In some embodiments, the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, or about 50 mM to about 60 mM) of a salt (e.g., NaCI); about 0.01 % w/v to about 0.2% w/v (e.g., about 0.01 % w/v to about 0.2% w/v, about 0.05% to about 0.2% w/v, about 0.1 % w/v to about 0.2% w/v, or about 0.15% w/v to about 0.2% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., human serum albumin); having a pH of about 5.5 to about 6.2 (e.g., about 6.0), and is free of a buffer and an organic co-solvent.
[00223] In some embodiments, the formulation can include aflibercept (e.g., at any of the final concentrations described herein); about 0.5% w/v to about 6% w/v (e.g., about 1.0% w/v to about 6% w/v, about 2.0% w/v to about 6% w/v, about 3.0% w/v to about 6.0% w/v, about 4.0% w/v to about 6.0% w/v, about 5.0% w/v to about 6.0% w/v) of a sugar (e.g., sucrose); about 1 mM to about 60 mM (e.g., about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, about 10 mM to about 20 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 20 mM to about 30 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 30 mM to about 40 mM, about 40 mM to about 60 mM, about 40 mM to about 50 mM, or about 50 mM to about 60 mM) of a salt (e.g., NaCI); about 0.01 % w/v to about 0.2% w/v (e.g., about 0.01 % w/v to about 0.2% w/v, about 0.05% to about 0.2% w/v, about 0.1% w/v to about 0.2% w/v, or about 0.15% w/v to about 0.2% w/v) of a stabilizer (e.g., any of the stabilizers described herein, e.g., any of the polysorbates described herein); about 0.5 mM to about 10 mM (e.g., about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM) potassium ion (e.g., provided as potassium chloride); about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM) calcium ion (e.g., provided as calcium chloride); about 0.1 mM to about 5 mM (e.g., about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, about 1 mM to about 2 mM, about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, or about 4 mM to about 5 mM) magnesium ion (e.g., provided as magnesium chloride); about 0.1 mM to about 10 mM (e.g., about 0.1 mM to about 8 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 8 mM, about 0.5 mM to about 6 mM, about 0.5 mM to about 4 mM, about 0.5 mM to about 2 mM, about 0.5 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 8 mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, about 1 mM to about 2 mM, about 2 mM to about 10 mM, about 2 mM to about 8 mM, about 2 mM to about 6 mM, about 2 mM to about 4 mM, about 4 mM to about 10 mM, about 4 mM to about 8 mM, about 4 mM to about 6 mM, about 6 mM to about 10 mM, about 6 mM to about 8 mM, or about 8 mM to about 10 mM) of a buffer (e.g., histidine or imidazole) having a pH of about 5.5 to about 6.2 (e.g., about 6.0).
[00224] In a further embodiment, exemplary aflibercept formulations are listed in Table 1 .2 below. A surfactant (e.g. polysorbate 80) is optionally included in the formulaitons of table 1.2. Formulations in Table 1.2 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co-solvent-free All formulations Table 1.2 contain 40 mg/mL aflibercept. In one embodiment, Captisol, if present in the formulation, is present at about 0.1 % to about 2% w/v. In another embodiment, CaCh, if present in the formulation, is present at about 1 to about 10 mM. In still another embodiment, hydroxypropyl-beta-cyclodextrin (HP-beta-CD), if present in the formulation, is present at about 0.1% to about 2% w/v. In another embodiment, human serum albumin (HSA), if present in the formulation, is present at about 0.01 % to about 2% w/v.
Table 1.2
Figure imgf000145_0001
[00225] In one embodiment, stable formulations comprise aflibercept and low concentration of specific salts (calcium chloride, magnesium chloride and potassium chloride) within the range present in human vitreous humor. In a futher embodiment, the formulation comprises a stabilizer such as a sugar (e.g. sucrose) or Human Serum Albumin (e.g. recombinant HAS). In another embodiment, the formulation is free of an organic co-solvent. The use of rHSA is the most common protein found in human plasma and is known for ligand binding and colloidal stabilization, properties that are also ideal for the stabilization of proteins and other biologies. In addition, because rHSA is a natural protein in the body, it is safer than alternative surfactant excipients, such as synthetic polymers, because it has a low risk for immunogenicity and there is a natural pathway for its elimination from the body.
[00226] In a further embodiment, exemplary aflibercept formulations are listed in Table 1.3 below. Formulations in Table 1.3 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co- solvent-free. All formulations Table 1 .3 contain 40 mg/mL aflibercept.
Table 1.3
Figure imgf000146_0001
[00227] In a further embodiment, exemplary aflibercept formulations are listed in Table 1.4 below. Formulations in Table 1.4 include formulations that are buffer free, organic co-solvent free, and buffer-free and organic co- solvent-free. All formulations Table 1 .4 contain 40 mg/mL aflibercept and 5%w/v sucrose.
Table 1.4
Figure imgf000146_0002
Figure imgf000147_0001
F. Analytical Methods
[00228] The chemical and physical stability of the aflibercept protein in a formulation is measured using, e.g., SEC, CEX, HPLC, RP, UV, pH, MFI, Flow-CAM, icIEF, CE-IEF and CE-SDS. However, other analytical methods may also be employed, for example, biophysical techniques such as those described by Jiskoot and Crommelin (Methods for Structural Analysis of Protein Pharmaceuticals, Springer, New York, 2005). Specific examples of such techniques include spectroscopic analyses (e.g., second derivative ultraviolet spectroscopy, circular dichroism, Fourier Transform infrared spectroscopy, Raman spectroscopy, fluorescence and phosphorescence spectroscopy), thermal analyses (e.g., differential scanning calorimetry), and size-based analyses (e.g., analytical ultracentrifuge, light scattering).
[00229] One of skill in the art can readily determine which of these or other suitable techniques may be used in specific situations when assessing the physical characteristics (e.g., stability, aggregation, oxidation, etc.) of the aflibercept protein in particular formulations.
G. Methods of Treatment
[00230] Formulations of the present invention may be used in methods of treating an ocular condition in a subject in need thereof. In one embodiment, the subject is a human patient. In another embodiment, treating comprises administering a therapeutically effective amount of the formulations of the invention to the subject. In another embodiment, the subject has an ocular disease or disorder that can be beneficially treated with aflibercept, for example any ocular disease or disorder that can benefit from an anti-VEGF injection. [00231] In one embodiment, the ocular disease or disorder is a vascular eye disease or disorder. Ocular diseases or disorders that can be treated with the formulations of the present invention includes, but is not limited to, those which can be treated with Eylea®, such as macular degeneration (AMD), diabetic macular edema (DME), retinal vein occlusion (RVO), diabetic retinopathy (DR).
[00232] In one embodiment, the formulations of the present invention are administered intravitreally.
[00233] As used herein, the terms "treat", "treating", or the like, mean to alleviate symptoms, eliminate the causation of symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of an eye disease. In certain embodiments, the present methods are useful for treating or ameliorating at least one symptom or indication including, but not limited to, retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 pm of the center of the fovea, hard, yellow exudates within 500 pm of the center of the fovea with adjacent retinal thickening, and at least 1 disc area of retinal thickening, any part of which is within 1 disc diameter of the center of the fovea, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision. In the context of methods for treating a vascular eye disease such as AMD or DME, the term means that, from the initiation of treatment, the patient exhibits gain of one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) letters on the Early Treatment Diabetic Retinopathy Study (EDTRS) visual acuity chart. In certain embodiments, the term means that, from initiation of treatment, vision loss of greater than or equal to 15 letters is prevented in the patient.
[00234] As used herein, the terms "prevent", "preventing", or the like, mean to prevent development of a symptom, indication or a complication of a vascular eye disease. In the context of methods for treating a vascular eye disease such as AMD or DME, the term means, from initiation of treatment, moderate or severe vision loss is prevented in a patient.
[00235] As used herein, a "vascular eye disease or disorder" refers to eye disease or disorders that affect blood vessels in the eye. The diseases may be caused due to abnormal angiogenesis (formation of new blood vessels) or occlusion or blockage of blood vessels. The term, as used herein, includes eye diseases or disorders associated with angiogenesis. The term includes, but is not limited to, eye disease or disorder selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, and choroidal neovascularization. In certain embodiments, the term "neovascular eye disease or disorder" may be used interchangeably with the term "eye disease or disorder associated with angiogenesis."
[00236] In certain embodiments, the present invention includes methods for treating, preventing, or ameliorating at least one symptom or indication of an eye disease or disorder associated with angiogenesis in a subject, wherein the disease or disorder is selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, polypoidal choroidal vasculopathy, and choroidal neovascularization.
[00237] “Diabetic Macular Edema" (DME), as used herein, refers to a serious eye condition that affects people with diabetes (type 1 or 2). Macular edema occurs when blood vessels in the retina leak into the macula and fluid and protein deposits collect on or under the macula of the eye (a yellow central area of the retina) and causes it to thicken and swell (edema). The swelling may distort a person's central vision, as the macula is near the center of the retina at the back of the eyeball. The primary symptoms of DME include, but are not limited to, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision. The pathology of DME is characterized by breakdown of the blood-retinal barrier, normally preventing water movement in the retina, thus allowing fluid to accumulate in the retinal tissue, and presence of retinal thickening. DME is presently diagnosed during an eye examination consisting of a visual acuity test, which determines the smallest letters a person can read on a standardized chart, a dilated eye exam to check for signs of the disease, imaging tests such as optical coherence tomography (OCT) or fluorescein angiography (FA) and tonometry, an instrument that measures pressure inside the eye. The following studies are also performed to determine treatment: optical coherence tomography (OCT), fluorescein angiography, and color stereo fundus photography. DME can be broadly characterized into two main categories-Focal and Diffuse. Focal DME is characterized by specific areas of separate and distinct leakage in the macula with sufficient macular blood flow. Diffuse DME results from leakage of the entire capillary bed surrounding the macula, resulting from a breakdown of the inner blood-retina barrier of the eye. In addition to Focal and Diffuse, DME is also categorized based on clinical exam findings into clinically significant macular edema (CSME), non-CSME and CSME with central involvement (CSME-CI), which involves the fovea. The present invention includes methods to treat the above-mentioned categories of DME.
[00238] Age-related macular degeneration (AMD), as used herein, refers to a serious eye condition when the small central portion of the retina, known as the macula, deteriorates. The wet form of AMD is characterized by the growth of abnormal blood vessels from the choroid underneath the macula. This is called choroidal neovascularization. These blood vessels leak blood and fluid into the retina, causing distortion of vision that makes straight lines look wavy, as well as blind spots and loss of central vision. These abnormal blood vessels eventually scar, leading to permanent loss of central vision. The symptoms of AMD include dark, blurry areas in the center of vision; and diminished or changed color perception. AMD can be detected in a routine eye exam. One of the most common early signs of macular degeneration is the presence of drusen-tiny yellow deposits under the retina-or pigment clumping.
[00239] As used herein, the expression "a subject in need thereof" means a human or non-human mammal that exhibits one or more symptoms or indications of, and/or who has been diagnosed with an eye disease or disorder associated angiogenesis. The term "a subject in need thereof" may also include, e.g., subjects who, prior to treatment, exhibit (or have exhibited) one or more indications of a neovascular eye disease such as, e.g., retinal angiogenesis, neovascularization, vascular leak, retinal thickening within 500 pm of the center of the fovea, hard, yellow exudates within 500 pm of the center of the fovea with adjacent retinal thickening, and at least 1 disc area of retinal thickening, any part of which is within 1 disc diameter of the center of the fovea, blurry vision, floaters, loss of contrast, double vision, and eventual loss of vision.
