(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`27 December 2007 (27.12.2007)
`
`
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`llllllIllllllllllllllllllllIllllllllllllllllllllllll|||l|llllllllllllllllllllllllllllllllllll
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`(10) International Publication Number
`
`W0 2007/149334 A2
`
`(51) International Patent Classification:
`
`Not classified
`
`(21) International Application Number:
`PCTIUSZDWIDIAIDSS
`
`[22) International Filing Date:
`
`14 Junezom (14.06.2ocrr}
`
`(25) Filing Language:
`
`[26) Publication Language:
`[30) Priority Data:
`5m14‘434
`
`English
`
`English
`
`16 June 2006 (16.05.2006) US
`
`[71) Applicant (for aii designated States except US): REGEN-
`ERON PHARMACEUTICALS, INC. [USJ'US]; 77? Old
`Saw Mill River Road, Tarrytown, NY 10591 (US).
`
`(81) Designated States (tiniest otherwise indicated for every
`kind of nationai protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK. DM, DO, DZ. EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM, GT, I-IN, HR, HU, ID, 11.,
`IN. IS. JP, ICE, KG. KM, KN, KP. KR. KZ, LA, LC. LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`ETEARTN, TR: TT: TZ, UA- UG— US, UZ: VC: VN: ZA~
`
`(84) Dmignatod States (unless otherwise indicated, for every
`kind of regional protection availabie): ARTPO ['BW, GH,
`GM, KE, LS, MW, LIZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW). Eurasian (AM, AZ,BY, KG. KZ. MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NIT, NL, PL,
`
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`[72) Inventors; and
`GA’ GN’ GQ’ GW, ML' MR’ NE’ SN‘ TD' TG)'
`[75) InventorsIAppllcants (for US only): FURFIINE, Eric
`Published:
`[USI'US]; 315 Harrington Avenue, Concord, MA 01742
`{US} DIX9 Daniel films]? 55 Memory Trail, La— — without international search report and to be repubiished
`grangeville, NY 12540 (US). GRAHAM, Kenneth, S.
`upon receipt afflict report
`[USIUS]; 213 RObbom Road. Pleasallt Valley. NY 12569 — with sequence iisting part of description published sepa—
`(US)- FRYE: Kelly [USN-TS]; 29 N— Ridge Road, Pomona,
`rateiy in electmnicform and available upon request frwn
`NY 10970 (US).
`the intemationai Bureau
`
`[‘74) Agent: GREGG, Vuleta; Regeneron Pharmaceuticals,
`Inc., 777 Old Saw Mill River Road, Thn'onwn, NY 10591
`(US).
`
`For ma-iettercades and other abbreviations, rgt'er to the "Guid—
`once Notes on Codes andAbbreviationr" appearing at the begin-
`ning ofeaeh regular issue afthe PCT Gazette,
`
`(54) Title: VEGF ANTAGONIST FORMJLA'ITONS SUITABLE FOR INTRAVITREAL ADMINISTRATION
`
`(57) Abstract: Ophthalmic formulations of a vascular endothelial growth factor (VEGfU-specific fusion protein antagonist are
`provided suitable for intravitreal administration to the eye. The ophthalmic formulations include a stable liquid formulation and a
`lyophilizable formuJation. Preferably, the protein antagonist has the amino acid sequence shown in SEQ 1D N014.
`
`Regeneron Exhibit 1021.001
`
`
`
`2007/149334A2lllllllllllllllllllllllllllIllllIlllllllllllllllllllIllllllllllllll|||||lllllllllllllllllllllll
`
`

