`
`(12) United States Patent
`Fur?ne et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,092,803 B2
`Jan. 10, 2012
`
`(54)
`
`(75)
`
`VEGF ANTAGONIST FORMULATIONS FOR
`INTRAVITREAL ADMINISTRATION
`
`Inventors: Eric Fur?ne, Concord, MA (US);
`Daniel Dix, LaGrangeville, NY (US);
`Kenneth S. Graham, Pleasant Valley,
`NY (US); Kelly Frye, Pomona, NY (US)
`
`(73)
`
`Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (US)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 7 days.
`
`(21)
`
`(22)
`
`(65)
`
`(60)
`
`(60)
`
`(51)
`
`Appl. N0.: 12/833,417
`
`Filed:
`
`Jul. 9, 2010
`
`Prior Publication Data
`
`US 2011/0257601A1
`
`Oct. 20, 2011
`
`Related US. Application Data
`
`Continuation of application No. 12/560,885, ?led on
`Sep. 16, 2009, now Pat. No. 7,807,164, which is a
`division of application No. 11/818,463, ?led on Jun.
`14, 2007, now Pat. No. 7,608,261.
`
`Provisional application No. 60/814,484, ?led on Jun.
`16, 2006.
`
`Int. Cl.
`A61K 38/18
`C07K14/71
`
`(2006.01)
`(2006.01)
`
`(52) US. Cl. ................. .. 424/134.1; 424/192.1; 514/1.1;
`514/8.1; 530/350
`(58) Field of Classi?cation Search ...................... .. None
`See application ?le for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`6,100,071 A
`8/2000 Davis-Smyth et a1.
`6,897,294 B2
`5/2005 Davis-Smyth et a1.
`7,052,691 B2
`5/2006 Sleeman et a1.
`2005/0281831 A1 12/2005 Davis-Smyth et a1.
`2006/0217311 A1
`9/2006 Dix et a1.
`
`FOREIGN PATENT DOCUMENTS
`W0 WO 2005/000895
`1/2005
`W0 WO 2006/047325
`5/2006
`W0 WO 2006/104852
`10/2006
`
`Primary Examiner * Christine J Saoud
`Assistant Examiner * Jon M Lockard
`(74) Attorney, Agent, or Firm * Joseph E. Zahner
`
`(57)
`
`ABSTRACT
`
`Ophthalmic formulations of a vascular endothelial growth
`factor (V EGF)-speci?c fusion protein antagonist are pro
`vided suitable for intravitreal administration to the eye. The
`ophthalmic formulations include a stable liquid formulation
`and a lyophilizable formulation. Preferably, the protein
`antagonist has the amino acid sequence shown in SEQ ID
`NO:4.
`
`19 Claims, No Drawings
`
`Mylan Exhibit 1160
`Mylan v. Regeneron, IPR2021-00881
`Page 1
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`US 8,092,803 B2
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`1
`VEGF ANTAGONIST FORMULATIONS FOR
`INTRAVITREAL ADMINISTRATION
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation application of US.
`patent application Ser. No. 12/560,885, ?led 16 Sep. 2009,
`which is a divisional application of US. patent application
`Ser. No. 11/818,463, ?led 14 Jun. 2007, which issued as US.
`Pat. No. 7,608,261 on 27 Oct. 2009, which claims the bene?t
`under 35 U.S.C. §119(e) of US. Provisional Application No.
`60/814,484, ?led 16 Jun. 2006, which applications are each
`hereby incorporated by reference.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`The present invention is directed to pharmaceutical formu
`lations suitable for intravitreal administration comprising
`agents capable of inhibiting vascular endothelial growth fac
`tor (V EGF), and to methods for making and using such for
`mulations. The invention includes liquid pharmaceutical for
`mulations having increased stability, as well as formulations
`that may be lyophilized and reconstituted for intravitreal
`administration.
`2. Statement of Related Art
`Vascular endothelial growth factor (VEGF) expression is
`nearly ubiquitous in human cancer, consistent with its role as
`a key mediator of tumor neoangio genesis. 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: 1 1399-404;
`Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99:11393-8),
`which applications are speci?cally incorporated by reference
`in their entirety.
`Ophthalmic formulations are known, see for example, US.
`Pat. Nos. 7,033,604 and 6,777,429. An ophthalmic formula
`tion of aVEGF antibody is described in US. Pat. No. 6,676,
`941 .
`
`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. Pat. No.
`6,436,897).