[00240] In the context of the invention, a "subject in need thereof" also includes human or non-human mammal who has a vascular eye disease or disorder selected from the group consisting of diabetic retinopathy, diabetic macular edema, age-related macular degeneration, retinal neovascularization, central retinal vein occlusion, branched retinal vein occlusion, polypoidal choroidal vasculopathy, and choroidal neovascularization.
[00241] In the context of the present invention, "a subject in need thereof" may include a subset of population which is more susceptible to DME or AMD or may show an elevated level of a DME-associated or an AMD- associated biomarker. For example, "a subject in need thereof" may include a subject suffering from diabetes for more than 10 years, have frequent high blood sugar levels or high fasting blood glucose levels. In certain embodiments, the term "a subject in need thereof" includes a subject who, prior to or at the time of administration of the formulations of the invention, has or is diagnosed with diabetes. In certain embodiments, the term "a subject in need thereof" includes a subject who, prior to or at the time of administration of the formulations of the invention, is more than 50 years old. In some embodiments, the term "a subject in need thereof" includes subjects who are smokers, or subjects with high blood pressure or high cholesterol.
[00242] The present invention includes methods for treating, preventing or reducing the severity of a vascular eye disease comprising administering a therapeutically effective amount of a formulation of the present invention to a subject in need thereof, wherein the formulation is administered to the subject as a single dose or in multiple doses, e.g., as part of a specific therapeutic dosing regimen. For example, the therapeutic dosing regimen may comprise administering multiple doses of the formulation to the subject at a frequency of about once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, or less frequently. In certain embodiments, the therapeutic dosing regimen may comprise administering multiple doses of the formulation to the subject at a frequency of once a day or 2 times a day or more.
[00243] In one embodiment, methods of treating include administering to a subject in need thereof an aflibercept composition of the present invention according to the dosing and administration regimen for Eylea®. See, Eylea® package insert.
[00244] In one embodiment, a subject with Neovascular (Wet) Age- Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 28 days, monthly) for the first 3 months, followed by 2 mg (0.05 ml_) via intravitreal injection once every 8 weeks (2 months). In antother embodiment, a subject with Neovascular (Wet) Age-Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 25 days, monthly). In antother embodiment, a subject with Neovascular (Wet) Age-Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 4 week (monthly) dosing after the first 12 weeks (3 months). In antother embodiment, a subject with Neovascular (Wet) Age-Related Macular Degeneration (AMD) is administered 2 mg of aflibercept by intravitreal injection every 12 weeks after one year of effective therapy.
[00245] In one embodiment, a subject with Macular Edema Following Retinal Vein Occlusion (RVO) is administered 2 mg of aflibercept by intravitreal injection once every 4 weeks (approximately every 25 days, monthly).
[00246] In one embodiment, a subject with Diabetic Macular Edema (DME) or Diabetic Retinopathy (DR) in Patients with Diabetic Macular Edema is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 28 days, monthly) for the first 5 injections followed by 2 mg via intravitreal injection once every 8 weeks (2 months). In another embodiment, a subject with Diabetic Macular Edema (DME) or Diabetic Retinopathy (DR) in Patients with Diabetic Macular Edema is administered 2 mg of aflibercept by intravitreal injection every 4 weeks (approximately every 25 days, monthly). In another embodiment, a subject with Diabetic Macular Edema (DME) or Diabetic Retinopathy (DR) in Patients with Diabetic Macular Edema is administered 2 mg of aflibercept by intravitreal injection every 4 week (monthly) dosing after the first 20 weeks (5 months).
[00247] In some embodiments of the invention, the formulations of the invention may be administered in combination with other ocular therapy, for example, laser treatment. As used herein, the phrase 'in combination with" means that the formulations of the invention may be administered to the subject at the same time as, just before, or just after other therapy including laser treatment.
[00248] The present invention also includes methods for inhibiting or reducing or suppressing vascular leak in a subject. In certain embodiments, the methods according to this aspect of the invention comprise administering to the subject one or more doses of a pharmaceutical formulation of the invention to reduce or inhibit vascular leak in the eye of a subject. In certain other embodiments, the methods comprise administering to the subject one or more doses of a formulation of the invention. In certain embodiments, the vascular leak is inhibited for more than 3 weeks, more than 4 weeks, more than 8 weeks, or more than 10 weeks than in a subject who has not been administered the formulations of the invention.
[00249] In certain embodiments, the methods comprise administering an initial dose of the pharmaceutical composition, followed by one or more secondary doses, wherein each secondary dose is administered 1 to 4 weeks after the immediately preceding dose. In certain embodiments, one or more tertiary doses of the pharmaceutical composition are administered, wherein each tertiary dose is administered 5 to 12 weeks after the immediately preceding dose. In certain embodiments, each dose of the pharmaceutical composition may comprise about 2 mg of the VEGF antagonist. [00250] The methods of the present invention are useful for treating or preventing vascular eye disorders in patients that have been diagnosed with or are at risk of being afflicted with a vascular eye disorder. Generally, the methods of the present invention demonstrate efficacy within 36 weeks of the initiation of the treatment regimen (with the initial dose administered at "week 0"), e.g., by the end of week 6, by the end of week 12, by the end of week 18, by the end of week 24, etc. In the context of methods for treating angiogenic eye disorders such as AMD, and DME, "efficacy" means that, from the initiation of treatment, the patient exhibits a loss of 10 or fewer letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart. In certain embodiments, "efficacy" means a gain of one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more) letters on the ETDRS chart from the time of initiation of treatment.
[00251] In one embodiment, the formulations of the invention may be intravitreally administered to a subject (e.g., a human) with an aflibercept concentration of about 0.01 mg/mL to about 200 mg/mL. In another embodiment, the concentration of aflibercept is about 0.1 mg/mL to about 100 mg/mL. In another embodiment, the concentration of aflibercept is about 1 mg/mL to about 60 mg/mL. In another embodiment, the concentration of aflibercept is about 40 mg/mL.
[00252] In certain embodiments, the formulations of the invention may be prepared in a bulk formulation, and as such, the components of the bulk formulation are adjusted to be higher than would be required for administration and then diluted appropriately prior to administration.
[00253] In one embodiment, the invention includes a container comprising a formulation of the present invention. In one embodiment the container is a vial. In another embodiment, the container is a syringe. In one embodiment, the syringe is silicone free. In another embodiment, the internal surface of the syringe is coated with baked-on silicone.
[00254] In some embodiments, the invention includes pre-filled syringes suitable for intravitreal administration wherein the pre-filled syringes comprise the formulations of the invention. In one embodiment the pre-filled syringe is filled a volume of a formulation that delivers 2mg of aflibercept upon administration. One one embodiment, the 2mg of aflibercept is delivered in 0.05 ml_. In a futher embodiment, the syringe is over-filled.
[00255] In another embodiment, the syringe is coupled to a needle. In one embodiment, the needle is a 30-gauge needle.
[00256] In another embodiment, the pre-filled syringe is a glass syringe. In another embodiment, the pre-filled syringe is a plastic syringe. In another embodiment, the pre-filled syringe is a glass syringe with an internal non glass coating.
[00257] Syringes, non-glass coatings, and syringes with intenral non glass coatings are known in the art. See, WO2017087798, US20180325728, W02017177120, WO2018194918, WO2018217995, W02018218013, and US20190000919 which are hereby incorporated by reference in their enterity.
[00258] In one embodiment, a formulation of the present invention is in a container that has no headspace. By providing the formulation in a closed container with no headspace interfacial stress on the formulation is reduced and the formulation is stabilized. Such containers and methods of preparing a container with no headspace that comprises a formulation are known in the art. See, US 20180043020 which is hereby incorporated by reference in its enterity.
[00259] In some embodiments, the methods of administration include administering the formulations of the invention to a subject in need thereof every four weeks for the first three months, and then once every eight weeks. In one embodiment, dose is about 2 mg of aflibercept (for example 0.05 ml_ of an aqueous solution containing 40 mg/mL aflibercept).
[00260] T reatment for longer periods may be necessary to induce the desired degree of improvement. For incurable chronic conditions the regimen may be continued indefinitely.
[00261] The pharmaceutical compositions may be administered as a sole therapeutic or in combination with additional therapies as needed. Thus, in one embodiment, the provided methods of treatment and/or prevention are used in combination with administering a therapeutically effective amount of another active agent. The other active agent may be administered before, during, or after administering the pharmaceutical compositions of the present invention. Another active agent may be administered either as a part of the provided compositions, or alternatively, as a separate formulation.
[00262] In some embodiments, the formulations of the invention can be administered in ways other than intravitreal administration, for example parenteral, oral, buccal, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, and topical application.
[00263] The pharmaceutical compositions may, if desired, be presented in a vial, pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. In one embodiment the dispenser device can comprise a syringe having a single dose of the liquid formulation ready for injection. The syringe can be accompanied by instructions for administration.
[00264] In another embodiment, the present invention is directed to a kit or container, which contains an aqueous pharmaceutical composition of the invention. The concentration of the polypeptide in the aqueous pharmaceutical composition can vary over a wide range, but is generally within the range of from about 0.05 to about 20,000 micrograms per milliliter (pg/ml) of aqueous formulation. The kit can also be accompanied by instructions for use.
[00265] The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention. EXAMPLES
[00266] Aflibercept formulations may be prepared as described more fully below. The formulations are typically grouped into blocks, where each block is described in the context of one Example or portion of one Example. The formulations are exposed to storage conditions, for example, 1 week at 40° C, 2 weeks at 25° C, and 4 weeks at 5° C. Storage at 40°C is an accelerated model which indicates that a formulation will be stable at lower temperatures for longer period of times. Aflibercept formulations of the present invention are compared to the aflibercept formulation for Elyea®. Eylea® has a pH of 6.2 and contains 40 mg/mL aflibercept, 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose.
[00267] For each time point, the chemical and physical stability of the aflibercept protein may be measured by, e.g., SEC, HIC, CEX, AF4, RP, UV, pH, CE-IEF and CE-SDS. However, studies using other durations, as well as other temperatures, may also be conducted following the guidance herein. Unless specified otherwise, all formulations contain 40 mg/ml aflibercept.
[00268] In the experimental disclosure which follows, the following abbreviations apply: SEC (size exclusion chromatography); HIC (Hydrophobic Interaction Chromatography); CEX (Cation Exchange Chromatography); AF4 (Asymmetrical flow field-flow fractionation); RP (reverse phase chromatography); UV (Ultra-violet spectroscopy); CE-IEF (Capillary Isoelectric Focusing); CE-SDS (Capillary Electrophoresis Sodium Dodecyl Sulfate); eq (equivalents); M (Molar); mM (micromolar); N (Normal); mol (moles); mmol (millimoles); gmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); pg (micrograms); L (liters); ml (milliliters); pi (microliters); cm (centimeters); mm (millimeters); pm (micrometers); nm (nanometers); °C (degrees Centigrade); h (hours); min (minutes); sec (seconds); msec (milliseconds).