`

`WO 2007/149334
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`PCT/U82007/014085
`
`VEGF ANTAGONIST FORMULATIONS SUITABLE
`FOR INTRAVITREAL ADMINISTRATION
`
`BACKGROUND OF INVENTION
`
`Field of the Invention
`
`[0001] The present invention is directed to pharmaceutical formulations suitable for intravitreal
`
`administration comprising agents capable of inhibiting vascular endothelial growth factor
`
`(VEGF), and to methods for making and using such formulations. The invention includes liquid
`
`pharmaceutical formulations having increased stability, as well as formulations that may be
`
`lyophilize and reconstituted for intravitreal administration.
`
`Statement of Related Art
`
`[0002] Vascular endothelial growth factor (VEGF) expression is nearly ubiquitous in human
`
`cancer, consistent with its role as a key mediator of tumor neoangiogenesis. Blockade of VEGF
`
`function, by binding to the molecule or its VEGFR—2 receptor, inhibits growth of implanted tumor
`
`cells in multiple different xenograft models (see, for example, Gerber et al. (2000) Cancer Res.
`
`60:6253-6258). A soluble VEGF-specific fusion protein antagonist, termed a "VEGF trap" has
`
`been described (Kim et al. (2002) Proc. Natl. Acad. Sci. USA 99:11399-404; Holash et al. (2002)
`
`Proc. Natl. Acad. Sci. USA 99:11393-8).
`
`[0003] Ophthalmic formulations are known, see for example. US. 7,033,604 and 6,777,429. An
`
`ophthalmic formulation of a VEGl= antibody is described in US 6,676,941.
`
`[0004] Lyophilization (freeze drying under controlled conditions) is commonly used for'long-term
`
`storage of proteins. The lyophilized protein is substantially resistant to degradation,
`
`aggregation, oxidation, and other degenerative processes while in the freeze-dried state (see,
`
`for example, US. 6.436.897).
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0005] Stable formulations of a VEGF-specific fusion protein antagonist are provided.
`
`Pharmaceutically acceptable formulations are provided that comprise a VEGF “trap” antagonist
`
`with a pharmaceutically acceptable carrier.
`
`In specific embodiments, liquid and lyophilized
`
`formulations are provided.
`
`[0006] in a first aspect, a stable liquid ophthalmic formulation of a VEGF-specific fusion protein
`
`antagonist is provided, comprising a fusion protein that comprises a receptor component
`
`consisting essentially of an immunoglobulin-like (lg) domain 2 of a first VEGF receptor and lg
`
`domain 3 of a second VEGF receptor, and a multimerizing component (also termed a “VEGF
`
`trap").
`
`In a specific embodiment of the VEGF-specific fusion protein antagonist, the first VEGF
`
`receptor is Flt‘l and the second VEGF receptor is Flk1 or Flt4.
`
`In a more specific embodiment
`
`the fusion protein has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. Preferably,
`
`Regeneron Exhibit 1021.002
`
`

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`WO 2007/149334
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`PCT/U52007/014085
`
`the VEGF antagonist is a dimer comprising two fusion proteins of SEQ ID NO:4.
`
`[0007] In one aspect, a stable liquid ophthalmic formulation is provided that comprises 1-100
`
`mglml VEGF-specific fusion protein antagonist, 0.01-5% of one or more organic co-solvent(s).
`
`30-150 mM of one or more tonicity agent(s), 5-40 mM of a buffering agent, and optionally, 1.0-
`
`7.5% of a stabilizing agent, pH between about 5.8-7.0.
`
`[0008]
`
`In one or more specific embodiments, the 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; the tonicity agent may be, for example, sodium
`
`chloride or potassium chloride; the stabilizing agent may be sucrose, sorbitol, glycerol,
`
`trehalose, or mannitol; and the buffering agent may be, for example, phosphate buffer.
`
`In a
`
`specific embodiment, the phosphate buffer is a sodium phosphate buffer.
`
`[0009]
`
`In various embodiments, the organic co—solvent is polysorbate and/or PEG, the
`
`stabilizing agent is sucrose, the buffering agent is phosphate buffer, and the tonicity agent is
`sodium chloride.
`
`[0010] More specifically, the stable liquid ophthalmic formulation comprises about 40-50 mg/ml
`
`of the VEGF antagonist (SEQ ID NO:4), about 10 mM phosphate buffer. 0.01-3% polysorbate
`
`and/or PEG, 40-135 mM sodium chloride, and optionally 5.0% sucrose, pH about 6.2-6.3.
`
`[0011]
`
`In a specific preferred embodiment, the stable liquid ophthalmic formulation comprises
`
`about 50 mglml of the VEGF antagonist (SEQ ID NO:4), 10 mM sodium phosphate buffer, 50
`
`mM sodium chloride, 0.1% polysorbate, and 5% sucrose, pH about 6.2-6.3.
`
`[0012]
`
`In a specific preferred embodiment, the stable liquid ophthalmic formulation comprises
`
`about 50 mg/ml of the VEGF antagonist (SEQ ID NO:4), 10 mM sodium phosphate buffer, 50
`
`mI‘vI sodium chloride, 3% PEG, and 5% sucrose, pH about 6.2-6.3.
`
`[0013]
`
`In a specific preferred embodiment, the stable liquid ophthalmic formulation comprises
`
`about 40 mg/ml of the VEGF antagonist (SEQ ID NO:4), 10 mM sodium phosphate buffer, 40
`
`mM sodium chloride, 0.03% polysorbate, and 5% sucrose, pH about 6.2-6.3.
`
`[0014]
`
`In a specific preferred embodiment, the stable liquid ophthalmic formulation comprises
`
`about 40 mg/ml of the VEGF antagonist (SEQ ID NO:4), 10 mM sodium phosphate buffer, 135
`
`mM sodium chloride, and 0.03% polysorbate, pH about 6.2-6.3.
`
`[0015] In another aspect, a stable liquid ophthalmic formulation is provided that comprises 1-
`
`100 mglml VEGF-specific fusion protein antagonist; 0.01-5% of one or more organic co-
`
`solvent(s); 5—40 mM of a buffering agent; and optionally 30-150 mM of one or more tonicity
`
`agent(s) and/or 1.0—7.5% of a stabilizing agent; having a pH between about 5.8-7.0.
`
`[0016] In various embodiments, the VEGF antagonist (SEQ ID N024) is present at a
`
`concentration of about 10 to about 80 mg/ml.
`
`In various embodiments, the VEGF antagonist
`
`(SEQ ID NO:4) is present at a concentration of about 10, about 20, about 30, about 40, about
`
`50, about 60, about 70, or about 80 mg/ml.
`
`In a preferred embodiment, the VEGF antagonist
`
`Regeneron Exhibit 1021.003
`
`

