(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PAlENT COOPERATION TREATY (PCO
`
`(19) World Intellectuul Property Organi:mtion
`International Bureau
`
`(43) International Publication Date
`27 December 2007 (27.12.2007)
`
`PCT
`
`(51) International Patent Classification:
`
`Not classified
`
`{21) Internatlunal Appllcallon Number.
`PCT/US'.!007/014085
`
`(22) International Filing Date:
`
`14 June 2007 {14.06.2007)
`
`(25) Filing Language:
`
`(26) PubUtatlun Language:
`
`(30) Priority Data:
`60/814,4&4
`
`English
`
`English
`
`16 June 2006 (16.06.2006) US
`
`(71) Applicant (for all designated States except US), REGEN(cid:173)
`ERON PHARMACEUTICALS, INC. [US/US]; 777 Old
`Saw Mill River Road, Tarrytown, NY 10591 (US).
`
`(72) Inventors; and
`(75) Inventors/Appllcants (for US only): FURFINE, Eric
`[US/US]; 315 Harrington Avenue, Concord, MA 01742
`(US). DIX, Daniel [US/US]; 55 Memory Trail, La(cid:173)
`grnngeville, NY 12540 (US). GRAHAM, Kenneth, S.
`[US/US]; 218 Robbout Road, Pleru;ant valley, NY 12569
`(US). FRYE, Kelly [USffiS]; 29 N. Ridge Read, Pomona,
`NY 10970 (US).
`
`(10) International Publication Number
`WO 2007/149334 A2
`(81) Designated States (unless othetwise indicated, for every
`kind of national 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, HN, HR, HU, ID, IL,
`IN, IS, JP, KE, 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,
`Pf, RO, RS, RU, SC, SD, SE, SO, SK, SL, SM, SY, SY,
`Tl, TM, TN, TR, TI, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, 'ZW.
`
`(84) Designated States (unless olhetwise indicated, for ew,ry
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, 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, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, Mr, NL, PL,
`Pf, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, Cl, CM,
`GA, ON, GQ, OW, ML, MR, NE, SN, TD, TO).
`
`Published:
`wiJhout international search report and to be republished
`upon receipt of that report
`with sequence listing part of description publi.ihed sepa(cid:173)
`rately in electronic form and available upon requesl from
`tire International Bureau
`
`(74) Agent: GREGG, Valela; Regeneron Pharmaceuticals.,
`Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591
`(US).
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbrevimums" appearing at 1he begin(cid:173)
`ning of each regular issue of the PCT Gaze tu.
`
`--=
`N <
`
`~
`ff')
`f"')
`
`i
`~ r=:
`----------------------------------------
`g (54) Title: VEGF ANTAGONIST FORMULATIONS SUITABLE FOR INTRAVITREAL ADMINISTRATION
`N O (57) Abstract: Ophthalmic formulations of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonist are
`
`:,. provided suitable for intravitreal administration to the eye. The ophthalmic formulations include a stable liquid fonnulation and a
`;,,- lyophilizable formulation. Preferably, the protein antagonist has the amino acid sequence shown in SEQ ID N0:4.
`
`Regeneron Exhibit 1021.001
`
`

`

`WO 2007/149334
`
`PCT/US2007 /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, U.S. 7,033,604 and 6,777,429. An
`ophthalmic formulation of a VEGF 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, U.S. 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 pharmaceµtically 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 Flt1 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 N0:2 or SEQ ID N0:4_ Preferably,
`
`Regeneron Exhibit 1021.002
`
`

`

`WO 2007/149334
`
`PCT /US2007/014085
`
`the VEGF antagonist is a dimer comprising two fusion proteins of SEQ ID N0:4.
`[0007] In one aspect, a stable liquid ophthalmic formulation is provided that comprises 1-100
`mg/ml 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 N0:4), 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 mg/ml of the VEGF antagonist (SEQ ID N0:4), 1 0 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 N0:4), 10 mM sodium phosphate buffer, 50
`mM 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 N0:4), 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 N0:4), 10 rnM 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 mg/ml VEGF-specific fusion protein antagonist; 0.01-5% of one or more organic co(cid:173)
`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 N0:4) is present at a
`concentration of about 10 to about 80 mg/ml. In various embodiments, the VEGF antagonist
`(SEQ ID N0:4) 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
`
`2
`
`Regeneron Exhibit 1021.003
`
`

`

`WO 2007/149334
`
`PCT/US2007/014085
`
`(SEQ ID N0:4) is present at a concentration of about 40 mg/ml.
`[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 potysorbate 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 N0:4) at 40 mg/ml, 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.
`[00241 In various embodiments, the VEGF antagonist (SEQ ID N0:4) is present at a
`concentration of about 1 D to about 80 mg/ml. In various embodiments, the VEGF antagonist
`(SEQ ID N0:4) 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 specific embodiment, the VEGF antagonist
`(SEQ ID N0:4) is present at a concentration of about 40 mg/ml.
`[0025] In one embodiment, the stable liquid ophthalmic formulation comprises 40 mg/ml of
`VEGF antagonist (SEQ ID N0:4), 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/ml of VEGF antagonist (SEQ ID N0:4), 10 mM sodium
`phosphate buffer, 0.03% polysorbate, and 135 mM sodium chloride at pH 6.2-6.3.
`[0026] In another aspect, a lyophilizable 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 lyophilizable formulation of a vascular endothelial growth factor
`
`3
`
`Regeneron Exhibit 1021.004
`
`