Materials and Methods
A. Representative Equipment Useful in Formulation Studies
Figure imgf000157_0001
Figure imgf000158_0001
B. Representative Chemicals and Materials Useful in Formulation
Studies
Figure imgf000158_0002
Figure imgf000159_0001
Figure imgf000160_0003
C. Representative Reagents Useful in Formulation Studies
Figure imgf000160_0001
D. Representative HPLC Columns Useful in Formulation Studies
Figure imgf000160_0002
Figure imgf000161_0001
E. Tangential Flow Filtration (TFF)
[00269] Tween 20 was removed from Zaltrap (comcerically available aflibercept formulation) using a protein A column. The processed material was buffer exchanged into 10 mM sodium phosphate at pH 6.2. TFF was used to buffer exchange the bulk material and the concentrate sample. The parameters below were used to process the material by TFF for buffer exchange and concentration.
[00270] The TFF system was stored in 0.1% NaoH, this was washed out with the exchange buffer which was 10 mM sodium phosphate, pH 6.2. To ensure that sodium hydroxide was been flushed from the system and the lines, the pH was checked in the tank and the waste line. After flushing the system the material was added to the tank and the flow was changed to pass through the Pellicon ® 3.
[00271] The processed material was added the tank and diluted with the dialysis buffer to 500ml_. After buffer exchange the sample volume was reduced to 100 ml_, concentrating aflibercept. The material was UF/DF vs 2000 mL of 10 mM phosphate, pH 6.2, using the following parameters: Stir 30%; Pump 30%; TMP target 14.1 pSI; DP target 28.1 pSI.
F. Fluorescence Micelle Assay (FMA)
[00272] FMA is a method that quantifies polysorbates (Tweens) by the interaction of a fluorescence dye with the surfactant micelles. Below is the basic method used for the quantification of polysorbate 20 (PS20). It turned out that this method is extremely dependent on the sample matrix. [00273] Stock NPN Solution was prepared by weighing out 219 mg of NPN in a glass volumetric container and 100 mL of Acetonitrile. The solution needs to be protected from light and is stable for 3 months at room temperature.
[00274] NPN Mobile Phase.
50 mM TRIS, 150 mM NaCI, 50 pg/mL Brij 35, 5 pM NPN.
7.9 g, Tris HCI, 8.8 grams NaCI dissolve in water and adjust to pH 8.
Q.S. to 1 L, Filter and then add 500 uL of 10 mM NPN stock solution and add 165 pL of Brij 35
[00275] HPLC Analysis Parameters.
Column Temperature: 25 °C
Flow Rate: 1 mL/min
Injection Vol: 50 uL
Fluorescence Parameters: Excitation Wavelength: 350 nm
Emission Wavelength: 395 nm
Separation Device: Supelco Knitted reaction coil, 5 m by 0.5 mm, Product ID 57405 (The coil is used to allow for enough time for the dye to interact with micelle)
G. Dialysis
[00276] The UF/DF processed bulk aflibercept material was formulated by dialysis using a Slide-A-Lyzer ® Mini Dialysis 20K MWCO, 2ML. The Slide- A-Lyzer® Mini Dialysis was prepared by hydrating the membrane using the formulation buffer. After the membrane was hydrated the liquid was removed and replaced UF/DF processed bulk aflibercpt material. Slide-A-Lyzer then shacked using an orbital rotator set to 300 rpm for 48 to 72 hours at 5-8 °C. The dialysis buffer was changed out 3 times during this procedure at 4, 24 and 48 hours. [00277]
H. Spin Concentration
[00278] After the pH of the sample was within ±0.1 pH units of the target value the sample was concentrated by spin concentrator. The spin concentrator was first hydrated by 4 mL of water pass through the membrane. The sample was then loaded into the spin concentrator and rotated at 2,000 g for 7 to 13 minutes. The sample was concentrated above the target concentration of 40 mg/mL and was then diluted to be within 10% of the target protein concentration for the formulation.
I. UV Analysis
[00279] In order to prevent sample loss and the error due to preparing the UV sample, the concentration of each formulation was measured using the Solo VP. The concentration of the sample was measured by added 50 to 100 pl_ of material into a SoloVP small UV disposable vessel. A new fibrette was installed and the sample absorbance was measured by the instrument, using an extinction coefficient of 1 .55 mL. mg 1 cm 1, and correcting background scattering. After analysis the sample is removed with a pipette the disposable vessel and fibrette are both disposed. This procedure is repeated for each sample.
J. pH Analysis
[00280] After sample preparation the pH will be checked for each formulation and be within ± 0.1 of the target pH. Before the start of analysis, the pH probe was calibrated with three pH standards ordered from fisher. The pH of the formulation will be measured by inserting the pH probe in to the sample and waiting until the measured value has stabilized, which can take up to 1 to 2 minutes. After the analysis the pH probe is washed with 18 W water for one minute and stored in the pH storage solution.
K. Osmotic Analysis
[00281] The osmotic analysis was performed using an Advanced Instruments, Osmo 1. At the start of analysis, a reference standard at 290 mOsm is analyzed to insure the instrument is working properly. After the reference standard has passed the samples are then analyzed. 20ul_ of material is removed and analyzed by the Osmo 1 , after analysis the chamber is cleared by a using a chamber cleaner. This procedure is repeated for each sample.
L. Formulating
[00282] The bulk processed aflibercept material was formulated following the procedures below. The aflibercept material was buffered exchanged following the dialysis procedure. After dialysis the osmotic pressure and the pH was checked, if the pH of the sample was not within ±0.1 pH units, the formulation buffer was made more acidic or basic, by repeat dialysis or by addition of HCI or NaOH, until the pH target was reached. Following pH adjustment if needed, the sample was concentrated above the target formulation concentration. The pH, osmotic pressure and the protein concentration was then measured once more. The sample was then diluted with the formulation buffer to reached the target formulation protein concentration within 10%. The protein concentration was then measured once more to insure the diluted sample was within 10% of the target concentration. The last step was the addition of the 20% PS 20 (diluted in water) to the sample based on weight, if required.
M. Sterile Filtering and Sample Filling
[00283] The samples were sterile filtered in a clean hood that was wiped down with 70% ethanol. Each formulation was loaded into a sterile syringe with sterile filter attached. The sample was then slowly pushed through filter into a sterile container. After the samples had been sterile filtered, they were loaded into autoclaved vials and caps
N. Size Exclusion Chromatography Method
[00284] Size exclusion chromatography (SEC) analyses were conducted on formulations to measure the chages in colloidal stability. SEC can quantify oligomers and soluble aggregates, as well as detect fragmentation of proteins. [00285] The SEC analysis parameters are described below
Column: ACQUITY UPLC BEH SEC (CAT. 186005225), 220A 1.7pm, 4.6 mm x 150 mm
Mobile Phase:20 mM Sodium Phosphate, 250 mM Sodium Chloride, pH 7.55*
Autosampler Temp : 5°C ± 3°C Column Temp.: 30°C ± 2°C Flow Rate: 0.25 mL/minute Injection Vol: 5 pl_
UV Setting: 280 nm Data Collection Time: 15 minutes O. Sub-Visible Particle Evaluation by MFI
[00286] In the examples below, screening studies were conducted to identify formulations with particle counts indicating that the formulation is sufficiently stable and therefore suitable as commercial aflibercept product. Formulations with exceptionally high particle counts (e.g. > 1 ,000,000) may not be sufficiently stable. The particle counts reported in the examples below for a formulation may not be the particle counts for a commercialized aflibercept product. For example, a commercialized product is manufactured under strict GMP conditions which is expected to reduce the number of particles found in the formulation. Moreover, USP guidelines (e.g. USP <788>, <789>, which are hereby incorporated by reference in their enterity) limit the number of particles that can be present in certain products. When manufactured and handled under GMP conditions, it is expected that the formulations herein that are identified to be sufficiently stable based on the screening test of sub-visible particles will be suitable for a commercial aflibercept product.
[00287] To evaluate sub-visible particles in formulations Micro Flow Imaging analysis was conducted. The MFI analysis was performed on a MFI 5200 Protein Simple system following the procedure described below.
[00288] MFI System Suitability. I n order to ensure the MFI was counting and assessing particle size accurately, 5uM size/concentration standards (5 um polystyrene beads for MFI system suitability Count-cal Cat#CC05) and 10uM size standards (10 um polystyrene beads for MFI system suitability Duke Std Cat#4210A) were used. Triplicate analyses for each standard were performed and the average of each standard were within 10% of true/given manufacture value.
[00289] Sample Preparation. Samples were placed in the BSC and allowed to equilibrate to room temperature for about 30 minutes. Using Neptune Barrier Tips (1000uL Barrier Tip, Cat#1000.96N), samples were diluted 1 :1 with MilliQ Water, specifically 190uL MilliQ water + 190uL Samples. All samples were degassed for 20min @ 70cmHg.
[00290] MFI Analysis. Prior to analysis, approximately 10mL of MilliQ water was flushed through the MFI system using a 100uM FlowCell (FlowCell, 100uM, 1.6mm, Silane Coating). For each sample, 350 pL was pipetted and sample-filled pipette was placed in the MFI inlet port. 0.03-0.05 ml_ manual purge (by volume) was performed followed by selecting Optimize illumination” to calibrate and set the background. Analysis was started by selecting “Start analysis”. -A total of 180 ul of sample was analyzed for each measurement. After each measurement, 1-4ml of MilliQ water was flushed through the flow-cell.
[00291] For the example bleow, the levels of subvisible particles (SVPs) were monitored for each of the formulations in Blocks A through G at all of the time points using micro-flow imaging (MFI). Total particle levels are reported, along with a corrected particle concentration once the circular particles likely due to air bubbles and oil droplets are removed. In addition, the levels of subvisible particles in four different size bins ( > 2 pm, > 5 pm, > 10 pm, and > 20 pm) are reported as well. Due to volume limitations, all samples were diluted two-fold to allow better quantitation. Particles > 2 pm in size are rarely associated with protein aggregates, so the focus of subsequent analysis was mostly on the amounts of larger particles and on the corrected total number of particles per ml_. P. Imaged Capillary Isoelectric Focusing (icIEF, ICE)
[00292] Imaged capillary isoelectric focusing was used to quantify the charge distribution for the formulations. The icIEF analysis was performed on aniCE3 Protein Simple system following the procedure described below.
[00293] Materials:
Figure imgf000167_0001
[00294] Working Sample Preparation. Samples were diluted with MilliQ Water to 5 mg/mL with a working volume of 20 uL (e.g. 2.5 uL of 40 mg/mL Sample + 17.5 uL MilliQ Water.
[00295] icIEF Matrix Preparation. The icIEF reagents were removed from 5°C storage and placed at ambient temperatures for 30 minutes. The following icIEF Matrix preparation was prepared for N+1 samples and all preparations were thoroughly mixed.
Figure imgf000167_0002
[00296] icIEF Sample Preparation and icIEF Analysis. For each sample and reference standard, 180uL of iciEF Matrix was added to 20uL of working sample. For blank preparation, 20uL of MilliQ water was combined with 180ul_ of icIEF matrix. Prepared sample was thoroughly mixed and centrifuge for at 3000g for 10-15 seconds. The sample was analyzed by the iCE3 instrument with a Pre-Focus of 1500V for 1 minute and a Focus of 3000V for 15minutes.
[00297] In the formulations evlauted by ICE below, twelve distinct features were observed. The measured relative areas of each of these twelve peaks for each sample are tabulated in the examples below. The average pi values for these twelve peaks within each block are determined. From these data, the average pi value for any given sample is calculated. Compared to the values T=0, the differences for each of the stored samples were calculated. These values are quite small, but fairly reproducible. This was the basis for the PLS model to evluate chemical stability of formulations.