`

`WO 2007/149334
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`PCT/US2007/014085
`
`(SEQ ID NO:4) is present at a concentration of about 40 mg/mI.
`
`[0017] In another embodiment, the stabilizing agent is selected from one or more of sucrose,
`sorbitol, glycerol, trehalose, and mannitol.
`
`[0018] In another embodiment, the organic co-solvent is selected from one or more of
`
`polysorbate, for example, polysorbate 20 or polysorbate 80, polyethylene glycol (PEG), for
`
`example, PEG 3350, and propylene glycol.
`
`[0019] In another embodiment, the buffer is a phosphate buffer, for example, sodium
`
`phosphate.
`
`[0020] In another embodiment, the tonicity agent is a salt, for example, sodium chloride.
`
`[0021] In one embodiment, the stable liquid ophthalmic formulation comprises 10 mM sodium
`
`phosphate buffer, about 0.03 to about 0.1% polysorbate and/or about 3% PEG or propylene
`
`glycol, about 40 mM sodium chloride, and about 5% sucrose.
`
`In a specific embodiment, the
`
`stable liquid ophthalmic formulation comprises 10 mM sodium phosphate buffer, about 0.03%
`
`polysorbate, about 40 mM sodium chloride, and about 5% sucrose.
`
`In another specific
`
`embodiment, the pH of the formulation is about 6.2 to about 6.3.
`
`In another specific
`
`embodiment, the pH is achieved by mixing mono— and dibasic sodium phosphate to the desired
`
`pH without acid/base titration.
`
`[0022] in a specific embodiment, the stable liquid ophthalmic formulation consists essentially of
`
`a VEGF antagonist (SEQ ID NO:4) at 40 mglml, 10 mM sodium phosphate buffer, polysorbate
`
`at 0.03%, sodium chloride at 40 mM, and sucrose at 5%, pH 6.2-6.3.
`
`[0023] In another aspect, a stable liquid ophthalmic formulation is provided that comprises
`
`about 10 to about 80 mg/ml VEGF antagonist, about 10 mM sodium phosphate buffer, about
`
`0.03% polysorbate, and about 135 mM sodium chloride, pH of 6.2 to 6.3.
`
`[0024] In various embodiments, the VEGF antagonist (SEQ ID N0:4) is present at a
`concentration of about 10 to about 80 mglml.
`In various embodiments, the VEGF antagonist
`
`(SEQ ID N024) is present at a concentration of about 10, about 20, about 30, about 40, about
`
`50, about 60. about 70, or about 80 mg/ml.
`
`In a specific embodiment, the VEGF antagonist
`
`(SEQ ID NO:4) is present at a concentration of about 40 mglml.
`
`[0025] In one embodiment, the stable liquid ophthalmic formulation comprises 40 mg/ml of
`
`VEGF antagonist (SEQ ID N024), 10 mM sodium phosphate buffer, 0.03% polysorbate, and 135
`
`mM sodium chloride at pH 6.2-6.3.
`
`In a specific embodiment, the stable liquid ophthalmic
`
`formulation consists essentially of 40 mg/mI of VEGF antagonist (SEQ ID N014), 10 mM sodium
`
`phosphate buffer, 0.03% polysorbate, and 135 mM sodium chloride at pH 6.2—6.3.
`
`[0026] In another aSpect, a Iyophilizable formulation of a VEGF antagonist is provided, wherein
`
`upon lyophilization followed by reconstitution, a stable liquid ophthalmic formulation as
`described herein is obtained.
`
`[0027] In another aspect, a Iyophilizable formulation of a vascular endothelial growth factor
`
`Regeneron Exhibit 1021.004
`
`

