(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0217311 A1
`Dix et al.
`(43) Pub. Date:
`Sep. 28, 2006
`
`US 20060217311A1
`
`(54) VEGF ANTAGONIST FORMULATIONS
`
`Related US. Application Data
`
`(76) Inventors: Daniel Dix, LaGrangeville, NY (US);
`Kelly Frye, Pomona, NY (US); Susan
`Kautz, Albany’ NY (Us)
`
`Correspondence Address:
`REGENERON PHARMACEUTICALS, INC
`777 OLD SAW MILL RIVER ROAD
`TARRYTOWN, NY 10591 (US)
`
`(21) Appl. No.:
`
`11/387,256
`
`(22) Filed:
`
`Mar. 22, 2006
`
`(60) Provisional application No. 60/665,125, ?led on Mar.
`25, 2005
`
`Publication Classi?cation
`
`(51) Int. Cl.
`(2006.01)
`A61K 38/1 7
`(2006.01)
`A61K 31/7012
`(200601)
`A 61K 31/4172
`(52) us. Cl. ............................. .. 514/12; 514/53; 514/400
`
`ABSTRACT
`(57)
`Formulations of a vascular endothelial growth factor
`(V EGF)-speci?c fusion protein antagonist are provided
`including a pre-lyophiliZed formulation, a reconstituted lyo
`philiZed formulation, and a stable liquid formulation. Pref
`erably, the fusion protein has the sequence of SEQ ID NO:4.
`
`Mylan Exhibit 1033
`Mylan v. Regeneron, IPR2021-00880
`Page 1
`
`Joining Petitioner: Apotex
`
`

`

`US 2006/0217311A1
`
`Sep. 28, 2006
`
`VEGF ANTAGONIST FORMULATIONS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`[0001] This application claims the bene?t under 35 USC §
`119(e) of US. Provisional 60/665,125 ?led 25 Mar. 2005,
`Which application is herein speci?cally incorporated by
`reference in its entirety.
`
`BACKGROUND OF INVENTION
`
`[0002] 1. Field of the Invention
`
`[0003] The present invention is directed to pharmaceutical
`formulations comprising agents capable of inhibiting vas
`cular endothelial groWth factor (V EGF), and to methods for
`making and using such formulations. The invention includes
`pharmaceutical formulations having increased stability.
`
`[0004] 2. Statement of Related Art
`
`[0005] Vascular endothelial groWth factor (V EGF) expres
`sion 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-speci?c 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), Which applications are spe
`ci?cally incorporated by reference in their entirety.
`
`[0006] LyophiliZation (freeZe drying under controlled con
`ditions) is commonly used for long term storage of proteins.
`The lyophiliZed protein is substantially resistant to degra
`dation, aggregation, oxidation, and other degenerative pro
`cesses While in the freeZe-dried state (see, for example, US.
`Pat. No. 6,436,897).
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0007] Stable formulations of a VEGF-speci?c fusion
`protein antagonist are herein provided. The pharmaceuti
`cally acceptable formulations of the invention comprise the
`VEGF “trap” antagonist With a pharmaceutically acceptable
`carrier. In speci?c embodiments, liquid and freeZe-dried, or
`lyophiliZed formulations are provided.
`
`[0008] In a ?rst aspect, the invention features a stable
`liquid formulation of a VEGF-speci?c fusion protein
`antagonist, comprising a fusion protein comprising a recep
`tor component consisting essentially of an immunoglobulin
`like (Ig) domain 2 of a ?rst VEGF receptor and Ig domain
`3 of a second VEGF receptor, and a multimeriZing compo
`nent, one or more buffers, and one or more thermal stabi
`liZers. In a speci?c embodiment of the VEGF-speci?c fusion
`protein antagonist, the ?rst VEGF receptor is Fltl and the
`second VEGF receptor is Flkl or Flt4. In a more speci?c
`embodiment the fusion protein has the amino acid sequence
`of SEQ ID NO:2 or SEQ ID NO:4. In one embodiment, the
`buffer is a phosphate buffer and/or citrate. More preferably,
`the buffers are phosphate and citrate. In one embodiment, the
`thermal stabiliZers are NaCl and/or sucrose. More prefer
`ably, the thermal stabiliZers are both NaCl and sucrose.