Q. Shipment Test
[00298] To evaluate the physical stability of aflibercept in formulations, a shipment test is performed. Formulations are filled into containers (e.g. a syringe) then packed into cardboard containers separated by corrugated cardboard. The containers are then shipped at least 1 ,000 miles by truck over paved roads. During shipment, the containers are kept at a constant temperature or within a suitable temperature range (e.g. +/- 10% of target temperature). The temperature may be 25°C, 2-8°C, 0°C, or freezing (i.e. below 0°C). After reaching their destination the formulations in the containers are inspected for particles (e.g. visible particles and sub-visible particles).
R. Drop Test
[00299] To evaluate the physical stability of aflibercept in formulations, a drop test is performed. Formulations are filled into containers (e.g. a syringe) then packed into cardboard containers separated by corrugated cardboard. The containers are then subjected to a dropping test. The dropping test may be conducted at 2-8°C or around room temperature (e.g. 25 °C). For the dropping test, the container is dropped in various orientations. In one example of a dropping test, a cardboard container holding a pre-filled syringe containing a formulation is dropped a total of 16 times from a height of 36 inches. The 16 drops include: 1) dropping the container on each of its 6 faces; 2) dropping the container on each of the 4 edges with the needle (or needle connector) pointing in the horizontal position (i.e. perpendicular to surface the container is being dropped on); 3) dropping the container on 1 edge with the needle pointing upward; 4) dropping the container on 1 edge with the needle pointing downward; 5) dropping the container on 2 separate corners with the needle generally pointing downward; and 6) dropping the container on 2 separate corners with the needle generally pointing upward. Following the dropping test the formulations in the containers are inspected for particles (e.g. visible particles and sub-visible particles).
S. Multivariate Statistical Modeling: Partial Least-Squares (PLS)
[00300] Partial least squares regression (PLS) was used to shed additional light on the effects of the various formulation parameters on stability of the formulations. For any large matrix of values, where there are a reasonable number of samples (together forming the so-called X-matrix), mathematical models can be constructed that explain the largest amount of variance in the dependent variable(s) of interest (the Y-matrix). The best single description of the relationship between the variation in the X-matrix and the endpoint (the Y matrix) is called the first principal component, PC1 . The next important (in terms of describing the variance in the Y-matrix) component is called the second principal component, PC2, and so on. Quite often, only one or two PCs are required to explain most of the variance in the Y-matrix. Each of these PCs contains some contribution from each of the variables in the X-matrix. If a variable within the X- matrix contributes heavily to the construction of a given PC, then it is ranked as being significant. In fact, regression coefficients can be calculated for each variable in the X-matrix for a given model, where a model is the composite of a certain number of PCs in order to provide an adequate description of the Y-matrix. In summary, PLS takes information from the X- matrix, calculates the desired number of PCs, and constructs a suitable model. The model that includes all of the samples is termed a calibration model. The overall coefficient of determination (r2) indicates the quality of the model. All PLS calculations were conducted using Unscrambler® software (CAMO, Corvallis, OR). A PLS analysis done with a single variable in the Y-matrix is termed PLS1 analysis. Building a model that fits multiple variables in the Y-matrix is called PLS2 analysis.
[00301] A full cross validation was performed on all calibration models using standard techniques. Briefly, one sample is removed at a time, the data set is recalibrated, and a new model is constructed. This process is repeated until all of the calibration samples are removed once and quantified as a validation model. Therefore, the first set, containing all samples is referred to as the calibration set and the one after cross-validation as the validation set. The jack-knife algorithm was used to determine statistical significance for any factor used in constructing the PLS models described above.
Stability Testing Conditions
[00302] Unless otherwise indicated, formulations may be maintained at an indicated temperature, e.g., 40° C, 25° C, 2-8° C, or a freezing temperature (e.g. 0 °C and below) for a period of time, e.g., days, weeks, or months, and assessed for stability and/or other parameters. For example, the pH, conductivity and/or viscosity of the samples may be measured at time zero and periodically throughout the experiment. The material may be analyzed for stability and other properties using, e.g., chromatography or other methods; non-limiting examples include SEC, RP, HIC, CEX, AF4 and/or CE-SDS. These analyses are typically carried out at time zero and then periodically during the experiment.
[00303] The samples may also be assessed by starting the experiment at one temperature, e.g., 40° C, and then stepping or ramping to a different temperature, e.g., 25° C, and/or 2-8° C, for a selected period of time. For example, the samples may be stored at -20° C or 2 months; and/or at 40° C for 1 and 2 weeks; and/or at 25° C for 2 and 4 weeks, and/or at 2-8° C for 2 months. A drop test can be carried out at 25° C.
Example 1 - Preparation of Aflibercept
[00304] Aflibercept may be prepared either de novo or through purification/isolation from a commercially available source. The steps below describe production of aflibercept by culturing cells transformed or transfected with a vector containing aflibercept nucleic acid. Alternative methods, which are well known in the art, may also be employed to prepare aflibercept. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et ai, Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of aflibercept may be chemically synthesized separately and combined using chemical or enzymatic methods to produce aflibercept.
A. Recombinant Production
[00305] DNA encoding aflibercept is inserted into an expression vector appropriate for the host cell to express aflibercept.
[00306] Host cells transfected or transformed with expression or cloning vectors described herein for aflibercept production is cultured in nutrient media modified as appropriate for inducing promoters, selecting transformants, and/or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, are selected by the skilled artisan using well-known approaches without undue experimentation. For example, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrookef a/., supra.
[00307] Methods of eukaryotic cell transfection and prokaryotic cell transformation, which means introduction of DNA into the host so that the DNA is replicable, either as an extrachromosomal or by chromosomal integrant, are known to the ordinarily skilled artisan, for example, CaCh, CaPC>4, liposome-mediated, polyethylene-gycol/DMSO and electroporation. [00308] Aflibercept may be expressed in a mammalian cell line, such as a CHO cell and may be modified post-translationally.
[00309] Aflibercept is recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g Triton-X 100) or by enzymatic cleavage. Cells employed in expression of aflibercept can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
[00310] It may be desired to purify aflibercept from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation- exchange resin such as DEAE; chromatofocusing; SD S-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982).
[00311] The aflibercept composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.
[00312] Following any preliminary purification step(s), the mixture comprising aflibercept and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5. The HIC may be performed at low salt concentrations (e.g., from about 0-0.25M salt).
B. Purifying Aflibercept from a Commercially-available Formulation
[00313] Alternatively, aflibercept is purified from a commercially available preparation such as Eylea® or Zaltrap®. Aflibercept is purified away from other formulation components in Eylea® or Zaltrap® using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, and/or any other applicable purification techniques, including but not limited to Protein A chromatography, fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.
[00314] In another alternative, compositions of the present invention are prepared by buffer exchange. Here, using techniques known to one of skill in the art, commercially available Eylea® or Zaltrap® is subjected to a buffer exchange wherein the ingredients, other than aflibercept, of the Eylea® or Zaltrap® formulation are removed and replaced with the ingredients of a formulation of the present invention.
Example 2 - Effects of pH and Buffers on Aflibercept Formulations
[00315] This example describes experiments to evaluate the effects of various pH levels and different buffering agents on aflibercept formulations (Block A). Block A formulations are listed in Table 2.
Table 2.
Figure imgf000173_0001
Concentration, pH, and Osmolality
[00316] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block A formulations were evaluated at to. Results are reported in Table 3 below.
Table 3.
Figure imgf000174_0001
Size Exclusion Chromatography
[00317] The stability of the Block A formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 4-7 below.
T able 4. SEC results for Block A formulations at T =0
Figure imgf000174_0002
Figure imgf000175_0001
Table 5. SEC results for Block A formulations after one week at 40° C
Figure imgf000175_0002
T able 6. SEC results for Block A formulations two weeks at 25° C
Figure imgf000175_0003
Figure imgf000176_0001
T able 7. SEC results for Block A formulations after four weeks at 5° C
Figure imgf000176_0002
[00318] Initially, at to, the monomer content by SEC was near 99% for these formulations. After one week at 40 °C, most formulations have monomer contents that have decreased to about 97%, although a couple (A- 2, A-17) are much less stable. Virtually all of the loss of monomer is due to aggregation (increases in higher molecular weight (HMW) pre-peaks) and none due to fragmentation, as evidenced by the lack of peaks after the main peak. A formulation consisting of the current Eylea formulation was also included in this block.
[00319] Likewise, loss at 25°C is due to aggregation, indicating that this is the primary degradation pathway that will limit shelf-life. Under these conditions (T=2 weeks, 25° C), almost all of the formulations retained > 98.5 % monomer. Another set of samples was held at 5° C for four weeks. These samples showed little loss of monomer, with slightly larger losses at the high pH conditions.
Micro-Flow Imaging
[00320] The stability, SVP levels by MFI, of the Block A formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. A few of the Block A samples appear to exhibit elevated particle counts, as noted below. Note that these formulations only contained buffer and no other stabilizers or tonicity modifiers. So, despite being frozen and shipped on dry ice, some damage may have occurred during sample preparation and shipping. Results are reported in Table 8-11 below.
Table 8. MFI results for Block A formulations at T=0
Figure imgf000177_0001
Figure imgf000178_0001
Note: (1) Schlieren Lines in middle during purge,
(2) Faint Schlieren line during analysis (Top of screen)- MVSS Not detected
(3) High Particle counts
(4) High Particle counts n.a. -not applicable
Table 9. MFI results for Block A formulations stored for one week at 40° C
Figure imgf000178_0002
Note: (1) Faint Schlieren line during analysis (Top of screen)- MVSS Not detected
(2) High Particle counts
(3) Faint Schlieren line during analysis (middle of screen)- MVSS Not detected
(4) Need time stamp after start after 26% Table 10. MFI results for Block A formulations stored for two weeks at 25° C
Figure imgf000179_0001
Note: (1) Schlieren Lines in middle during purge
(2) Faint Schlieren line during analysis (Top of screen)- MVSS Not detected
(3) Faint Schlieren line during analysis (Top of screen)- MVSS Not detected
(4) Faint Schlieren line during analysis (middle of screen)- MVSS Not detected
Table 11. MFI results for Block A formulations stored for four weeks at 5° C
Figure imgf000179_0002
Figure imgf000180_0001
Note: (1) Faint Schlieren line during analysis (Top of screen)- MVSS Not detected
(2) Faint Schlieren line during analysis (middle of screen)- MVSS Not detected
Imaging Capillary Isoelectric Focusing (icIEF)
[00321] The chemical stability of the Block A formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. The relative areas for each of the twelve features detected by icIEF are listed for samples from Block A. These samples were frozen and shipped before analysis. Results are reported in Table 12-15 below.
Table 12. icIEF results for Block A formulations at T=0
Figure imgf000180_0002
Table 13. icIEF results for Block A formulations after storage for one week at 40° C
Figure imgf000181_0001
Table 14. icIEF results for Block A formulations after storage for two weeks at 25° C
Figure imgf000181_0002
Table 15. icIEF results for Block A formulations after storage for four weeks at 5° C
Figure imgf000182_0001
Discussion
[00322] The aflibercept formulations of Block A are physically and chemically stable.