`

`WO 2007/149334
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`PCT/U52007/014085
`
`(VEGF)-specific fusion protein antagonist is provided, comprising 5-50 mglml of the VEGF
`
`antagonist, 5-25 mM buffer, such as phosphate buffer, 0.01 to 0.15% of one or more of an
`
`organic co-solvent. such as polysorbate, propylene glycol and/or PEG, and optionally 1—10% of
`
`a stabilizing agent such as sucrose, sorbitol, trehalose, glycerol, or mannitol, pH about 5.8—7.0.
`
`In various embodiments, the VEGF antagonist (SEQ ID NO:4) is present at about 5. about 10,
`
`about 20, about 30, or about 40 mglml.
`
`In a specific embodiment, the Iyophilizable ophthalmic
`
`formulation of the invention comprises 20 mglml of the VEGF antagonist, 10 mM sodium
`
`phosphate buffer, 0.03% polysorbate, 0.1% PEG, and 2.5% sucrose. pH about 6.2—6.3.
`
`In
`
`further embodiments, the Iyophilizable formulation further comprises sodium chloride.
`
`In a
`
`specific embodiment, the sodium chloride is present at a concentration of about 20 mM.
`
`In
`
`another specific embodiment, the sodium chloride is present at a concentration of about 67.5
`mM.
`
`[0028] In another specific embodiment, the Iyophilizable ophthalmic formulation of the invention
`
`comprises 20 mglml of the VEGF antagonist. 5 mM sodium phosphate buffer, 0.015%
`
`polysorbate, 20 mM sodium chloride, and 2.5% sucrose, pH about 6.2-6.3.
`
`[0029] In another embodiment, the Iyophilizable ophthalmic formulation comprises 5 mglml, 10
`
`mglml, or 40 mglml VEGF antagonist, 5 mM sodium phosphate buffer, 0.015% polysorbate, 20
`
`mM sodium chloride, and 2.5% sucrose, at pH 6.2—6.3.
`
`In a specific embodiment, the
`
`Iyophilizable ophthalmic formulation consists essentially of 5 mglml, 1O mglml, or 40 mglml
`
`VEGF antagonist (SEQ ID NO:4), 5 mM sodium phosphate buffer, 0.015% polysorbate, 20 mM
`
`sodium chloride, and 2.5% sucrose, at pH 6.2-6.3.
`
`[0030] In another specific embodiment, the lyophilizable ophthalmic formulation comprises 20
`
`mglml of the VEGF antagonist. 5 mM sodium phosphate buffer, 0.015% polysorbate, and 67.5
`
`mM sodium chloride, pH about 6.2—6.3.
`
`In a more specific embodiment, the Iyophilizable
`
`ophthalmic formulation consists essentially of 20 mglml of the VEGF antagonist (SEQ ID N014),
`
`5 mM sodium phosphate buffer, 0.015% polysorbate, and 67.5 mM sodium chloride, pH 6.2-6.3.
`
`[0031] In another specific embodiment, the lyophilizable ophthalmic formulation comprises 5
`
`mglml, 10 mglml, or 40 mglml VEGF antagonist, 5 mM sodium phosphate buffer, 0.015%
`
`polysorbate, and 67.5 mM sodium chloride, pH about 6.2-6.3.
`
`In a more specific embodiment,
`
`the Iyophilizable ophthalmic formulation consists essentially of 5 mglml, 10 mglml, or 40 mglml
`
`VEGF antagonist (SEQ ID NO:4), 5 mM sodium phosphate buffer, 0.015% polysorbate, and
`
`67.5 mM sodium chloride, pH about 6.2-6.3.
`
`[0032] Generally. the reconstituted formulation is about 2 times the concentration of the pre-
`
`Iyophilized formulation, e.g., a 20 mg fusion protein/ml pre-Iyophilized formulation is
`
`reconstituted to a final formulation of 40 mg fusion protein/ml.
`
`[0033] Generally, the Iyophilized formulation is reconstituted with sterile water suitable for
`
`injection.
`
`In one embodiment, the reconstitution liquid is bacteriostatic water.
`
`Regeneron Exhibit 1021.005
`
`