`
`[0009] In a speci?c embodiment, the stable liquid formu
`lation of a VEGF-speci?c fusion protein antagonist com
`prises 1-10 mM phosphate buffer, 1-10 mM citrate, 25-150
`mM NaCl, 5-30% sucrose, 10-50 mg/ml of the fusion
`protein, at a pH of about 6-6.5. In a more speci?c embodi
`ment, the stable liquid formulation comprises 5 mM phos
`phate buffer, 5 mM citrate buffer, 100 mM NaCl, 20%
`sucrose, 25 mg/ml of the fusion protein, at a pH of about 6.0.
`Additionally, polysorbate may be present, for example 0.05
`0.15% polysorbate 20. The stable liquid formulation of the
`VEGF-speci?c fusion protein antagonist of the invention
`exhibits little or no precipitation after storage of a 25 mg/ml
`VEGF formulation for about 6 months at —80° C. and little
`or no precipitation after storage for 6 months at 50 C.
`
`[0010] In a second aspect, the invention features a high
`concentration stable liquid formulation of a VEGF antago
`nist comprising 1-50 mM histidine, 25-150 mM NaCl,
`5-30% sucrose, 50-100 mg/ml of the fusion protein, at a pH
`of about 6-6.5, and either 0.1-0.5% polysorbate or 1-5%
`PEG. In a more speci?c embodiment, the high concentration
`stable liquid formulation comprises 10 mM histidine, 50
`mM NaCl, 5-20% sucrose, 50-100 mg/ml of the fusion
`protein, at a pH of about 60-65, With either 0.1% polysor
`bate (e.g., polysorbate 20) or 3% PEG (e.g., PEG 3350). The
`high concentration stable liquid formulation of the VEGF
`speci?c fusion protein antagonist of the invention exhibits
`less than about 3% degradation after 15 months of storage at
`50 C. (75 or 100 mg/ml VEGF trap protein) or less than
`about 1.5% degradation after 24 months (50 mg/ml).
`
`[0011] In a third aspect, the invention features a pre
`lyophiliZed formulation of a vascular endothelial groWth
`factor (VEGF)-speci?c fusion protein antagonist, compris
`ing a (i) fusion protein comprising a receptor component
`consisting essentially of an immunoglobulin-like (Ig)
`domain 2 of a ?rst VEGF receptor and Ig domain 3 of a
`second VEGF receptor, and a multimeriZing component, (ii)
`a buffer, (iii) an organic co-solvent or bulking agent, and (iv)
`one or more lyoprotectants. In various embodiments, the
`buffer is histidine, the organic co-solvent or bulking agent is
`PEG, and the lyoprotectant(s) is at least one of glycine and
`sucrose. In one embodiment, the pre-lyophiliZed formula
`tion of the invention does not contain a preservative.
`
`[0012] In one embodiment of the pre-lyophiliZed formu
`lation of the invention, the formulation comprises 5-50 mM
`histidine, 0.1-3.0% PEG, 0.25-3.0% glycine, 0.5-6.0%
`sucrose, and 5-75 mg/ml of the fusion protein, at a pH of
`about 60-65. In any embodiment, the pre-lyophiliZed for
`mulation may further comprise up to 0.05 mM citrate and/or
`0.003-0.005% polysorbate. The polysorbate present may be,
`for example, polysorbate 20.
`
`[0013] In a more speci?c embodiment, the pre-lyophiliZed
`formulation comprises about 10 mM histidine, about 1.5%
`PEG 3350, about 0.75% glycine, about 2.5% sucrose, and
`about 12.5 to 75 mg/ml VEGF-speci?c fusion protein, at a
`pH of about 6.25. In speci?c embodiments, the fusion
`protein comprises the protein sequence of SEQ ID NO:4,
`present as a multimer, e.g., a dimer. In separate embodi
`ments, the reconstituted formulation is 2 times the concen
`tration of the pre-lyophiliZed formulation, e.g., a 20 mg
`fusion protein/ml pre-lyophiliZed formulation is reconsti
`tuted to a ?nal formulation of 60 mg fusion protein/ml.