`
`BRIEF SUMMARY OF THE INVENTION
`
`Stable formulations of a VEGF-speci?c fusion protein
`antagonist are provided. Pharmaceutically acceptable formu
`lations are provided that comprise a VEGF “trap” antagonist
`with a pharmaceutically acceptable carrier. In speci?c
`embodiments, liquid and lyophilized formulations are pro
`vided.
`In a ?rst aspect, a stable liquid ophthalmic formulation of a
`VEGF-speci?c fusion protein antagonist is provided, com
`prising a fusion protein that comprises 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 (also termed a
`“VEGF trap”). In a speci?c embodiment of the VEGF-spe
`ci?c fusion protein antagonist, the ?rst VEGF receptor is Fltl
`and the second VEGF receptor is Flkl or Flt4. In a more
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`speci?c embodiment the fusion protein has the amino acid
`sequence of SEQ ID NO:2 or SEQ ID NO:4. Preferably, the
`VEGF antagonist is a dimer comprising two fusion proteins
`of SEQ ID NO:4.
`In one aspect, a stable liquid ophthalmic formulation is
`provided that comprises 1-100 mg/ml VEGF-speci?c fusion
`protein antagonist, 0.01-5% of one or more organic co-sol
`vent(s), 30-150 mM of one or more tonicity agent(s), 5-40
`mM of a buffering agent, and optionally, 10-75% of a sta
`biliZing agent, pH between about 5.8-7.0.
`In one or more speci?c embodiments, the organic co-sol
`vent 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 potas
`sium chloride; the stabiliZing agent may be sucrose, sorbitol,
`glycerol, trehalose, or mannitol; and the buffering agent may
`be, for example, phosphate buffer. In a speci?c embodiment,
`the phosphate buffer is a sodium phosphate buffer.
`In various embodiments, the organic co-solvent is polysor
`bate and/or PEG, the stabiliZing agent is sucrose, the buffer
`ing agent is phosphate buffer, and the tonicity agent is sodium
`chloride.
`More speci?cally, 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% polysor
`bate and/or PEG, 40-135 mM sodium chloride, and option
`ally 5.0% sucrose, pH about 6.2-6.3.
`In a speci?c preferred embodiment, the stable liquid oph
`thalmic formulation comprises about 50 mg/ml 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.
`In a speci?c preferred embodiment, the stable liquid oph
`thalmic formulation comprises about 50 mg/ml of the VEGF
`antagonist (SEQ ID NO:4), 10 mM sodium phosphate buffer,
`50 mM sodium chloride, 3% PEG, and 5% sucrose, pH about
`6.2-6.3.
`In a speci?c preferred embodiment, the stable liquid oph
`thalmic 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.
`In a speci?c preferred embodiment, the stable liquid oph
`thalmic 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.
`In another aspect, a stable liquid ophthalmic formulation is
`provided that comprises 1-100 mg/ml VEGF-speci?c fusion
`protein antagonist; 0.01-5% of one or more organic co-sol
`vent(s); 5-40 mM of a buffering agent; and optionally 30-150
`mM of one or more tonicity agent(s) and/or 10-75% of a
`stabiliZing agent; having a pH between about 5.8-7.0.
`In various embodiments, the VEGF antagonist (SEQ ID
`NO:4) 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
`(SEQ ID NO:4) is present at a concentration of about 40
`mg/ml.
`In another embodiment, the stabiliZing agent is selected
`from one or more of sucrose, sorbitol, glycerol, trehalose, and
`mannitol.
`In another embodiment, the organic co-solvent is selected
`from one or more of polysorbate, for example, polysorbate 20
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`or polysorbate 80, polyethylene glycol (PEG), for example,
`PEG 3350, and propylene glycol.
`In another embodiment, the buffer is a phosphate buffer, for
`example, sodium phosphate.
`In another embodiment, the tonicity agent is a salt, for
`example, sodium chloride.
`In one embodiment, the stable liquid ophthalmic formula
`tion 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 speci?c embodiment, the stable liquid ophthalmic for
`mulation comprises 10 mM sodium phosphate buffer, about
`0.03% polysorbate, about 40 mM sodium chloride, and about
`5% sucrose. In another speci?c embodiment, the pH of the
`formulation is about 6.2 to about 6.3. In another speci?c
`embodiment, the pH is achieved by mixing mono- and dibasic
`sodium phosphate to the desired pH without acid/base titra
`tion.
`In a speci?c embodiment, the stable liquid ophthalmic
`formulation consists essentially of a VEGF antagonist (SEQ
`ID N014) 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.
`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
`6.2 to 6.3.
`In various embodiments, the VEGF antagonist (SEQ ID
`N014) is present at a concentration of about 10 to about 80
`mg/ml. In various embodiments, the VEGF antagonist (SEQ
`ID N014) 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 speci?c embodiment, the VEGF antagonist (SEQ
`ID N014) is present at a concentration of about 40 mg/ml.