`

`WO 2007/149334
`
`PCT/US2007/014085
`
`(VEGF)-specific fusion protein antagonist is provided, comprising 5-50 mg/ml 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 N0:4) is present at about 5, about 10,
`
`about 20, about 30, or about 40 mg/ml. In a specific embodiment. the lyophilizable ophthalmic
`formulation of the invention comprises 20 mg/ml 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 lyophilizable 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 lyophilizable ophthalmic formulation of the invention
`comprises 20 mg/ml 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 lyophilizable ophthalmic formulation comprises 5 mg/ml, 10
`mg/ml, or40 mg/ml 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
`lyophilizable ophthalmic formulation consists essentially of 5 mg/ml, 10 mg/ml, or 40 mg/ml
`VEGF antagonist (SEQ ID N0:4), 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
`mg/ml 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 lyophilizable
`ophthalmic formulation consists essentially of 20 mg/ml of the VEGF antagonist (SEQ ID N0:4),
`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
`mg/ml, 10 mg/ml, or 40 mg/ml 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 lyophilizable ophthalmic formulation consists essentially of 5 mg/ml, 10 mg/ml, or 40 mgfml
`VEGF antagonist (SEQ ID N0:4}, 5 mM sodium phosphate buffer, 0.015% palysorbate, 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(cid:173)
`lyophilized formulation, e.g., a 20 mg fusion protein/ml pre-lyophilized formulation is
`reconstituted to a final formulation of 40 mg fusion protein/ml.
`[0033] Generally, the lyophilized formulation is reconstituted with sterile water suitable for
`injection. In one embodiment, the reconstitution liquid is bacteriostatic water.
`
`4
`
`Regeneron Exhibit 1021.005
`
`

`

`WO 2007/149334
`
`PCT/US2007/014085
`
`[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 pre-lyophilized formulation, e.g., the method of the
`invention comprises: (a) producing a pre-lyophilized 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.
`
`5
`
`Regeneron Exhibit 1021.006
`
`

`

`WO 2007/149334
`
`PCT/US2007/014085
`
`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
`excipients 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 lyophilization, 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 (VEGF), and includes fusion proteins capable of trapping
`VEGF. In a preferred embodiment, the VEGF antagonist is the fusion protein of SEQ ID N0:2
`or 4; more preferably, SEQ ID N0:4. In specific embodiments, the VEGF antagonist is
`expressed in a mammalian cell line such as a CHO cell and may be modified post(cid:173)
`translationally. In a specific embodiment, the fusion protein comprises amino acids 27-457 of
`SEQ ID N0:4 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 N0:4.
`[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 o/o 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
`
`6
`
`Regeneron Exhibit 1021.007
`
`

`

`WO 2007/149334
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`PCT/US2007/014085
`
`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 NaCl, 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-e><clusion 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.
`Lyophilizable 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
`
`7
`
`Regeneron Exhibit 1021.008
`
`

`

`WO 2007/149334
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`PCT/US2007/014085
`
`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 WFI
`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 mg/ml 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 N0:4), 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 OD405
`nm; and percent recovered protein and purity by size exclusion HPLC.
`
`8
`
`Regeneron Exhibit 1021.009
`
`

`

`WO 2007/149334
`
`PCT/US2007/014085
`
`Table 1. Stability of 50 mg/ml VEGF Trap Pro1ein (VGFT-SS065)
`
`Months
`
`Visual
`Appearance
`
`Turbidity
`(OD4os nm)
`
`0
`
`3
`
`6
`
`9
`
`12
`18
`24
`
`Pass
`
`Pass
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`0.00
`
`0.00
`0.01
`
`0.01
`
`0.01
`0.01
`
`0.01
`
`pH
`
`6.2
`
`6.2
`6.3
`6.3
`6.3
`6.3
`6.3
`
`% VEGF Trap
`Recovered
`100
`
`% VEGFTrap
`Native Configuration
`98.8
`
`101
`100
`101
`
`104
`96
`
`105
`
`98.7
`98.3
`98.3
`
`98.4
`98.1
`
`98.1
`
`Example 2. Stability of 50 mg/ml VEGF Trap Liquid Formulation Stored at S"C in 3 ml
`Glass Vials.
`[0057] A liquid formulation containing 50 mg/ml VEGF Trap (SEQ ID N0:4), 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
`Table 2. Turbidity, percent ,ecovered protein and purity was determined as described above.
`
`Table 2. Stability' of 50 mg/ml VEGF Trap Protein (VGFT-SS065)
`
`Months
`
`0
`
`3
`6
`
`9
`12
`
`18
`
`24
`
`Visual
`Appearance
`Pass
`
`Pass
`
`Pass
`
`Pass
`Pass
`
`Pass
`
`Pass
`
`Turbidity
`
`0.00
`
`0.00
`0.01
`0.00
`0.01
`
`0.00
`
`0.00
`
`pH
`
`6.2
`
`6.1
`
`6.3
`
`6.3
`6.3
`
`6.3
`
`6.2
`
`% VEGFTrap
`Recovered
`100
`
`% VEGF Trap
`Native Configuration
`98.9
`
`104
`99
`102
`103
`
`113
`
`106
`
`98.5
`
`98.3
`97.6
`98.0
`
`97.7
`
`97.6
`
`Example 3. Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at sac in 3 ml
`Glass Vials.
`[0058] A liquid formulation containing 40 mg/ml VEGF Trap (SEQ ID N0:4), 10 mM phosphate,
`40 mM NaCl, 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.
`
`9
`
`Regeneron Exhibit 1021.010
`
`