`Generally, the lyophiliZed formulation is reconstituted With
`
`Mylan Exhibit 1033
`Mylan v. Regeneron, IPR2021-00880
`Page 2
`
`Joining Petitioner: Apotex
`
`

`

`US 2006/0217311A1
`
`Sep. 28, 2006
`
`sterile Water suitable for injection. In one embodiment, the
`reconstitution liquid may be bacteriostatic Water.
`
`[0014] In a preferred embodiment, the pre-lyophiliZed
`formulation consists essentially of about 10 mM histidine,
`about 1.5% PEG 3350, about 0.75% glycine, about 2.5%
`sucrose, and about 50 mg/ml of the fusion protein having the
`sequence of SEQ ID NO:4 as a dimer, at a pH of about 6.25.
`Citrate (less than or equal to about 0.02 mM) and/or polysor
`bate (less than or equal to about 0.0005%) may be present.
`Optionally, the pre-lyophiliZed formulation does not contain
`a preservative, a phosphate buffer, and/or more than trace
`amounts of NaCl. In one embodiment, the pre-lyophiliZed
`formulation consists of about 10 mM histidine, about 1.5%
`PEG 3350, about 0.75% glycine, about 2.5% sucrose, and
`about 50 mg/ml of the VEGF trap protein (SEQ ID NO:4),
`pH 6.3, and upon reconstitution contains 20 mM histidine,
`3% PEG, 1.5% glycine, about 5% sucrose, and about 100
`mg/ml VEGF trap protein.
`
`[0015] In a fourth aspect, the invention features a method
`of producing a lyophiliZed formulation of a VEGF-speci?c
`fusion protein antagonist, comprising subjecting the pre
`lyophiliZed formulation of the invention to lyophiliZation to
`generate a lyophiliZed formulation. The lyophiliZed formu
`lation may be lyophiliZed by any method knoWn in the art
`for lyophiliZing a liquid.
`
`[0016] In a ?fth related aspect, the invention features a
`method of producing a reconstituted lyophiliZed formulation
`of a VEGF-speci?c fusion protein antagonist, comprising
`reconstituting the lyophiliZed formulation of the invention to
`a reconstituted formulation. In one embodiment, the recon
`stituted 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-speci?c fusion protein antagonist, (b) subjecting the
`pre-lyophiliZed formulation of step (a) to lyophiliZation; and
`(c) reconstituting the lyophiliZed formulation of step (b).
`[0017] In speci?c embodiments of the method of produc
`ing a reconstituted lyophiliZed formulation, a pre-lyo
`philiZed solution is present in a vial as a 25 mg VEGF
`speci?c fusion protein antagonist per ml solution of pre
`lyophiliZed formulation, Which is
`lyophiliZed and
`reconstituted to an 50 mg/ml solution. In another embodi
`ment, a 30 mg/ml pre-lyophiliZed solution is lyophiliZed and
`reconstituted to a 60 mg/ml solution. In another embodi
`ment, a 40 mg/ml pre-lyophiliZed solution is lyophiliZed and
`reconstituted to a 80 mg/ml solution. In another embodi
`ment, a 12.5 mg/ml pre-lyophiliZed solution is lyophiliZed
`and reconstituted to a 25 mg/ml solution. In another embodi
`ment, a 50 mg/ml pre-lyophiliZed solution is lyophiliZed and
`reconstituted to a 100 mg/ml solution. In another embodi
`ment, a 75 mg/ml pre-lyophiliZed solution is lyophiliZed and
`reconstituted to a 150 mg/ml solution. Preferably, the recon
`stituted lyophiliZed formulation does not contain a preser
`vative.
`
`[0018] Other objects and advantages Will become apparent
`from a revieW of the ensuing detailed description.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0019] 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.
`[0020] As used in this speci?cation and the appended
`claims, the singular forms “a”, “an”, and “the” include plural
`references unless the context clearly dictates otherWise.