`In one embodiment, the stable liquid ophthalmic formula
`tion comprises 40 mg/ml 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. In a speci?c
`embodiment, the stable liquid ophthalmic formulation con
`sists essentially of 40 mg/ml 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.
`In another aspect, a lyophilizable formulation of a VEGF
`45
`antagonist is provided, wherein upon lyophilization followed
`by reconstitution, a stable liquid ophthalmic formulation as
`described herein is obtained.
`In another aspect, a lyophilizable formulation of a vascular
`endothelial growth factor (V EGF)-speci?c 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, treha
`lose, glycerol, or mannitol, pH about 5.8-7.0. In various
`embodiments, the VEGF antagonist (SEQ ID N014) is
`present at about 5, about 10, about 20, about 30, or about 40
`mg/ml. In a speci?c embodiment, the lyophilizable oph
`thalmic 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 formula
`tion further comprises sodium chloride. In a speci?c embodi
`ment, the sodium chloride is present at a concentration of
`about 20 mM. In another speci?c embodiment, the sodium
`chloride is present at a concentration of about 67.5 mM.
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`In another speci?c embodiment, the lyophilizable oph
`thalmic 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.
`In another embodiment, the lyophilizable ophthalmic for
`mulation comprises 5 mg/ml, 10 mg/ml, or 40 mg/ml VEGF
`antagonist, 5 mM sodium phosphate buffer, 0.015% polysor
`bate, 20 mM sodium chloride, and 2.5% sucrose, at pH 6.2
`6.3. In a speci?c embodiment, the lyophilizable ophthalmic
`formulation consists essentially of 5 mg/ml, 10 mg/ml, or 40
`mg/ml VEGF antagonist (SEQ ID N014), 5 mM sodium
`phosphate buffer, 0.015% polysorbate, 20 mM sodium chlo
`ride, and 2.5% sucrose, at pH 6.2-6.3.
`In another speci?c embodiment, the lyophilizable oph
`thalmic formulation comprises 20 mg/ml of the VEGF
`antagonist, 5 mM sodium phosphate buffer, 0.015% polysor
`bate, and 67.5 mM sodium chloride, pH about 6.2-6.3. In a
`more speci?c embodiment, the lyophilizable ophthalmic for
`mulation consists essentially of 20 mg/ml 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.
`In another speci?c embodiment, the lyophilizable oph
`thalmic 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 speci?c embodiment, the lyophilizable
`ophthalmic formulation consists essentially of 5 mg/ml, 10
`mg/ml, or 40 mg/leEGF antagonist (SEQ ID N014), 5 mM
`sodium phosphate buffer, 0.015% polysorbate, and 67.5 mM
`sodium chloride, pH about 6.2-6.3.
`Generally, the reconstituted formulation is about 2 times
`the concentration of the pre-lyophilized formulation, e.g., a
`20 mg fusion protein/ml pre-lyophilized formulation is
`reconstituted to a ?nal formulation of 40 mg fusion protein/
`ml.
`Generally, the lyophilized formulation is reconstituted
`with sterile water suitable for injection. In one embodiment,
`the reconstitution liquid is bacteriostatic water.
`In another aspect, the invention features a method of pro
`ducing a lyophilized formulation of a VEGF-speci?c 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.
`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-lyo
`philized formulation of a VEGF-speci?c fusion protein
`antagonist, (b) subjecting the pre-lyophilized formulation of
`step (a) to lyophilization; and (c) reconstituting the lyo
`philized formulation of step (b).
`The invention further features ophthalmic formulations
`provided in a pre-?lled syringe or vial, particularly suitable
`for intravitreal administration.
`Other objects and advantages will become apparent from a
`review of the ensuing detailed description.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present invention is not limited to particular methods,
`and experimental conditions described, as such methods and
`
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`Mylan v. Regeneron, IPR2021-00881
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`5
`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 limit
`ing unless indicated, since the scope of the present invention
`will be limited only by the appended claims.
`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 simi
`lar 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
`Safe handling and administration of formulations compris
`ing proteins represent signi?cant challenges to pharmaceuti
`cal formulators. Proteins possess unique chemical and physi
`cal 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.
`Chemical and physical stability can be promoted by
`removing water from the protein. Lyophilization (freeze-dry
`ing under controlled conditions) is commonly used for long
`term storage of proteins. The lyophilized protein is substan
`tially 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., ben
`zyl alcohol) prior to administration.