`

`WO 2007/149334
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`PCT/USZiNl7/014085
`
`[0034] In another aspect, the invention features a method of producing a lyophilized formulation
`
`of a VEGF-specific fusion protein antagonist, comprising subjecting the lyophilizable formulation
`
`of the invention to lyophilization to generate a lyophilized formulation. The lyophilized
`
`formulation may be lyophilized by any method known in the art for lyophilizing a liquid.
`
`[0035] In another related aspect, the invention features a method of producing a reconstituted
`
`lyophilized formulation of a VEGF antagonist. comprising reconstituting the lyophilized
`formulation of the invention to a reconstituted formulation.
`In one embodiment, the reconstituted
`
`formulation is twice the concentration of the ore—lyophilized formulation. e.g.. the method of the
`
`invention comprises: (a) producing a pre-Iyophilized formulation of a VEGF—specific fusion
`
`protein antagonist, (b) subjecting the pre-lyophilized formulation of step (a) to lyophilization: and
`
`(c) reconstituting the lyophilized formulation of step (b).
`
`[0036] The invention further features ophthalmic formulations provided in a pre—filled syringe or
`
`vial, particularly suitable for intravitreal administration.
`
`[0037] Other objects and advantages will become apparent from a review of the ensuing
`
`detailed description.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0038] The present invention is not limited to particular methods, and experimental conditions
`
`described. as such methods and conditions may vary.
`
`It is also to be understood that the
`
`terminology used herein is for the purpose of describing particular embodiments only, and is not
`
`intended to be limiting unless indicated, since the scope of the present invention will be limited
`
`only by the appended claims.
`
`[0039] Unless stated otherwise, all technical and scientific terms and phrases used herein have
`
`the same meaning as commonly understood by one of ordinary skill in the art to which the
`
`invention belongs. 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 now described.
`
`General Description
`
`[0040] Safe handling and administration of formulations comprising proteins represent
`
`significant challenges to pharmaceutical formulators. Proteins possess unique chemical and
`
`physical properties that present stability problems: a variety of degradation pathways exist for
`
`proteins, implicating both chemical and physical instability. Chemical instability includes
`
`deamination, aggregation. clipping of the peptide backbone, and oxidation of methionine
`
`residues. Physical instability encompasses many phenomena, including, for example,
`
`aggregation and/or precipitation.
`
`[0041] Chemical and physical stability can be promoted by removing water from the protein.
`
`Regeneron Exhibit 1021.006
`
`

`

`W0 2007/149334
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`PCT/U52007/014085
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`Lyophilization (freeze-drying under controlled conditions) is commonly used for long-term
`
`storage of proteins. The lyophilized protein is substantially resistant to degradation,
`
`aggregation, oxidation, and other degenerative processes while in the freeze-dried state. The
`
`lyophilized protein may be reconstituted with water optionally containing a bacteriostatic
`
`preservative (e.g., benzyl alcohol) prior to administration.
`
`Definitions
`
`[0042] The term "carrier" includes a diluent, adjuvant, excipient, or vehicle with which a
`
`composition is administered. Carriers can include sterile liquids, such as, for example, water
`
`and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example.
`
`peanut oil, soybean oil, mineral oil, sesame oil and the like.
`
`[0043] The term “excipient” includes a non-therapeutic agent added to a pharmaceutical
`
`composition to provide a desired consistency or stabilizing effect. Suitable pharmaceutical
`
`exoipiehts include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
`
`silica gel, sodium stearate, glycerol monostearate. talc, sodium chloride, dried skim milk,
`
`glycerol, propylene, glycol. water. ethanol and the like.
`
`[0044] The term “lyophilized” or “freeze—dried" includes a state of a substance that has been
`
`subjected to a drying procedure such as Iyophilization, where at least 90% of moisture has been
`removed.
`
`VEGF Antagonists
`
`[0045] A VEGF antagonist is a compound capable of blocking or inhibiting the biological action
`
`of vascular endothelial growth factor (VEG F), and includes fusion proteins capable of trapping
`
`VEGF.
`
`In a preferred embodiment, the VEGF antagonist is the fusion protein of SEQ ID NO:2
`
`or 4; more preferably, SEQ ID NO:4.
`
`ln specific embodiments. the VEGF antagonist is
`
`expressed in a mammalian cell line such as a CHO cell and may be modified post-
`
`translationally.
`
`In a specific embodiment, the fusion protein comprises amino acids 27-457 of
`
`SEQ ID N04 and is glycosylated at Asn residues 62, 94, 149, 222 and 308. Preferably, the
`
`VEGF antagonist is a dimer composed of two fusion proteins of SEQ ID NO:4.
`
`[0046] The VEGF antagonist of the methods and formulations of the invention can be prepared
`
`by any suitable method known in the art, or that comes to be known. The VEGF antagonist is
`
`preferably substantially free of protein contaminants at the time it is used to prepare the
`
`pharmaceutically acceptable formulation. By "substantially free of protein contaminants" is
`
`meant, preferably, that at least 90 % of the weight of protein of the VEGF-specific fusion protein
`
`antagonist preparation used for making a formulation is VEGF fusion protein antagonist protein,
`
`more preferably at least 95%, most preferably at least 99%. The fusion protein is preferably
`
`substantially free of aggregates. "Substantially free of aggregates" means that at least 90% of
`
`Regeneron Exhibit 1021.007
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`