`Thus for example, references to “a method” include one or
`more methods, and/or steps of the type described herein
`and/or Which Will become apparent to those persons skilled
`in the art upon reading this disclosure.
`
`[0021] Unless stated otherWise, all technical and scienti?c
`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. All
`publications mentioned herein are incorporated herein by
`reference.
`General Description
`[0022] Safe handling and administration of formulations
`comprising proteins represent signi?cant challenges to phar
`maceutical formulators. Proteins possess unique chemical
`and physical properties that present stability problems: a
`variety of degradation pathWays exist for proteins, impli
`cating 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, includ
`ing, for example, aggregation.
`[0023] Chemical and physical stability can be promoted
`by removing Water from the protein. LyophiliZation (freeZe
`drying under controlled conditions) is commonly used for
`long-term storage of proteins. The lyophiliZed protein is
`substantially resistant to degradation, aggregation, oxida
`tion, and other degenerative processes While in the freeZe
`dried state. The lyophiliZed protein is normally reconstituted
`With Water optionally containing a bacteriostatic preserva
`tive (e.g., benZyl alcohol) prior to administration.
`De?nitions
`
`[0024] The term “carrier” includes a diluent, adjuvant,
`excipient, or vehicle With Which a composition is adminis
`tered. 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.
`
`[0025] The term “excipient” includes a non-therapeutic
`agent added to a pharmaceutical composition to provide a
`desired consistency or stabiliZing effect. Suitable pharma
`ceutical excipients include, for example, starch, glucose,
`lactose, sucrose, gelatin, malt, rice, ?our, chalk, silica gel,
`sodium stearate, glycerol monostearate, talc, sodium chlo
`ride, dried skim milk, glycerol, propylene, glycol, Water,
`ethanol and the like.
`
`[0026] 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 50% of
`moisture has been removed.
`
`Mylan Exhibit 1033
`Mylan v. Regeneron, IPR2021-00880
`Page 3
`
`Joining Petitioner: Apotex
`
`

`

`US 2006/0217311A1
`
`Sep. 28, 2006
`
`[0027] The phrase “bulking agent” includes a compound
`that is pharmaceutically acceptable and that adds bulk to a
`lyo cake. Generally, acceptable bulking agents known to the
`art include, for example, carbohydrates, including simple
`sugars such as dextrose, ribose, fructose and the like, alcohol
`sugars such as mannitol, inositol and sorbitol, disaccharides
`including trehalose, sucrose and lactose, naturally occurring
`polymers such as starch, dextrans, chitosan, hyaluronate,
`proteins (e.g., gelatin and serum albumin), glycogen, and
`synthetic monomers and polymers. In the formulations of
`the invention, PEG 3350 is an organic co-solvent Which is
`used to stabiliZe the fusion protein When agitated, mixed, or
`handled, and as a bulking agent to help produce an accept
`able bulk.
`
`[0028] The term “lyoprotectant” includes a substance that
`may be added to a freeZe-dried or lyophiliZed formulation to
`help maintain protein structure When freeZe-dried or lyo
`philiZed.
`[0029] A “preservative” includes a bacteriostatic, bacte
`riocidal, fungistatic or fungicides compound that is gener
`ally added to formulations to retard or eliminate groWth of
`bacteria or other contaminating microorganisms in the for
`mulations. Preservatives include, for example, benZyl alco
`hol, phenol, benZalkonium chloride, m-cresol, thimerosol,
`chlorobutanol, methylparaben, propylparaben and the like.
`Other examples of pharmaceutically acceptable preserva
`tives can be found in the USP.
`VEGF Antagonists
`[0030] An VEGF antagonist is a compound capable of
`blocking or inhibiting the biological action of vascular
`endothelial groWth factor (V EGF), and includes fusion pro
`teins 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 N014. In speci?c embodi
`ments, the VEGF antagonist is expressed in a mammalian
`cell line such as a CHO cell and may be modi?ed post
`translationally. In a speci?c embodiment, the fusion protein
`comprises amino acids 27-457 of SEQ ID NO4 and is
`glycosylated at Asn residues 62, 94, 149, 222 and 308.