`
`DEFINITIONS
`
`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 syn
`thetic origin, such as, for example, peanut oil, soybean oil,
`mineral oil, sesame oil and the like.
`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, ?our, chalk, silica gel, sodium
`stearate, glycerol monostearate, talc, sodium chloride, dried
`skim milk, glycerol, propylene, glycol, water, ethanol and the
`like.
`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
`AVEGF antagonist is a compound capable of blocking or
`inhibiting the biological action of vascular endothelial growth
`factor (V EGF), and includes fusion proteins capable of trap
`ping 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. In speci?c embodiments, the VEGF antago
`nist 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 NO: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 NO:4.
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`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-speci?c fusion protein antagonist preparation
`used for making a formulation is VEGF fusion protein
`antagonist protein, more preferably at least 95%, most pref
`erably at least 99%. The fusion protein is preferably substan
`tially 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 effective formulation. Unless
`stated otherwise, the phosphates employed are sodium phos
`phates and a desired buffering pH is achieved by mixing
`appropriate amounts of mono- and dibasic sodium phosphate.
`Stable Liquid Ophthalmic Formulations
`In one aspect, the invention provides a stable pharmaceu
`tically acceptable formulation comprising a VEGF antago
`nist, wherein the formulation is a liquid formulation suitable
`for ophthalmic use. Preferably, the liquid formulation com
`prises 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 pharmaceuti
`cally 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.
`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 con
`tent by methods known in the art, e. g., UV spectroscopy, and
`purity is determined by, for example, SDS-PAGE, size-exclu
`sion HPLC, bioassay determination of activity, isoelectric
`focusing, and isoaspartate 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
`VEGF165 binding by the VEGF antagonist of the invention.
`Liquid formulations can be stored in an oxygen-deprived
`environment. Oxygen-deprived environments can be gener
`ated by storing the formulations under an inert gas such as, for
`example, nitrogen or argon. Liquid formulations are prefer
`ably stored at about 5° C.
`Ophthalmic Lyophilized Formulations
`In one aspect of the invention, an ophthalmically accept
`able formulation comprising a VEGF antagonist is provided,
`wherein the formulation is a lyophilizable formulation. Lyo
`philizable formulations can be reconstituted into solutions,
`suspensions, emulsions, or any other suitable form for admin
`istration or use. Lyophilizable 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 ordinary skill in the art, and typically includes sub
`limation of water from a frozen formulation under controlled
`conditions.
`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 tem
`perature (e.g., approximately 25° C.). Preferably, lyophilized
`formulations are stored below about 25° C., more preferably,
`at about 4-20° C.; below about 4° C.; below about —20° C.;
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`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.
`Lyophilized formulations are typically reconstituted for
`use by addition of an aqueous solution to dissolve the lyo
`philized formulation. A wide variety of aqueous solutions can
`be used to reconstitute a lyophilized formulation. Preferably,
`lyophilized formulations are reconstituted using water. Lyo
`philized 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 pharmaceuti
`cally acceptable carries can also be used.
`Freeze-dried or lyophilized formulations are typically pre
`pared from liquids, that is, from solutions, suspensions, emul
`sions, and the like. Thus, the liquid that is to undergo freeze
`drying or lyophilization preferably comprises all components
`desired in a ?nal reconstituted liquid formulation. As a result,
`when reconstituted, the freeze-dried or lyophilized formula
`tion will render a desired liquid formulation upon reconstitu
`tion.
`
`EXAMPLES
`
`Before the present methods are described, it is to be under
`stood 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 limit
`ing, since the scope of the present invention will be limited
`only to the appended claims.
`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.
`Unless de?ned otherwise, all technical and scienti?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 prac
`tice 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 50 mg/ml VEGF Trap Liquid
`Formulation Stored at 5° C. in 3 ml Glass Vials
`
`20
`
`25
`
`30
`
`35
`
`40
`
`50
`
`55
`
`An ophthalmic liquid formulation containing 50 mg/ml
`60
`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
`OD405 nm; and percent recovered protein and purity by size
`exclusion HPLC.
`
`65
`
`8
`TABLE 1
`
`Stability of50 rug/ml VEGF Trap Protein (VGFT—SS065)
`
`Visual
`Months Appearance
`
`Turbidity
`(OD405
`nm)
`
`% VEGF Trap
`Recovered
`
`pH
`
`% 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
`0.01
`0.01
`
`6.2
`6.2
`6.3
`6.3
`6.3
`6.3
`6.3
`
`100
`101
`100
`101
`104
`96
`105
`
`98.8
`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 5° C. in 3 ml Glass Vials
`
`A liquid formulation containing 50 mg/ml VEGF Trap
`(SEQ ID NO:4), 10 mM phosphate, 50 mM NaCl, 3% poly
`ethylene glycol 3350, 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 results are shown in Table 2. Turbid
`ity, percent recovered protein and purity was determined as
`described above.