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`WO 2007/149334
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`PCT/U82007/014085
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`the weight of fusion protein is not present in an aggregate at the time the fusion protein is used
`
`to prepare the pharmaceutically effective formulation. Unless stated otherwise, the phosphates
`
`employed are sodium phosphates and a desired buffering pH is achieved by mixing appropriate
`
`amounts of mono— and dibasic sodium phosphate.
`
`Stable Liquid Ophthalmic Formulations
`
`[0047]
`
`In one aspect, the invention provides a stable pharmaceutically acceptable formulation
`
`comprising a VEGF antagonist, wherein the formulation is a liquid formulation suitable for
`
`ophthalmic use. Preferably, the liquid formulation comprises a pharmaceutically effective
`
`amount of the VEGF antagonist. The formulation can also comprise one or more
`
`pharmaceutically acceptable carriers, buffers, tonicity agents, stabilizers, and/or excipients. An
`
`example of a pharmaceutically acceptable liquid formulation comprises a VEGF antagonist in a
`
`pharmaceutically effective amount, a buffer, an organic co-solvent such as polysorbate, a
`
`tonicity agent such as NaCi, and optionally, a stabilizer such as sucrose or trehalose.
`
`[0048] Stability is determined in a number of ways at specified time points, including
`
`determination of pH, visual inspection of color and appearance. determination of total protein
`
`content by methods known in the art. e.g.. UV spectroscopy, and purity is determined by, for
`
`example, SDS-PAGE, size-exclusion HPLC, bioassay determination of activity, isoelectric
`
`focusing, and isoaspartate quantification.
`
`in one example of a bioassay useful for determining
`
`VEGF antagonist activity, a BAF/3 VEGFR1/EPOR cell line is used to determine VEGF165
`
`binding by the VEGF antagonist of the invention.
`
`[0049] Liquid formulations can be stored in an oxygen-deprived environment. Oxygen—deprived
`
`environments can be generated by storing the formulations under an inert gas such as, for
`
`example, nitrogen or argon. Liquid formulations are preferably stored at about 5°C.
`
`Ophthalmic Lyophilized Formulations
`
`[0050] in one aspect of the invention, an ophthalmically acceptable formulation comprising a
`
`VEGF antagonist is provided, wherein the formulation is a lyophilizable formulation.
`
`Lyophiiizable formulations can be reconstituted into solutions, suspensions, emulsions, or any
`
`other suitable form for administration or use. Lyophilizable formulations are typically first
`
`prepared as liquids, then frozen and lyophilized. The total liquid volume before lyophilization
`
`can be less, equal to, or more than, the final reconstituted volume of the lyophilized formulation.
`
`The lyophilization process is well known to those of ordinary skill in the art, and typically
`includes sublimation of water from a frozen formulation under controlled conditions.
`
`[0051] Lyophilized formulations can be stored at a wide range of temperatures. Lyophilized
`
`formulations may be stored below 25°C, for example, refrigerated at 2-8°C, or at room
`
`temperature (e.g., approximately 25°C). Preferably, lyophilized formulations are stored below
`
`Regeneron Exhibit 1021.008
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`