`
`[0031] The VEGF antagonist of the methods and formu
`lations 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 pharma
`ceutically acceptable formulation. By “substantially free of
`protein contaminants” is meant, preferably, that at least 90%
`of the Weight of protein of the VEGF-speci?c 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 the Weight of
`fusion protein is not present in an aggregate at the time the
`fusion protein is used to prepare the pharmaceutically effec
`tive formulation. The fusion protein of the methods and
`formulations of the invention may contain loW or trace
`amounts of compounds as a results of the puri?cation
`process, for example, loW or trace amounts of citrate and/or
`polysorbate. In one embodiment of the pre-lyophiliZed for
`mulation of the invention containing about 50 mg of fusion
`protein/ml, citrate may be present at a concentration of about
`0.02 mM and/or polysorbate may be present at a concen
`
`tration of about 0.0005%. If the pre-lyophiliZed formulation
`is reconstituted after lyophiliZation to half of the original
`volume (e.g., 100 mg/ml of fusion protein), the resulting
`concentrations may be 0.04 mM citrate and/or 0.001%
`polysorbate.
`LyophiliZation and LyophiliZed Formulations
`
`[0032] In one aspect of the invention, a pharmaceutically
`acceptable formulation comprising a VEGF-speci?c fusion
`protein antagonist is provided, Wherein the formulation is a
`freeZe-dried or lyophiliZed formulation. LyophiliZed formu
`lations can be reconstituted into solutions, suspensions,
`emulsions, or any other suitable form for administration or
`use. LyophiliZed formulations are typically ?rst prepared as
`liquids, then froZen and lyophiliZed. The total liquid volume
`before lyophiliZation can be less, equal to, or more than, the
`?nal reconstituted volume of the lyophiliZed formulation.
`The lyophiliZation process is Well knoWn to those of ordi
`nary skill in the art, and typically includes sublimation of
`Water from a froZen formulation under controlled condi
`tions.
`
`[0033] LyophiliZed formulations can be stored at a Wide
`range of temperatures. LyophiliZed formulations may be
`stored beloW 25° C., for example, refrigerated at 40 C., or at
`room temperature (e.g., approximately 250 C.). Preferably,
`lyophiliZed formulations are stored beloW about 250 C.,
`more preferably, at about 4-200 C.; beloW about 40 C.; beloW
`about —20° C.; about —400 C.; about —70° C., or about —80°
`C.
`
`[0034] LyophiliZed formulations are typically reconsti
`tuted 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 formula
`tion. Preferably, lyophiliZed formulations are reconstituted
`using Water. LyophiliZed formulations are preferably recon
`stituted 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, solu
`tions comprising bulfers and/or excipients and/or one or
`more pharmacetically acceptable carries can also be used.
`
`[0035] Freeze-dried or lyophiliZed formulations are typi
`cally prepared from liquids, that is, from solutions, suspen
`sions, emulsions, and the like. Thus, the liquid that is to
`undergo freeZe-drying or lyophiliZation preferably com
`prises all components desired in a ?nal reconstituted liquid
`formulation. As a result, When reconstituted, the freeZe-dried
`or lyophiliZed formulation Will render a desired liquid
`formulation upon reconstitution. A preferred liquid formu
`lation used to generate a freeZe-dried or lyophiliZed formu
`lation comprises a VEGF-speci?c fusion protein antagonist
`in a pharmaceutically effective amount, a buffer, a stabiliZer,
`and a bulking agent. Freeze-dried or lyophiliZed formula
`tions preferably comprise histidine, since histidine, in com
`parison to phosphate, is more effective at stabiliZing the
`fusion protein When the fusion protein is lyophiliZed.
`Organic cosolvents, such as PEG 3350, are used to stabiliZe
`the fusion protein When agitated, mixed, or handled. A
`lyoprotectant is preferably used in freeZe-dried or lyo
`philiZed formulations. Lyoprotectants help to maintain the
`secondary structure of proteins When freeZe-dried or lyo
`philiZed. TWo preferred example lyoprotectants are glycine
`and sucrose, Which are preferably used together.