[00323] In addition to the formulations listed in Block A, an aflibercept formulation may have a succinate buffer or a tartrate buffer at a pH of 3.2-5.2 or 2.0-5.4, respectively. Thus, a stable formulation for aflibercept can include these buffers within the pH range. Further, an aflibercept formulation with a buffer may further include an excipient such as a stabilizer or a tonicity modifier. An exemplary aflibercept formulation includes a buffer, sucrose, sodium chloride, and a surfactant.
Example 3 - Buffer-Free Aflibercept Formulations
[00324] This example describes experiments to evaluate aflibercept formulations that are buffer-free (Block B). The target pH ranged from 5.5 to 7.0 and different levels of co-solvents (e.g. surfactants or water-miscible nonaqueous solvents) were included. Table 16, below, shows an exemplary set of such formulations. Table 16
Figure imgf000183_0001
Concentration, pH, and Osmolality
[00325] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block B formulations were evaluated at to. Results are reported in Table 17 below.
Table 17.
Figure imgf000183_0002
Size Exclusion Chromatography
[00326] The stability of the Block B formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Table 18-21 below.
Table 18. SEC results for Block B formulations at T=0
Figure imgf000184_0001
Table 19. SEC results for Block B formulations after one week at 40° C
Figure imgf000184_0002
Figure imgf000185_0001
Table 20. SEC results for Block B formulations two weeks at 25° C
Figure imgf000185_0002
Table 21. SEC results for Block B formulations after four weeks at 5° C
Figure imgf000185_0003
Figure imgf000186_0001
[00327] The initial monomer contents were 98-99%. After storage for one week at 40° C, monomer contents decreased to about 97%, with small differences being apparent. The losses were entirely due to aggregation and not due to fragmentation, again emphasizing the nature of the primary degradation pathway.
[00328] After two weeks at 25° C, the monomer contents were mostly near 98%. The two nominal pH 7 formulations showed the lowest levels of monomer. For samples stored at 5° C for four weeks, the losses were similar to those seen after two weeks at 25° C.
Micro-Flow Imaging
[00329] The stability of the Block B formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. Block B samples, along with those in Block C were shipped on blue ice. This means that they were exposed to interfacial stress during the shipping process. While Block B formulation do not contain a buffer, all of the formulations contain some co-solvent, usually a surfactant, which appears to have provided some level of protection. A couple formulations (e.g., B-10 and B-14) did display elevated levels of SVPs, likely due to shipping stress. Results are reported in Table 22-25 below.
Table 22. MFI results for Block B formulations at T=0
Figure imgf000186_0002
Figure imgf000187_0001
Note: (1) Schlieren Line
(2) transparent particles, more particles — Possible Contamination
(3) Time stamp Applied
(4) High count-possible contamination n.a.- not applicable
Table 23. MFI results for Block B formulations stored for one week at 40° C
Figure imgf000187_0002
Note: (1) Schlieren Line/ Time Stamp Applied
(2) transparent particles, more particles
(3) Stop run at 86% Table 24. MFI results for Block B formulations stored for two weeks at 25° C
Figure imgf000188_0001
Note: (1) Schlieren Line
(2) transparent particles, more particles
(3) Time stamp Applied
(4) High Count- Possible contamination
Table 25. MFI results for Block B formulations stored for four weeks at 5° C
Figure imgf000188_0002
Figure imgf000189_0001
Note: (1) Schlieren lines observed
(2) Stop run at 86%
(3) Time stamp Applied
(4) Stop run at 60%
Imaging Capillary Isoelectric Focusing (icIEF)
[00330] The chemical stability of the Block B formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. Results are reported in Table 26-29 below.
Table 26. icIEF results for Block B formulations at T=0
Figure imgf000189_0002
Table 27. icIEF results for Block B formulations after storage for one week at 40° C
Figure imgf000189_0003
Figure imgf000190_0001
Table 28. icIEF results for Block B formulations after storage for two weeks at 25° C
Figure imgf000190_0002
Table 29. icIEF results for Block B formulations after storage forfourweeks at 5° C
Figure imgf000190_0003
Discussion
[00331] The aflibercept formulations of Block B are physically and chemically stable.
[00332] In addition to the formulations listed in Block B, an aflibercept formulation may include additional excipients, including but not limited to, sorbitol and/or mannitol.
Example 4 -Aflibercept Formulations Free of Organic Co-Solvents
[00333] This example describes experiments to evaluate aflibercept formulations that are free of an organic co-solvent (Block C). The design of Block C centered on removing the co-solvents (e.g., PS 20, PEG 3350) used in Block B, while using buffers, such as acetate, histidine (His), and phosphate across the same pH range of 5.5 to 7.0. Table 30, below, shows an exemplary set of such formulations.
Table 30.
Figure imgf000191_0001
Concentration, pH, and Osmolality
[00334] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block C formulations were evaluated at to. Results are reported in Table 31 below.
Table 31.
Figure imgf000192_0001
Size Exclusion Chromatography
[00335] The stability of the Block C formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 32-35 below.
Table 32. SEC results for Block C formulations at T=0
Figure imgf000192_0002
Figure imgf000193_0001
Table 33. SEC results for Block C formulations after one week at 40° C
Figure imgf000193_0002
Table 34. SEC results for Block C formulations two weeks at 25° C
Figure imgf000193_0003
Figure imgf000194_0001
Table 35. SEC results for Block C formulations after four weeks at 5° C
Figure imgf000194_0002
[00336] At T=0, comparable monomer contents were observed for all formulations, but the T =0 sample for C-15 was lost.
[00337] After one week at 40° C, for all but one formulation (C-6, which does not cotain a sugar) the monomer contents only decreased to 97-98%, suggesting that sucrose was a potent stabilizer even in the absence of co solvents across the entire pH range. Similar behavior was seen for the T=2 weeks/25° C samples, with the pH 7 formulations showing the largest decrease in monomer content.
[00338] After four weeks at 5° C, there was little loss in monomer by SEC across nearly all of the formulations. However, in these samples, this is the first time that some measurable amount of fragmentation was observed. This may be due to the quality of the SEC columns, which were found to degrade relatively quickly with these multiple formulations of aflibercept, suggesting that it was mostly due to the inherent properties of the protein. Micro-Flow Imaging
[00339] The stability of the Block C formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. A number of the Block C samples were damaged due to shipping at a refrigerated temperature (5°C), as they lacked co-solvents (especially surfactants). Most likely, the interfacial stress of agitation with a head space on blue ice led to the dramatic increases in particle counts, especially for formulations C-3 through C-10. Interestingly, there was almost no damage at the pH extremes of 5.5 (formulations C-1 and C-2) and pH 7.0 (formulations C-11) and C-12). It is quite possible that the colloidal stability is improved at these pH conditions, thereby modulating interfacial stress. Results are reported in Table 36-39 below.
Table 36. MFI results for Block C formulations at T=0
Figure imgf000195_0001
Note:
(1) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(2) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles)-MFI stop counting at 29% completion due max counting limit reached (3)
(3) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 68% completion due max counting limit reached
(4) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(5) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 29% completion due max counting limit reached
(6) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 80% completion due max counting limit reached
(7) Possible Contamination- High Particle Count (Semi transparent irreg. particles) // Schlieren Line-at bottom throughout analysis (8) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 56% completion due max counting limit reached
(9) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(10) Possible Contamination- High Particle Count (Semi transparent irreg. particles) n.a.- not applicable
Table 37. MFI results for Block C formulations stored for one week at 40° C
Figure imgf000196_0001
Note:
(1) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(2) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particle s)-MFI stop counting at 68% completion due max counting limit reached
(3) Possible Contamination- High Particle Count (Semi transparent irreg. particles
(4) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particle s)-MFI stop counting at 54% completion due max counting limit reached
(5) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(6) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(7) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particle s)-MFI stop counting at 61% completion due max counting limit reached
(8) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
Table 38. MFI results for Block C formulations stored for two weeks at 25° C
Figure imgf000196_0002
Figure imgf000197_0001
(1) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(2) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 71% completion due max counting limit reached
(3) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(4) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(5) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 31% completion due max counting limit reached
(6) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(7) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(8) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
Table 39. MFI results for Block C formulations stored for four weeks at 5° C
Figure imgf000197_0002
Note:
(1) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(2) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(3) Possible Contamination- High Particle Count (Semi transparent irreg. particles)
(4) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 55% completion due max counting limit reached
(5) Possible Contamination- >1,000,000 Particle Count (Semi transparent irreg. particles) -MFI stop counting at 40% completion due max counting limit reached Imaging Capillary Isoelectric Focusing (icIEF)
[00340] The chemical stability of the Block C formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were shipped on blue ice pior to analysis. Results are reported in Tables 40-43 below.
Table 40. icIEF results for Block C formulations at T=0
Figure imgf000198_0001
Table 41. icIEF results for Block C formulations after storage for one week at 40° C
Figure imgf000198_0002
Table 42. icIEF results for Block C formulations after storage for two weeks at 25° C
Figure imgf000199_0001
Table 43. icIEF results for Block C formulations after storage for four weeks at 5° C
Figure imgf000199_0002
Discussion
[00341] The aflibercept formulations of Block C are physically and chemically stable. Example 5 - Aflibercept Formulations Free of Organic Co-Solvents with
Alternative Stabilizers
[00342] This example describes experiments to evaluate buffer-free aflibercept formulations that have a stabilizer other than sucrose such as amino acids (Gly, Pro, ArgHCI) as well as electrolytes, such NaCI and MgCh. (Block D). In these samples, no surfactant or co-solvent was included. In addition, dextran, a large polymer was investigated. Table 44, below, shows an exemplary set of such formulations.
Table 44. All formulations include 10 mM His buffer
Figure imgf000200_0001
Concentration, pH, and Osmolality
[00343] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block D formulations were evaluated at to. Results are reported in Table 45 below.
Table 45.
Figure imgf000200_0002
Figure imgf000201_0001
Size Exclusion Chromatography
[00344] The stability of the Block D formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 46-49 below.
Table 46. SEC results for Block D formulations at T=0
Figure imgf000201_0002
Table 47. SEC results for Block D formulations after one week at 40° C
Figure imgf000201_0003
Figure imgf000202_0001
Table 48. SEC results for Block D formulations two weeks at 25° C
Figure imgf000202_0002
Table 49. SEC results for Block D formulations after four weeks at 5° C
Figure imgf000202_0003
[00345] All of these formulations exhibit monomer contents near 99% at TO, as was seen with other blocks.
[00346] After one week at 40° C, the monomer content decreased to 97- 98%, similar to what was observed for previous blocks. No fragmentation is noted and the losses are solely due to aggregation. At 25° C storage there is only slight changes from the initial levels, suggesting that these types of excipients (amino acids, salts) are good stabilizers for aflibercept. For samples stored at 5° C for four weeks, little degradation is seen as well.
Micro-Flow Imaging
[00347] The stability of the Block D formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. These samples were shipped frozen for MFI analysis and seem to have survived the shipping without any significant damage. Results are reported in Tables 50-53 below.
Table 50. MFI results for Block D formulations at T=0
Figure imgf000203_0001
Table 51. MFI results for Block D formulations stored for one week at 40° C
Figure imgf000204_0001
Table 52. MFI results for Block D formulations stored for two weeks at 25° C
Figure imgf000204_0002
Table 53. MFI results for Block D formulations stored for four weeks at 5° C
Figure imgf000204_0003
Figure imgf000205_0001
Note:
(1) Need Time stamp, 90%- insufficent sample caused bottom right screen schlieren lines due to illumination error
(2) Need Time stamp, 80%- insufficent sample caused bottom right screen schlieren lines due to illumination error
Imaging Capillary Isoelectric Focusing (icIEF)
[00348] The chemical stability of the Block D formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Table 54-57 below.