`

`WO 2007/149334
`
`PCT/US2007/014085
`
`Table 3. Stability of 40 mg/ml VEGF Trap Protein (VGFT-SS207)
`
`Months
`
`0
`0.5
`
`1
`2
`
`3
`
`4
`
`Visual
`Appearance
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Turbidity
`
`pH
`
`% VEGF Trap
`Recovered
`
`0.00
`
`0.00
`0.00
`
`0.00
`0.01
`0.01
`
`6.3
`6.3
`6.2
`
`6.2
`
`6.4
`6.3
`
`100
`99
`
`98
`95
`
`% VEGF Trap
`Native Configuration
`99.5
`99.4
`
`99.5
`99.2
`
`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 N0:4), 10 mM phosphate,
`
`40 mM NaCl, 0.03% polysorbate 20, 5% sucrose, and pH 6.3, was stored at 5 °C in 1 ml
`prefilled luer 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 mg/ml VEGF Trap Protein {VGFT-SS207)
`
`Months
`
`Visual
`Appearance
`
`Turbidity
`
`pH
`
`% VEGFTrap
`Recovered
`
`% VEGFTrap
`Native Configuration
`
`0
`
`0.5
`1
`2
`3
`
`4
`
`Pass
`
`Pass
`Pass
`
`Pass
`Pass
`
`Pass
`
`0.00
`
`0.00
`0.00
`
`0.00
`0.01
`
`0.01
`
`6.3
`6.3
`6.3
`
`6.3
`
`6.4
`
`6.3
`
`100
`
`100
`100
`
`97
`
`99.4
`
`99.3
`99.4
`99.1
`
`Example 5. Stability of 40 mg/ml 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 N0:4), 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/US2UU7/U14085
`
`Table 5. Stability of 40 mg/ml VEGF Trap Protein (VGFT-SS203)
`
`Months
`
`Visual
`Appearance
`
`Turbidity
`
`pH
`
`% VEGFTrap
`Recovered
`
`% VEGFTrap
`Native
`Configuration
`
`0
`
`0.5
`
`1
`
`2
`
`3
`
`4
`5
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`
`0.00
`
`0.00
`
`0.00
`0.00
`
`0.00
`0.00
`
`6.3
`
`6.2
`
`6.2
`
`6.3
`6.3
`6.2
`
`6.3
`
`100
`87
`88
`
`103
`88
`85
`84
`
`99.3
`
`99.2
`99.1
`
`99.2
`
`99.0
`98.9
`99.0
`
`Example 6. Stability of 40 mg/ml VEGF Trap Liquid Formulation Stored at 5°C in 1 ml Pre(cid:173)
`filled Glass Syringe.
`[0061] A liquid formulation containing 40 mg/ml VEGF trap {SEQ ID N0:4), 10 mM phosphate,
`135 mM NaCl. 0.03% polysorbate 20, and pH 6.3, was stored at 5 ·c in 1 ml prefilled 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 mgfml VEGF Trap Protein (VGFT-55203)
`
`Months
`
`0
`0.5
`1
`2
`
`3
`
`4
`
`5
`
`Visual
`Appearance
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Pass
`
`Turbidity
`
`pH
`
`0.00
`
`0.01
`0.00
`0.00
`
`0.01
`
`0.01
`
`0.00
`
`6.3
`6.3
`6.3
`6.3
`
`6.3
`6.3
`
`6.3
`
`% VEGF Trap
`Recovered
`100
`101
`101
`-
`102
`103
`99
`
`% VEGF Trap
`Native Configuration
`99.2
`99.2
`99.2
`-
`99.1
`98.8
`
`98.9
`
`Example 7. Stability of Lyophilized 20 mgfml VEGF Trap Formulation Stored at 5°C in 3
`
`ml Glass Vials and Reconstituted to 40 mg/ml.
`[0062] 0.8 ml of a liquid formulation containing 20 mg/ml VEGF trap (SEQ ID N0:4). 5 mM
`phosphate, 20 mM NaCl, 0.015% polysorbate 20, 2.5% sucrose, and pH 6.3, were lyophiliz