`
`Mylan Exhibit 1033
`Mylan v. Regeneron, IPR2021-00880
`Page 4
`
`Joining Petitioner: Apotex
`
`

`

`US 2006/0217311Al
`
`Sep. 28, 2006
`
`Stable Liquid Formulations
`
`[0036] In one aspect, the invention provides a stable
`pharmaceutically acceptable formulation comprising a
`VEGF-speci?c fusion protein antagonist, Wherein the for
`mulation is a liquid formulation. Preferably, the liquid
`formulation comprises a pharmaceutically effective amount
`of the fusion protein. The formulation can also comprise one
`or more pharmaceutically acceptable carriers, buffers, bulk
`ing agents, stabiliZers, preservatives, and/or excipients. An
`example of a pharmaceutically acceptable liquid formula
`tion comprises a VEGF-speci?c fusion protein antagonist in
`a pharmaceutically effective amount, a buffer, a co-solvent,
`and one or more stabiliZers.
`[0037] A preferred liquid formulation comprises phos
`phate buffer, an organic co-solvent, and one or more thermal
`stabiliZers to minimiZe formation of aggregates and loW
`molecular Weight products When stored, and about 10 mg/ml
`to about 50 mg/ml fusion protein, Wherein the formulation
`is from about pH 6.0-6.5. A preferred liquid formulation
`comprises about 5 mM phosphate buffer, about 5 mM
`citrate, about 100 mM NaCl, about 25% sucrose, and about
`l0-50 mg/ml fusion protein, Wherein the formulation is at a
`pH of about 6.0; optionally polysorbate may be present (e. g.,
`0.1% polysorbate 20). Although either NaCl or sucrose can
`be used as a stabiliZer, a combination of NaCl and sucrose
`has been established to stabiliZe the fusion protein more
`effectively than either individual stabiliZer alone.
`
`[0038] Stability is determined in a number of Ways at
`speci?ed 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, SDS-PAGE, siZe-exclusion HPLC, bioassay
`determination of activity, isoelectric focusing, and isoaspar
`tate quanti?cation. In one example of a bioassay useful for
`determining VEGF antagonist activity, a BAF/3 VEGFRl/
`EPOR cell line is used to determine VEGFl65 binding by
`the VEGF-speci?c fusion protein antagonist of the inven
`tion.
`
`[0039] Formulations, Whether liquid or freeZe-dried and
`lyophiliZed, can be stored in an oxygen-deprived environ
`ment. Oxygen-deprived environments can be generated by
`storing the formulations under an inert gas such as, for
`example, argon, nitrogen, or helium.
`
`EXAMPLES
`[0040] 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.
`[0041] As used in this speci?cation and the appended
`claims, the singular forms “a”, “an”, and “the” include plural
`references unless the context clearly dictates otherWise.
`Thus for example, a reference to “a method” includes one or
`more methods, and/or steps of the type described herein
`and/or Which Will become apparent to those persons skilled
`in the art upon reading this disclosure and so forth.
`[0042] Unless de?ned otherWise, all technical and scien
`ti?c 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. All
`publications mentioned herein are incorporated herein by
`reference in their entirety.
`
`Example 1
`
`Stability of a 50 mg/ml Liquid Formulation of
`VEGF Trap
`[0043] A liquid formulation containing 10 mM phosphate,
`50 mM NaCl, 0.1% polysorbate 20, 20% sucrose, and 50
`mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, Was stored at
`5° C. and samples tested at 3, 6, 9, l2, l8 and 24 months.
`Stability Was determined by SE-HPLC. The results, shoWn
`in Table 1, show that 98.6% and 98.3% of VEGF trap protein
`remain undegraded at 12 and 24 months, respectively.
`Turbidity Was measured at OD405 nm; and percent recovered
`protein by siZe exclusion HPLC.