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`WO 2007/149334
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`PCT/U82007/014085
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`about 25°C, more preferably, at about 4-20°C; below about 4°C; below about -20°C; about -
`
`40°C: about —70°C, or about -80°C. Stability of the lyophilized formulation may be determined in
`
`a number of ways known to the art, for example, by visual appearance of the cake and/or by
`moisture content.
`
`[0052] Lyophilized formulations are typically reconstituted for use by addition of an aqueous
`
`solution to dissolve the lyophilized formulation. A wide variety of aqueous solutions can be used
`
`to reconstitute a lyophilized formulation. Preferably, lyophilized formulations are reconstituted
`
`using water. Lyophilized formulations are preferably reconstituted with a solution consisting
`
`essentially of water (e.g., USP WFI, or water for injection) or bacteriostatic water (e.g., USP WFl
`
`with 0.9% benzyl alcohol). However. solutions comprising buffers and/or excipients and/or one
`
`or more pharmaceutically acceptable carries can also be used.
`
`[0053] Freeze-dried or lyophilized formulations are typically prepared from liquids, that is. from
`
`solutions. suspensions, emulsions, and the like. Thus, the liquid that is to undergo freeze—drying
`
`or lyophilization preferably comprises all components desired in a final reconstituted liquid
`
`formulation. As a result, when reconstituted, the freeze-dried or lyophilized formulation will
`
`render a desired liquid formulation upon reconstitution.
`
`EXAMPLES
`
`[0054] Before the present methods are described, it is to be understood that this invention is
`
`not limited to particular methods, and experimental conditions described, as such methods and
`
`conditions may vary.
`
`It is also to be understood that the terminology used herein is for the
`
`purpose of describing particular embodiments only, and is not intended to be limiting, since the
`
`scope of the present invention will be limited only to the appended claims.
`
`[0055] Unless defined otherwise, 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. 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 now described.
`
`Example 1. Stability of 50 mglml VEGF Trap Liquid Formulation Stored at 5°C in 3 ml
`Glass Vials.
`
`[0056] An ophthalmic liquid formulation containing 50 mg/ml VEGF Trap (SEQ ID NO:4), 10
`
`mM phosphate, 50 mM NaCl, 0.1% polysorbate 20, 5% sucrose, and pH 6.25, was stored at 5
`
`°C in 3 ml glass vials and samples tested at 3, 6, 9, 12, 18 and 24 months. Stability was
`
`determined by SE-HPLC. The results are shown in Table 1. Turbidity was measured at ODm
`
`rim; and percent recovered protein and purity by size exclusion HPLC.
`
`Regeneron Exhibit 1021.009
`
`

`

`WO 2007/149334
`
`PCT/U52007/014085
`
`
`Table 1. Stability of 50 mglml VEGF Trap Protein (VGFT—SSOBS)
`Visual
`
`% VEGF Trap
`Turbidity
`% VEGF Trap
` (00405 rim)
`
`Rec overed
`Native Configuration
`
`Appearance ‘
`
`98.8
`100
`Pass
`
`
`
`
`
`
`
`
`
`
`
`Example 2. Stability of 50 mg/ml VEGF Trap Liquid Formulation Stored at 5°C in 3 ml
`Glass Vials.
`
`[0057] A liquid formulation containing 50 mg/ml VEGF Trap (SEQ ID N024). 10 mM phosphate.
`50 mM NaCl, 3% polyethylene glycol 3350, 5% sucrose, and pH 6.25, was stored at 5 “C in 3 nil
`
`glass vials and samples tested at 3, 6, 9, 12, 18 and 24 months. Stability results are shown in I
`Table 2. Turbidity, percent recovered protein and purity was determined as described above.
`
`Table 2. Stability of 50 mglml VEGF Trap Protein (VGFT-SSOB5)
`Visual
`“A VEGF Trap
`Turbidity
`Recovered
`Appearance
`
`% VEGF Trap
`Native Configuration
`
`
`
`
`
`98.3
`
`Example 3. Stability of 40 mglml VEGF Trap Liquid Formulation Stored at 5°C in 3 ml
`Glass Vials.
`
`[0058] A liquid formulation containing 40 mg/ml VEGF Trap (SEQ ID NO:4), 10 mM phosphate.
`
`40 mM NaCI, 0.03% polysorbate 20. 5% sucrose, and pH 6.3, was stored at 5 °C in 3 ml glass
`
`vials and samples tested at 0.5. 1, 2. 3, and 4 months. Stability results are shown in Table 3.
`Turbidity. percent recovered protein and purity was determined as described above.
`
`Regeneron Exhibit 1021.010
`
`

`

`WO 2007/149334
`
`PCT/U82007/014085
`
`Table 3. Stability of 40 mglrhl VEGF Trap Protein (VGFT-SSZOT)
`
`°/n VEGF Trap
`Recovered
`
`Native Configuration
`
`% VEGF Trap
`
`Example 4. Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at 5°C in Pre-filled
`
`Glass Syringe.
`
`[0059] A liquid formulation containing 40 mg/ml VEGF trap (SEQ ID NO:4), 10 mM phosphate,
`
`40 mM NaCI, 0.03% polysorbate 20. 5% sucrose, and pH 6.3. was stored at 5 °C in 1 ml
`
`prefilled Iuer glass syringe with 4023/50 FluroTec coated plunger and samples tested at 0.5. 1,
`
`2, 3, and 4 months. Stability results are shown in Table 4. Turbidity, percent recovered protein
`
`and purity was determined as described above.
`
`Table 4. Stability of 40 mglml VEGF Trap Protein (VGFT—SSZO7)
`
`Visual
`Appearance
`
`Turbidity
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native Configuration
`
`
`
`“—
`
`Example 5. Stability of 40 mglml VEGF Trap Liquid Formulation Stored at 5°C in 3 ml
`Glass Vials.
`
`[0060] A liquid formulation containing 40 mg/ml VEGF trap (SEQ ID N04), 10 mM phosphate,
`
`135 mM NaCl, 0.03% polysorbate 20, and pH 6.3, was stored at 5 °C in 3 ml glass vials and
`
`samples tested at 0.5. 1, 2, 3, and 4 months. Stability results are shown in Table 5. Turbidity.
`
`percent recovered protein and purity was determined as described above.
`
`10
`
`Regeneron Exhibit 1021.011
`
`