`
`TABLE 2
`
`Stability of50 rug/ml VEGF Trap Protein (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
`0.00
`
`6.2
`6.1
`6.3
`6.3
`6.3
`6.3
`6.2
`
`100
`104
`99
`102
`103
`113
`106
`
`98.9
`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 5° C. in 3 ml Glass Vials
`
`A liquid formulation containing 40 mg/ml VEGF Trap
`(SEQ ID NO: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.
`
`TABLE 3
`
`Stability of40 rug/ml VEGF Trap Protein (VGFT—SS207)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`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
`6.2
`6.2
`6.4
`6.3
`
`100
`99
`98
`95
`
`99.5
`99.4
`99.5
`99.2
`
`Mylan Exhibit 1160
`Mylan v. Regeneron, IPR2021-00881
`Page 5
`
`
`
`US 8,092,803 B2
`
`9
`Example 4
`
`10
`TABLE 6
`
`Stability of 40 mg/ml VEGF Trap Liquid
`Formulation Stored at 5° C. in Pre-Filled Glass
`Syringe
`
`A liquid formulation containing 40 mg/ml VEGF trap
`(SEQ ID NO: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 pre?lled luer glass syringe with 4023/ 50 FLUROTECTM
`coated (elastomer coated) plunger and samples tested at 0.5,
`l, 2, 3, and 4 months. Stability results are shown in Table 4.
`Turbidity, percent recovered protein and purity was deter
`mined as described above.
`
`Stability of40 rug/ml VEGF Trap Protein (VGFT—SS203)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`0
`0.5
`1
`2
`3
`4
`5
`
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`Pass
`
`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
`
`100
`101
`101
`i
`102
`103
`99
`
`99.2
`99.2
`99.2
`i
`99.1
`98.8
`98.9
`
`TABLE 4
`
`Example 7
`
`Stability of 40 mg/ml VEGF Trap Protein (VGFT—SS207)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`20
`
`Stability of Lyophilized 20 mg/ml VEGF Trap
`Formulation Stored at 5° C. in 3 ml Glass Vials and
`Reconstituted to 40 mg/ml
`
`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
`
`A liquid formulation containing 40 mg/ml VEGF trap
`(SEQ ID NO: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, l, 2, 3, and 4 months. Stability
`results are shown in Table 5. Turbidity, percent recovered
`protein and purity was determined as described above.
`
`TABLE 5
`
`Stability of 40 mg/ml VEGF Trap Protein (V GFT—SS203)
`
`Visual
`Months Appearance Turbidity pH
`
`% VEGF Trap
`Recovered
`
`% VEGF Trap
`Native
`Con?guration
`
`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
`
`0.8 ml of a liquid formulation containing 20 mg/ml VEGF
`trap (SEQ ID NO:4), 5 mM phosphate, 20 mM NaCl, 0.015%
`polysorbate 20, 2.5% sucrose, ande 6.3, were lyophilized in
`3 ml glass vials. Samples were stored at 5° C. and tested at l,
`and 2 months. VEGF trap was reconstituted to a ?nal concen
`tration of 40 mg/ml VEGF Trap (?nal volume of 0.4 ml).
`Stability results are shown in Table 7 (t?ime in months;
`*q/isual appearance; **:reconstitution time). Turbidity, per
`cent recovered protein and purity was determined as
`described above.
`
`TABLE 7
`
`Stability of Lyophilized 20 mg/ml VEGF Trap Protein (V GFT—SS216)
`
`Vis.
`App.*
`
`Pass
`Pass
`Pass
`
`t
`
`0
`1
`2
`
`Recon.
`Time**
`(min)
`
`Vis. App. *
`Reconst’d
`Liquid
`
`% VEGF % VEGF
`Trap Re— Trap Native
`Tur-
`bidity pH covered
`Con?g.
`
`0.6
`0.6
`0.4
`
`Pass
`Pass
`Pass
`
`0.00 6.3
`0.01
`6.3
`0.01
`6.2
`
`100
`106
`103
`
`99.5
`99.4
`99.3
`
`25
`
`30
`
`35
`
`40
`
`Example 8
`
`Stability of Lyophilized 20 mg/ml VEGF Trap
`Formulation Stored at 5° C. in 3 ml Glass Vials
`
`0.8 ml of a liquid formulation containing 20 mg/ml VEGF
`trap (SEQ ID N