`
`TABLE 1
`
`Stability of 50 mgml VEGF Trap Protein When
`Stored at 5° C. (VGFT-SS065)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`0
`3
`6
`9
`12
`18
`24
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`0.00
`0.01
`0.01
`0.01
`0.00
`0.00
`
`6.2
`6.2
`6.2
`6.3
`6.3
`6.3
`6.2
`
`100
`102
`103
`102
`106
`103
`93
`
`99.0
`98.8
`98.7
`98.2
`98.6
`98.4
`98.3
`
`[0044] A liquid formulation containing 10 mM phosphate,
`50 mM NaCl, 3% PEG 3350, 20% sucrose, and 50 mg/ml
`VEGF trap (SEQ ID NO:4), pH 6.25, Was stored at 50 C. and
`samples tested at 3, 6, 9, l2, l8 and 24 months. Stability
`results are shoWn in Table 2.
`
`TABLE 2
`
`Stability of 50 mgml VEGF Trap Protein When
`Stored at 5° C. (VGFT-SS065)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`0
`3
`6
`9
`12
`18
`24
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`0.00
`0.01
`0.00
`0.01
`0.00
`0.01
`
`6.2
`6.2
`6.3
`6.3
`6.3
`6.3
`6.2
`
`100
`100
`103
`103
`110
`113
`90
`
`99.0
`98.8
`98.5
`98.3
`98.3
`98.0
`97.8
`
`Example 2
`
`Stability of a 75 mg/ml Liquid Formulation of
`VEGF Trap
`[0045] A liquid formulation containing 10 mM phosphate,
`50 mM NaCl, 0.1% polysorbate 20, 20% sucrose, and 75
`
`Mylan Exhibit 1033
`Mylan v. Regeneron, IPR2021-00880
`Page 5
`
`Joining Petitioner: Apotex
`
`

`

`US 2006/0217311A1
`
`Sep. 28, 2006
`
`mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, Was stored at
`5° C. and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months.
`Stability results are shown in Table 3.
`
`TABLE 3
`
`TABLE 5-continued
`
`Stability of 100 mgml VEGF Trap Protein
`Stored at 5° C. (VGFT-SS101)
`
`Stability of 75 rng/rnl VEGF Trap Protein When
`Stored at 5° C. (VGFT-SS101)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`% VEGF Trap
`Visual
`Months Appearance Turbidity pH Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`12
`15
`
`Pass
`Pass
`
`—0.01
`0.00
`
`6.2
`6.3
`
`110
`108
`
`93.9
`94.8
`
`0
`1
`2.3
`3
`9
`12
`15
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`0.00
`0.00
`0.00
`—0.01
`0.00
`0.00
`
`6.2
`6.2
`6.2
`6.2
`6.0
`6.3
`6.3
`
`100
`96
`98
`97
`101
`110
`92
`
`97.1
`97.0
`96.7
`96.1
`96.0
`94.5
`95.6
`
`[0048] A liquid formulation containing 10 mM phosphate,
`50 mM NaCl, 3% PEG 3350, 20% sucrose, and 100 mg/ml
`VEGF trap (SEQ ID NO:4), pH 6.25, Was stored at 50 C. and
`samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months. Stability
`results are shoWn in Table 6.
`
`[0046] A liquid formulation containing 10 mM phosphate,
`50 mM NaCl, 3% PEG 3350, 20% sucrose, and 75 mg/ml
`VEGF trap (SEQ ID NO:4), pH 6.25, Was stored at 50 C. and
`samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months. Stability
`results are shoWn in Table 4.
`
`TABLE 4
`
`Stability of 75 rng/rnl VEGF Trap Protein When
`Stored at 5° C. (VGFT-SS101)
`
`% VEGF Trap
`Visual
`Months Appearance Turbidity pH Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`0
`1
`2.3
`3
`9
`12
`15
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`0.00
`0.00
`0.00
`—0.01
`—0.01
`0.00
`
`6.2
`6.2
`6.2
`6.2
`6.2
`6.3
`6.3
`
`100
`99
`97
`89
`98
`112
`98
`
`96.8
`96.7
`96.3
`95.6
`95.4
`94.1
`94.8
`
`Example 3
`
`Stability of a 100 mg/ml Liquid Formulation of
`VEGF Trap
`
`[0047] A liquid formulation containing 10 mM phosphate,
`50 mM NaCl, 0.1% polysorbate 20, 20% sucrose, and 100
`mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, Was stored at
`50 C. and samples tested at 0, 1, 2.3, 3, 9, 12 and 15 months.