Table 54. icIEF results for Block D formulations at T=0
Figure imgf000205_0002
Table 55. icIEF results for Block D formulations after storage for one week at 40° C
Figure imgf000206_0001
Table 56. icIEF results for Block D formulations after storage for two weeks at 25° C
Figure imgf000206_0002
Table 57. icIEF results for Block D formulations after storage for four weeks at 5° C
Figure imgf000206_0003
Figure imgf000207_0001
Discussion
[00349] The aflibercept formulations of Block D are physically and chemically stable.
[00350] It is further contemplated that in the formulations of Block D, phosphate could be used instead of histidine buffer. It is also contemplated that CaC could be used instead of, or in addition to, MgCh.
Example 6 - Buffer-Free Aflibercept Formulations with Alternative
Stabilizers
[00351] This example describes experiments to evaluate buffer-free aflibercept formulations that have a stabilizer other than sucrose (Block E). The design mirrored Block D, except all of these compositions include 0.03% PS 20. This will allow for a direct and detailed examination of the importance PS 20 (and co-solvents in general) for their impact on storage stability. Table 58, below, shows an exemplary set of such formulations.
Table 58. All formulations include 0.03% PS 20.
Figure imgf000207_0002
Concentration, pH, and Osmolality
[00352] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block E formulations were evaluated at to. Results are reported in Table 59 below.
Table 59.
Figure imgf000208_0001
Size Exclusion Chromatography
[00353] The stability of the Block E formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Table 60-63 below.
Table 60. SEC results for Block E formulations at T=0
Figure imgf000208_0002
Figure imgf000209_0001
Table 61. SEC results for Block E formulations after one week at 40° C
Figure imgf000209_0002
Table 62. SEC results for Block E formulations two weeks at 25° C
Figure imgf000209_0003
Table 63. SEC results for Block E formulations after four weeks at 5° C
Figure imgf000209_0004
Figure imgf000210_0001
[00354] As with nearly all of the previous samples, aggregation is the dominant degradation pathway and no fragmentation is observed.
[00355] The SEC results for Block E are comparable to those in Block D, suggesting that PS 20 has only a modest effect on storage stability. The levels of aggregates seem to be slightly higher than for Block D when stored at 5 °C, but these differences may be within the error of the SEC method. Micro-Flow Imaging
[00356] The stability of the Block E formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. As with Block D samples, they were shipped for MFI analysis frozen on dry ice. Results are reported in Table 64-67 below.
Table 64. MFI results for Block E formulations at T=0
Figure imgf000210_0002
Table 65. MFI results for Block E formulations stored for one week at 40° C
Figure imgf000211_0001
Note:
(1) High Particle Count, semi transparent particles
(2) High Particle Count, semi transparent particles
(3) High Particle Count, semi transparent particles
Table 66. MFI results for Block E formulations stored for two weeks at 25° C
Figure imgf000211_0002
Note:
(1) High Particle Count, semi transparent particles Table 67. MFI results for Block E formulations stored for four weeks at 5° C
Figure imgf000212_0001
Note:
(1) Need Time stamp, 79%- insufficent sample caused bottom right screen Schlieren lines due to illumination error and bubble in beginning-
Imaging Capillary Isoelectric Focusing (icIEF)
[00357] The chemical stability of the Block E formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Tables 68-71 below.
Table 68. icIEF results for Block E formulations at T=0
Figure imgf000212_0002
Table 69. icIEF results for Block E formulations after storage for one week at 40° C
Figure imgf000213_0001
Table 70. icIEF results for Block E formulations after storage for two weeks at 25° C
Figure imgf000213_0002
Table 71. icIEF results for Block E formulations after storage for four weeks at 5° C
Figure imgf000213_0003
Discussion
[00358] The aflibercept formulations of Block E are physically and chemically stable.
Example 7 - Aflibercept Formulations Free of Both Buffers and Organic
Co-Solvents
[00359] This example describes experiments to evaluate aflibercept formulations that are free of a buffer and free of an organic co-solvent (Block F). Additionally, it was intedned to to investigate the behavior of mixtures of sucrose and NaCI across the same pH range of 5.5 to 7.0 in terms of aflibercept stability. Table 72, below, shows an exemplary set of such formulations.
Table 72.
Figure imgf000214_0001
Concentration, pH, and Osmolality
[00360] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block F formulations were evaluated at to. Results are reported in Table 73 below. Table 73.
Figure imgf000215_0001
Size Exclusion Chromatography
[00361] The stability of the Block F formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 74-77 below.
Table 74. SEC results for Block F formulations at T=0
Figure imgf000215_0002
Figure imgf000216_0001
T able 75. SEC results for Block F formulations after one week at 40° C
Figure imgf000216_0002
Table 76. SEC results for Block F formulations two weeks at 25° C
Figure imgf000216_0003
Table 77. SEC results for Block F formulations after four weeks at 5° C
Figure imgf000217_0001
[00362] The initial monomer levels were near 99, as it was for the other blocks. At 40° C, some of the formulations show somewhat larger increases in aggregation, but these are the formulations containing only NaCI.
[00363] After two weeks at 25° C, the losses were again fairly minimal, as was observed for the previous blocks. Slightly lower monomer levels were seen near pH 7. At 5°C, the aggregate contents were about 1 %, reflecting only sight losses from T=0.
Micro-Flow Imaging
[00364] The stability of the Block F formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. These samples were also shipped frozen on dry ice prior to evaluation. Results are reported in Tables 78-81 below. Table 78. MFI results for Block F formulations at T=0
Figure imgf000218_0001
Note: (1) Due to low sample volume- stopped at 70%
(2) Bubble during illumination»Lowsample volume n.a.- not applicable
Table 79. MFI results for Block F formulations stored for one week at 40° C
Figure imgf000218_0002
Table 80. MFI results for Block F formulations stored for two weeks at 25° C
Figure imgf000219_0001
Note: (1) Schileren Lines observed detected post-MVSS analysis, Time stamp fdter used.
Table 81. MFI results for Block F formulations stored for four weeks at 5° C
Figure imgf000219_0002
Imaging Capillary Isoelectric Focusing (icIEF)
[00365] The chemical stability of the Block F formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Tables 82-85 below.
Table 82. icIEF results for Block F formulations at T=0
Figure imgf000220_0001
Table 83. icIEF results for Block F formulations after storage for one week at 40° C
Figure imgf000220_0002
Table 84. icIEF results for Block F formulations after storage for two weeks at 25° C
Figure imgf000221_0001
Table 85. icIEF results for Block F formulations after storage for four weeks at 5° C
Figure imgf000221_0002
Discussion
[00366] The aflibercept formulations of Block F are physically and chemically stable. Example 8 - Aflibercept Formulations [00367] This example describes experiments to evaluate aflibercept formulations with various excipient combinations (Block G). Formulations in Block G include those that are free of a buffer; free of an organic co-solvent; include a sugar other than sucrose, include a polyol; include a surfactant other than polysorbate 20 (e.g. in buffer-free formulations); include CaCh as a stabilizer; and have a high concentration of aflibercept. Table 86, below, shows an exemplary set of such formulations.
Table 86.
Figure imgf000222_0001
Concentration, pH, and Osmolality
[00368] The aflibercept concentration (mg/mL), pH, and osmolality (mOSm) of the Block G formulations were evaluated at to. Results are reported in Table 87 below.
Table 87.
Figure imgf000223_0001
Size Exclusion Chromatography
[00369] The stability of the Block G formulations was evaluated by measuring the amount of non-degraded aflibercept present in the formulation at time 0 (tO) and after being subjected to storage conditions. Size Exclusion Chromatography was conducted on the formulations and the percentage of protein present in the main peak (non-degraded aflibercept) in each formulation is reported in Tables 88-91 below. Table 88. SEC results for Block G formulations at T=0
Figure imgf000224_0001
Table 89. SEC results for Block G formulations after one week at 40° C
Figure imgf000224_0002
Figure imgf000225_0001
Table 90. SEC results for Block G formulations two weeks at 25° C
Figure imgf000225_0002
Table 91. SEC results for Block G formulations after four weeks at 5° C
Figure imgf000226_0001
[00370] At t=0 monomer contents are near 99%. Upon 40° C storage, the monomer contents varied around 98%. After two weeks at 25° C, there appears some higher level of aggregation at higher protein concentrations. As with other blocks, the losses at 5° C after four weeks are insignificant.
Micro-Flow Imaging
[00371] The stability of the Block G formulations was evaluated by measuring the particles present in the formulation after being subjected to storage conditions. These samples were also shipped frozen on dry ice prior to evaluation. Results are reported in Tables 92-95 below. Table 92. MFI results for Block G formulations at T=0
Figure imgf000227_0001
Note: for most samples non-detected MVSS Schlieren lines were observed for most samples.
High concentration samples (G3 and G6, @ 120mgmL) had detectable shlieren lines which is expected.
Table 93. MFI results for Block G formulations stored for one week at 40° C
Figure imgf000227_0002
Figure imgf000228_0001
Table 94. MFI results for Block G formulations stored for two weeks at 25° C
Figure imgf000228_0002
Table 95. MFI results for Block G formulations stored for four weeks at 5° C
Figure imgf000229_0001
Imaging Capillary Isoelectric Focusing (icIEF)
[00372] The chemical stability of the Block G formulations was evaluated by measuring the charge variants present in the formulation after being subjected to storage conditions. These samples were frozen and shipped on dry ice prior to analysis. Results are reported in Tables 96-99 below.
Table 96. icIEF results for Block G formulations at T=0
Figure imgf000229_0002
Figure imgf000230_0001
Table 97. icIEF results for Block G formulations after storage for one week at 40° C
Figure imgf000230_0002
Table 98. icIEF results for Block G formulations after storage for two weeks at 25° C
Figure imgf000231_0001
Table 99. icIEF results for Block G formulations after storage for four weeks at 5° C
Figure imgf000231_0002
Figure imgf000232_0001
Discussion
[00373] The aflibercept formulations of Block G are physically and chemically stable.
Example 9 - Factors Contributing to SEC Stability at 40°C for 1 Week
[00374] A PLS1 model used the monomer content after storage at 40° C for one week as the endpoint. Two of the 112 formulations (A-2 and F-13) were found to be outliers. The correlation coefficient for the calibration set is 0.89 and the r-value for the validation set is 0.74, which is reasonable for a data set that is this large and diverse. Significant factors were determined to be sucrose, NaCI, PS 20, PEG 3350, His, Gly, MgCh, CaCh, protein, and pH.
[00375] The model predicts that the optimal pH for a buffer-free formulation would be 5.0 to 6.0 (see front edge of surface in Figure 1). The addition of acetate has little impact on stability, except a slight narrowing of the optimal pH range to about 5.0 to 5.5. Adding NaCI has no effect on the pH-acetate profile (Figure 2), suggesting that the degree of ionization (i.e., buffering capacity) is not impacting stability.
[00376] Using His as the buffer, one obtains a broader pH optimum, extending from about 5.0 to about 6.5 (Figure 3). If one fixes the pH at 5.5 in 20 mM His buffer, the effects of NaCI and sucrose can be observed (Figure 4). There is substantial stabilization by sucrose, but only a modest improvement with NaCI.