`

`WO 2007/149334
`
`PCT/U52007/014085
`
`Table 5. Stability of 40 mglml VEGF Trap Protein (VGFT—SSZO3)
`
`.
`Visual
`Appearance
`
`% VEGF Trap
`.
`% VEGF Trap
`.
`.
`6“$31;on
`Recovered
`Turbidity
`m-——
`
`103
`
`99.1
`
`99.2
`
`
`
`“-3—— 000
`
`6.2
`
`6.3
`
`000
`
`
`
`Example 6. Stability of 4D mglml VEGF Trap Liquid Formulation Stored at 5°C in 1 ml Pre-
`
`filled Glass Syringe.
`
`[0061] A liquid formulation containing 40 mglml VEGF trap (SEQ lD NO:4), 10 mM phosphate,
`
`135 mM NaCl, 0.03% polysorbate 20, and pH 6.3, was stored at 5 ”C in 1 ml prefiiied glass luer
`
`syringe with 4023/50 FluroTec coated plunger and samples tested at 0.5. 1, 2, 3, 4. and 5
`
`months. Stability results are shown in Table 6. Turbidity, percent recovered protein and purity
`was determined as described above.
`
`Table 6. Stability of 40 mglml VEGF Trap Protein (VGFT-SSZDS)
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native Configuration
`
`
`
`Example 7. Stability of Lyophilized 20 mglml VEGF Trap Formulation Stored at 5°C in 3
`
`ml Glass Vials and Reconstituted to 40 mglml.
`
`[0062] 0.8 ml of a liquid formulation containing 20 mglml VEGF trap (SEQ lD N024). 5 mM
`
`phosphate. 20 mM NaCI, 0.015% polysorbate 20, 2.5% sucrose, and pH 6.3, were Iyophilized in
`
`3 ml glass vials. Samples were stored at 5°C and tested at 1, and 2 months. VEGF trap was
`
`reconstituted to a final concentration of 40 mglml VEGF Trap (final volume of 0.4 ml). Stability
`
`ll
`
`Regeneron Exhibit 1021.012
`
`

`

`WO 2007/149334
`
`PCT/U82007/014085
`
`results are shown in Table 7 (t = time in months; * = visual appearance; ** = reconstitution time).
`
`Turbidity, percent recovered protein and purity was determined as described above.
`
`Table 7. Stability of Lyophilized 20 mglml VEGF Trap Protein (VGFT-SS216)
`
`
`
`% VEGF Trap
`Native Config.
`
`
`
`
`
`
`M“
`
`°/n VEGF
`Vis. App.*
`Vis
`Trap
`Reconst’d ‘ Turbidity
`A ',
`Recovered
`Liquid
`(min)
`pp.
`6.3
`0.00
`0
`100
`Pass
`0.6
`Pass
`
`Pass
`0.6
`Pass
`0.01
`6.3
`106
`
`1
`
`Example 8. Stability of Lyophilized 20 mglml VEGF Trap Formulation Stored at 5°C in 3
`ml Glass Vials.
`
`[0063] 0.8 ml ofa liquid formulation containing 20 mg/ml VEGF trap (SEQ ID NO:4), 5 mM
`phosphate, 67.5 mM NaCI, 0.015% polysorbate 20, and pH 6.3, were lyophilized in 3 ml glass
`
`vials. Samples were stored at 5°C and tested at 1 . 2, and 3 months. VEGF trap was
`
`reconstituted to a final concentration of 40 mg/ml VEGF trap (final volume of 0.4 mi). Stability
`
`results are shown in Table 8 (t = time in months; "' = visual appearance; ** = reconstitution time).
`
`Table 8. Stability of Lyophilized 20 mg/ml VEGF Trap Protein (VGFT-SSZ16)
`
`Vis. App.
`Reconst’d
`
`
`
`% VEGF
`Trap
`Recovered
`
`°/a VEGF Trap
`Native Config.
`
`12
`
`Regeneron Exhibit 1021.013
`
`

`

`WO 2007/149334
`
`PCT/USZOO7/014085
`
`We claim:
`
`1. An oph