`Stability results are shoWn in Table 5.
`
`TABLE 5
`
`Stability of 100 mgml VEGF Trap Protein
`Stored at 5° C. (VGFT-SS101)
`
`% VEGF Trap
`Visual
`Months Appearance Turbidity pH Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`0
`1
`2.3
`6
`9
`
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`0.00
`0.00
`0.00
`—0.01
`
`6.3
`6.2
`6.2
`6.2
`6.2
`
`100
`92
`92
`99
`92
`
`96.7
`96.6
`96.2
`95.5
`95.5
`
`TABLE 6
`
`Stability of 100 mg/ml VEGF Trap Protein
`Stored at 5° C. (VGFT-SS101)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`0
`1
`2.3
`6
`9
`12
`15
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`0.00
`0.01
`0.01
`0.01
`0.00
`0.00
`0.01
`
`6.3
`6.2
`6.2
`6.2
`6.2
`6.3
`6.3
`
`100
`94
`93
`102
`95
`96
`102
`
`96.5
`96.2
`95.7
`94.6
`94.6
`92.8
`93.9
`
`Example 4
`
`Further Embodiments of Stable VEGF Trap
`Formulations
`
`[0049] In one embodiment, the invention provides a stable
`liquid VEGF-binding fusion protein (VEGF trap) formula
`tions comprising 5 mM phosphate, 5 mM citrate, 100 mM
`NaCl, 0.1% Polysorbate 20, 20% sucrose, 25 mg/ml VEGF
`trap protein, pH 6.0. This formulation can either be delivered
`subcutaneously or diluted and delivered by intravenous
`infusion. Due to the high osmolality of this formulation, it is
`diluted 3-fold to achieve an iso-osmolar solution for intra
`venous administration. Stability studies shoWed less than
`about 1% degradation Was detected after 3 years of storage
`at 2-8° C.
`
`[0050] In one embodiment, the invention features a lyo
`philiZed formulation Which is preferably concentrated tWo
`fold from the pre-lyophiliZed to the post-lyophiliZed formu
`lation, e.g., 50 to 100 mg/ml; 75 to 150 mg/ml, or 100 to 200
`mg/ml VEGF trap protein. In one speci?c embodiment, the
`pre-lyophiliZed formulation comprises 10 mM histidine,
`1.5% PEG 3350, 0.75% glycine, 2.5% sucrose, 50 mg/ml
`VEGF trap protein, pH 6.3, and is reconstituted to a formu
`lation comprisisng 20 mM histidine, 3% PEG 3350, 1.5%
`glycine, 5% sucrose, 100 mg/ml VEGF trap protein, pH 6.3.
`Stability studied shoWed no degradation of the VEGF trap
`Was detected after 6 months of storage at 2-8° C.
`
`[0051] In one embodiment of a liquid formulation, the
`formulation comprises 10 mM histidine, 50 mM NaCl,
`5-20% sucrose, 50-100 mg/ml VEGF trap, and one of 0.1%
`
`Mylan Exhibit 1033
`Mylan v. Regeneron, IPR2021-00880
`Page 6
`
`Joining Petitioner: Apotex
`
`

`

`US 2006/0217311Al
`
`Sep. 28, 2006
`
`polysorbate 20 or 3% PEG 3350. One advantage of this
`liquid formulation is that it provides a higher concentration
`of VEGF trap Without requiring the manufacture of a lyo
`philiZed product. Thus, this formulation provides ease for
`subcutaneous delivery, for example, by allowing provision
`of a liquid pre-?lled syringe at a concentraion higher than
`that delivered by IV infusion. Also, this formulation could
`advantageously be used to provide loWer infusion volumes
`and shorter infusion times. The amount of degradation
`determined by SE-HPLC folloWing incubation at 5° C. for
`up to 15 or 24 months is summarized in Table 7.
`
`TABLE 7
`
`Stability of Liquid Formulation With 50e100 rng/rnl VEGF
`T