[00377] The impact of co-solvents is an important consideration, given the overall formulation strategy. Here the effects of PS 20 and PS 80 can be seen (Figure 5). There is a slight stabilization predicted for PS 80, but a slight destabilization for PS 20. On the other hand, PEG 3350 does not contribute to stabilization and is likely destabilizing, while glycerol was found to provide a sizable improvement in storage stability, at least at higher concentrations (Figure 6).
[00378] The effects of amino acids are seen in Figure 7, where glycine (Gly) appears to be a good stabilizer for aflibercept, while the stabilizing effect of proline (Pro) is only slight. Finally, a response surface for trehalose and CaC is present (Figure 8). CaCh was found to be a good stabilizer, even when added at only 10 mM concentration. The trend for trehalose is similar to what was seen for sucrose, suggesting that sugars may be good choices for stabilization of aflibercept, as evidenced by storage at elevated temperature.
[00379] Overall, the PLS analysis of the t1w/40 °C SEC data found that the optimal pH for a buffer-free formulation would be 5.0 to 6.0. In the presence of acetate or His buffer, this narrows to 5.0 to about 5.5. Of the two, His appears to be the best buffer. Of the stabilizers, sucrose appears to be quite effective, but other sugars, like trehalose may work just as well. Amino acids, especially, Gly, could also be effective stabilizers. There is evidence that some of the co-solvents tested (glycerol, polysorbate 80) may improve storage stability. PEG 3350 and PS 20 were actually destabilizing to some degree.
Example 10 - Factors Contributing to SEC Stability at 25°C for 2 Weeks [00380] A PLS1 model used the monomer content by after two weeks at 25° C as the endpoint. All of the 112 formulations were included and the model indicates that the significant factors are sucrose, PEG 3350, His, ArgHCI, CaCh, and pH. The correlation coefficient for the calibration set is 0.82, while the r-value for the validation set is 0.73, so the model quality is similar, but slightly lower than the last model.
[00381] This PLS1 model predicts that the optimal pH range for a buffer- free formulation is from 5.0 to 6.0 (Figure 9). In the presence of 20 mM His, this range expands upwards to 6.8. As with the previous model, the presence of 100 mM NaCI has virtually no effect on the pH-buffer stability profile (Figure 10). Using acetate as the buffer has a more limited effect than when using His (Figure 11), although the slightly larger pH range in the presence of buffer is observed
[00382] The stability in the presence of NaCI is nearly constant until the concncentation exceeds about 100mM or 120 mM (Figure 12). Similarly, stability with sucrose is maintained until greater 180 mM. Both PS 20 and PS 80 are predicted to have a modest stabilization effect on aflibercept during storage at 25° C (Figure 13). On the other hand, PEG 3350 is destabilizing (Figure 14). The amino acid, ArgHCI, displays some ability to stabilize the protein (Figure 14), while the effect of Gly is relatively weak (Figure 15).
Again, CaCh was found to be a good stabilizer during storage. In the last response surface, glycerol was found to be comparable to ArgHCI as a stabilizer (Figure 16), while MgCh was a weaker stabilizer.
[00383] The PLS analysis of the 25° C SEC data indicates that the optimal pH for a buffer-free formulation is between about 5.0 and about 6.0. The range appears to broaden if one uses 20 mM His buffer. His appears to be superior to acetate as a buffer system in this model. Stability is maintained in the presence of sucrose concentrations up to about 180 mM. There appears to be storage stabilization by different co-solvents, like glycerol and polysorbates (but not PEG 3350). Of the amino acids, ArgHCI is the most effective stabilizer. Finally, increased stability is seen with the levels of CaCh evlauted (and higher amounts may also be stabilizing) and with increasing amounts of MgCh.
Example 11 - Factors Contributing to SEC Stability at 5°C for 4 Weeks [00384] A PLS1 model employed the monomer content after storage for four weeks at 5° C (a suitable storage condition of aflibecpt according to the Eylea package insert) as the endpoint. All formulations were included and the significant factors were determined to be sucrose, PS 20, PS 80, PEG 3350, tris, ArgHCI, Pro, MgCh, dextran, and pH. The r-value for the calibration set was 0.88, while the correlation coefficient for the validation set was 0.79.
[00385] The PLS1 model indicates that the optimal pH for a buffer-free formulation is 5.0 to 5.5 under these conditions, with a slightly extended range (maybe to 5.7) in the presence of 20 mM His (Figure 17). The pH-His response surface is expended to remove the clearly unstable conditions > pH 7 (Figure 18). This zoomed in view suggests that the best pH conditions may be < 5.5. With acetate as the buffer, the profile is quite similar to that with His, suggesting that either buffer may be sufficient (Figure 19). The model also predicts tris as having a significant effect, but this is in relation to stabilization at elevated pH, where the stability profile is the poorest (Figure 20). It would not be suitable for a formulation between pH 5 and 6.
[00386] Sucrose is found to be a stabilizer, especially at concentrations between 50-200 mM, while destabilization is predicted for NaCI, especially at concentrations above 50 mM (Figure 21). The effects of the two polysorbates, PS 20 and PS 80, are shown in Figure 22. The range is very small, so effectively neither excipient impacts the stability profile of aflibercept at 5° C. The effect of PEG 3350 is again detrimental to storage stability (Figure 23), while Gly has only a small effect, primarily at concentrations below 200 mM. The effects of other amino acids (Pro, ArgHCI) are similar to what was seen for Gly (Figure 24).
[00387] The storage stability is decreased in the presence of MgCh according this model, while CaCh provide stabilization at 10 mM (Figure 25), similar to what previous models are forecast. The impact of the co-solvents PG and glycerol are seen in Figure 26. Both were found to increase stability during storage at 5 C. Finally, the effect of protein concentration was examined, as it was marked as being a significant factor. Not surprisingly, the stability decreases slightly at higher protein concentrations (Figure 27), which would be expected for an aggregation process. However, the losses are quite small.
[00388] Overall, the PLS1 model for the 5°C SEC data on storage stability find the best stability is < pH 6.0, preferably below 5.5. Both His and acetate appears to be good buffer choices. Sucrose is an effective stabilizer, while salts and amino acids contribute less to stability. The one ionic compound that appears to be of value in stabilizing the protein is CaCh. Most of the co-solvents tested appear to provide stabilization, except PEG 3350, which is destabilizing, and polysorbates that have little effect.
Example 12 - Factors Contributing to Particle Stability [00389] A PLS2 model was constructed using the total particles concentration (counts per ml_) at all three time points (t1w/40° C, t2w/25° C, t4w/5° C). All of the formulations were considered, but a number were identified as outliers, especially within Block C, where many of the samples were damaged due to agitation upon shipping on blue ice. The model has some scatter, with a correlation coefficient of 0.74 for the calibration set and 0.56 for the validation set, but it still shows a clear trend of particle levels as a function of formulation composition.
[00390] The response surface for pH and His shows a clear minimum corresponding to the optimal pH of about 6.2 for minimizing particle formation (Figure 28). Addition of His results in a slight increase in particle levels, as is seen for acetate (Figure 29), but the increases are quite small for both buffers. Use of phosphate buffer shows a sizable increase in particles when employed at higher pH conditions (Figure 30), but little, if any effect at lower pH except for shifting the pH optimum towards pH 5.0.
[00391] Addition of both sucrose and NaCI result in modest decreases in the levels of SVPs at pH 6.0 (Figure 31). Reductions in particle counts are even greater when using polysorbate surfactants (PS 20, PS 80) (Figure 32). In general, amino acids were not very effective at reducing the amounts of SVPs. While Gly showed some degree of stabilization, ArgHCI has essentially no effect (Figure 33), while Pro actually increased particle levels (Figure 34). Like its effect on aggregate levels measured by SEC, PEG 3350 reduced particle levels somewhat (Figure 34). The non-ionic, polymeric surfactant Pluronic F-68 (poloxamer 188) also reduced particle levels, while MgCI2 has no impact (Figure 35).
[00392] According to this model, trehalose and glycerol both reduced particle levels somewhat (Figure 36), comparable to what was seen with sucrose and NaCI. By comparison, propylene glycol (PG) did not impact SVP levels, while CaCh did provide some degree of stabilization (Figure 37).
[00393] Overall, the PLS2 model for total particles concentration (counts per ml_) indicates that the optimal pH is near 6.3, although the range is roughly from 5.5. to 7.0. Both acetate and His were seen to increase particle levels slightly, but neither altered the optimal pH significantly. Co solvents/surfactants were mostly effective at lowering SVP levels. PS 20 and PS 80 were more effective than F-68 and glycerol, which were more effective than PEG 3350, which was more effective than PG). Of the amino acids, only Gly showed any appreciable effect on reducing particle formation. Sucrose and trehalose, along with NaCI and CaCh efectively reduced SVP levels to some degree. Other sugars and polyols are expected to have similar stabilizing effects.
Example 13 - Factors Contributing to Chemical Stability [00394] The average measured isoelectric points for each of the 12 peaks in the icIEF data for the formulations in the blocks above is listed in Table 100 below. The measured pi values for each feature were used, along with the relative areas listed above, to calculate the average pi value for each sample. Then, for each stored sample, these were compared to the values at T=0. This was the metric used for the PLS modeling. The best model used data from samples stored at 5° C. In all cases, a number of formulations were marked as outliers..
Table 100.
Figure imgf000237_0001
[00395] A PLS1 model was constructed using the change or difference in average pi value for samples stored at 5° C as the endpoint. The model quality was lower than for the previous models, but the results still are sufficient to allow some indication of the effect of various factors, especially pH and buffers. Likely, these would be the most influential on chemical processes leading to differences in pi value.
[00396] Figure 38 shows the response surface for pH and acetate. A line has been drawn in to mark the line where no change in pi (D) value occurs. In a buffer-free system, the model predicts the optimal pH is close to 5.6. In the presence of 20 mM acetate, this value shifts all the way to 7.4. A similar trend is seen for using citrate buffer (Figure 40). However, with His buffer, the effect of pH on D is evened out, but does not reach 0 at a concentration of 20 mM (Figure 39).
[00397] In the response surfaces of Figures 42-46, the pH is set at 5.6, the point where D is effectively zero. This allows the relative impact of an excipient to be evaluated quantitatively (at least within the error of the model). For sucrose and NaCI, each lead to a slight, but almost imperceptible rise in D (Figure 41). By comparison, both PS 20 and PS 80 exhibit a much bigger effect on D, although they trend in opposite directions (Figure 42). PEG 3350 has an even larger effect on D (Figure 43), indicating that co-solvents, in general, facilitate chemical degradation seen by icIEF. MgCh also leads to a relatively sizable change in D (Figure 43), unlike NaCI. The effect of Pro is nearly identical to that of MgCh (Figure 44), while Gly trends in the opposite direction.
[00398] Glycerol appears to have the smallest effect on D of the co solvents, while CaCl2 has little impact as well (Figure 45), much like the effects seen with sucrose and NaCI. Finally, trehalose appears to have a larger impact on D than sucrose, while ArgHCI has an effect much like that of Pro (Figure 46).
Example 14 - Discussion of Formulations and Stability [00399] The stability of the formulations from the blocks were elvauated.
Physical Stability by Size Exclusion Chromatography [00400] An examination of the most stable formulations after one week at 40° C shows that 49 of the 110 novel formulations surpassed the stability exhibited on average by the Eylea formulation. Among these, the two most stable (F-2 and F-4) were buffer-free and co-solvent-free. Formulation B-2 is buffer-free, while formulations D-1 and D-2 were co-solvent-free. This summary indicates that both general strategies for obtaining a stable, formulation are viable, at least in terms of storage stability. Most of these formulations are based on sucrose, but Gly and Pro both were found to provide significant stabilization.
T able 101. List of fifteen most stable formulations after one week at 40° C based on monomer content by SEC
Figure imgf000239_0001
Table 102. List of fifteen most stable formulations after four weeks at 5° C based on monomer content by SEC
Figure imgf000239_0002
Figure imgf000240_0001
[00401 ] When one considers 5° C storage for four weeks, 47 of the 110 formulations outperform the average of the two Eylea compositions. Interestingly, two of the most stable at 5° C (F-1 and F-2) were also one of the most stable at 40° C. Inlcuded in this list, are seven formulations that are buffer-free and co-solvent-free. The optimal pH is mostly 6.0 to 6.3 in these preparations. Again, sucrose is a good stabilizer, although the addition of 10 mM CaC appears to be beneficial. On this list, formulations from Block A were not listed, as they contained no tonicity modifiers, but many of them exhibited relatively high stability indicating that an aflibercept formulation can be buffered with one the buffers in Block A.
[00402] The results of storage at 5° C are particularly relevant since the Eylea package insert states ’’EYLEA should be refrigerated at 2°C to 8°C (36°F to 46°F). Do Not Freeze.” Thus, formulations that are stable at 5° C, including a biosimilar of Eylea, are suitable for commercial use.
Discussion
[00403] The resuls of the stability studies show that the pH range at which aflibercept is physically stable also provides chemical stability.
[00404] Seven blocks of formulations, encompassing over 110 discrete formulations of aflibercept were prepared and evaluated for storage stability at various temperatures in comparison with the current Eylea formulation. Many of the new formulations appear to exhibit greater stability than the Eylea composition. Stability was measured using SEC, MFI, and icIEF. The stability as determined by SEC was examined using PLS modeling for each of the storage temperatures. While the outcomes from the three models differed to some degree, they provided a mostly coherent description of the most stable formulations. The optimal pH appears to be between 5.0 and 6.0. [00405] For buffered formulations, both acetate and His appear to be suitable choices for buffers, based on their effects on stability and their buffering capacities in this pH range.
[00406] Sucrose appears to be potent stabilizer, while many of the co solvents tested appear to provide some stabilization as well in terms of recovery of monomer as measured by SEC upon storage. Of the co-solvents, PEG 3350 appears to be the least favorable, while PS 20, PS 80, and glycerol would be the better choices. However, PEG provided stability at lower temperatures (e.g. 5 °C) indicating that it may suitable for aflibercept since Eylea requires storage at 2-8°C. Trehalose may also be a good choice for a stabilizer. Amino acids provide some degree of stabilization, with Gly being the most consistent in terms of stabilization performance. CaCh, at a concentration of 10 mM, appears to provide a measurable improvement in storage stability. Without being limited to any particular theory, this stability is a result of Ca2+ binding to aflibercept and providing conformational stability.
[00407] In terms of subvisible particle formation, the optimal pH range was from 5.5 to 7.0, more preferably at about 6.3. So, the range overlaps with optimal range determed by SEC analysis. Again, for buffered formulations, acetate and His appear to be favorable buffer choices. Co-solvents appear to reduce SVP formation upon storage, along with sucrose, trehalose, NaCI, and CaCI2.
[00408] Chemical stability was assessed using icIEF, where small, but discernible differences in pi were observed upon storage. PLS analysis of these differences produced a poorer quality model, but still allowed one to discern that the optimal pH was near 5.6, at least for a buffer-free system. Inclusion of a buffer has a small effect, but tends to shift the optimal pH upward. Sucrose, NaCI, and CaCh appears to be favorable stabilizers, while glycerol is predicted to be the most effective co-solvent.
[00409] Overall, these studies provide a basis for the conclusion that the formulations of aflibercept disclosed herein will be commercially viable. In addition, these results indicate that an aflibercept formulation that is buffer- free, a co-solvent-free, or both buffer-free and a co-solvent-free is technically achievable.
[00410] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

What is claimed is:
1. An aqueous formulation comprising: a. aflibercept; b. a stabilizer; and c. an organic co-solvent, wherein:
(i) the formulation is free of buffer, and
(ii) the organic co-solvent comprises one or more of a block copolymer, an alkyl saccharide, or FM1000; and/or the stabilizer comprises one or more of trehalose, human serum albumin, Recombumin®, betaine, tyrosine, phenylalanine, or tryptophan.
2. The aqueous formulation of claim 1 , wherein the pH of the formulation is about 5.0 to about 7.0.
3. The aqueous formulation of claim 1 , wherein the pH of the formulation is about 5.0 to about 5.8.
4. The aqueous formulation of claim 1 , wherein the pH of the formulation is about 5.5.
5. The aqueous formulation of claim 1 , wherein the aflibercept is present at a concentration of about 1 mg/mL to about 150 mg/mL.
6. The aqueous formulation of claim 1 , wherein the aflibercept is present at a concentration of about 10 mg/mL to about 80 mg/mL.
7. The aqueous formulation of claim 1 , wherein the aflibercept is present at a concentration of about 40 mg/mL.
8. The aqueous formulation of claim 1 , wherein the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
9. The aqueous formulation of claim 1 , wherein the organic co-solvent is selected from the group consisting of a surfactant, polyethylene glycol (PEG), and propylene glycol.
10. The aqueous formulation of claim 9, wherein the organic co-solvent is a surfactant.
11. The aqueous formulation of claim 10, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, SDS,
DDM and poloxamer 188 (Pluronic F-68).
12. The aqueous formulation of claim 11 , wherein the surfactant is polysorbate 20.
13. The aqueous formulation of claim 1 further comprising a salt.
14. The aqueous formulation of claim 13, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
15. The aqueous formulation of claim 1 , wherein the stabilizer is sucrose.
16. The aqueous formulation of claim 1 , wherein the stabilizer is sucrose and the organic co-solvent is polysorbate 20 and the pH of the formulation is about 5.0 to about 7.0.
17. The aqueous formulation of claim 1 , wherein the stabilizer is an amino acid.
18. The aqueous formulation of claim 17, wherein the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof.
19. The aqueous formulation of claim 17, wherein the amino acid is present at a concentration of about 25 mM to about 300 mM.
20. The aqueous formulation of claim 15, wherein the formulation further comprises a salt selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
21. The aqueous formulation of claim 20, wherein the salt is present at a concentration of about 10 mM to about 100 mM.
22. The aqueous formulation of claim 1 , wherein the formulation is suitable for ophthalmic administration.
23. An aqueous formulation comprising: a. aflibercept; b. a stabilizer; and c. a buffer, wherein:
(i) the formulation is free of an organic co-solvent; and
(ii) the stabilizer comprises one or more of trehalose, human serum albumin, Recombumin®, betaine, tyrosine, phenylalanine, or tryptophan; and/or the buffer comprises imidazole.
24. The aqueous formulation of claim 23, wherein the pH of the formulation is about 5.0 to about 7.0.
25. The aqueous formulation of claim 23, wherein the pH of the formulation is about 5.5 to about 6.3.
26. The aqueous formulation of claim 23, wherein the pH of the formulation is about 6.2.
27. The aqueous formulation of claim 23, wherein the aflibercept is present at a concentration of about 1 mg/mL to about 150 mg/mL.
28. The aqueous formulation of claim 23, wherein the aflibercept is present at a concentration of about 10 mg/mL to about 80 mg/mL.
29. The aqueous formulation of claim 23, wherein the aflibercept is present at a concentration of about 40 mg/mL.
30. The aqueous formulation of claim 23, wherein the stabilizer is selected from the group consisting of amino acids, sugars, polyols, polymers, and combinations thereof.
31. The aqueous formulation of claim 23, wherein the buffer is selected from the group consisting of acetate, histidine, phosphate, citrate, succinate, tartrate and maleate.
32. The aqueous formulation of claim 31 , wherein the buffer is acetate.
33. The aqueous formulation of claim 31 , wherein the buffer is histidine.
34. The aqueous formulation of claim 31 , wherein the buffer is phosphate.
35. The aqueous formulation of claim 23, wherein the buffer is present at a concentration of about 1 mM to 150 mM.
36. The aqueous formulation of claim 35, wherein the buffer is present at a concentration of about 5 mM to 20 mM.
37. The aqueous formulation of claim 36, wherein the buffer is present at a concentration of about 10 mM.
38. The aqueous formulation of claim 23 further comprising a salt.
39. The aqueous formulation of claim 38, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
40. The aqueous formulation of claim 23, wherein the stabilizer is sucrose.
41. The aqueous formulation of claim 23, wherein the stabilizer is sucrose and the buffer is acetate and the pH of the formulation is about 5.5 to about 7.0.
42. The aqueous formulation of claim 23, wherein the stabilizer is sucrose and the buffer is histidine and the pH of the formulation is about 5.5 to about 7.0.
43. The aqueous formulation of claim 23, wherein the stabilizer is sucrose and the buffer is phosphate and the pH of the formulation is about 5.5 to about 7.0.
44. The aqueous formulation of claim 23, wherein the stabilizer is an amino acid.
45. The aqueous formulation of claim 44, wherein the amino acid is selected from the group consisting of glycine, arginine, and proline or combinations thereof.
46. The aqueous formulation of claim 44, wherein the amino acid is present at a concentration of about 25 mM to about 300 mM.
47. The aqueous formulation of any one of claims 40-46, wherein the formulation further comprises a salt.
48. The aqueous formulation of claim 47, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
49. The aqueous formulation of claim 48, wherein the salt is present at a concentration of about 10 mM to about 100 mM.
50. The aqueous formulation of claim 23, wherein the formulation is suitable for ophthalmic administration.
51. An aqueous formulation comprising: a. aflibercept; and b. a stabilizer; wherein:
(i) the formulation is free of an organic co-solvent and is free of a buffer; and
(ii) the stabilizer comprises one or more of trehalose, human serum albumin, Recombumin®, betaine, tyrosine, phenylalanine, or tryptophan.
52. The aqueous formulation of claim 51 , wherein the pH of the formulation is about 5.0 to about 7.0.
53. The aqueous formulation of claim 51 , wherein the pH of the formulation is about 5.0 to about 5.8.
54. The aqueous formulation of claim 51 , wherein the pH of the formulation is about 5.5.
55. The aqueous formulation of claim 51 , wherein the aflibercept is present at a concentration of about 1 mg/mL to about 100 mg/mL.
56. The aqueous formulation of claim 51 , wherein the aflibercept is present at a concentration of 10 mg/mL to about 80 mg/mL.
57. The aqueous formulation of claim 51 , wherein the aflibercept is present at a concentration of about 40 mg/mL.
58. The aqueous formulation of claim 51 further comprising a salt.
59. The aqueous formulation of claim 58, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride and calcium chloride.
60. The aqueous formulation of claim 59, wherein the salt is present at a concentration of 10 mM to about 100 mM.
61. The aqueous formulation of claim 51 , wherein the formulation is suitable for ophthalmic administration.
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