AU2012202859C1 - VEGF antagonist formulations suitable for intravitreal administration - Google Patents

Abstract

#$%^&*AU2012202859C120250807.pdf##### ABSTRACT Ophthalmic formulations of a vascular endothelial growth factor (VEGF)-specific fusion protein antagonists are provided suitable for intravitreal administration to the eye. The ophthalmic formulations include a stable liquid formulation and a lyophilizable formulation. 5 Preferably, the protein antagonist has the amino acid sequence shown in SEQ ID NO:4. 2293065.1 ABSTRACT Ophthalmic formulations of a vascular endothelial growth factor (VEGF)-specific fusion 2012202859 16 May 2012 protein antagonists are provided suitable for intravitreal administration to the eye. The ophthalmic formulations include a stable liquid formulation and a lyophilizable formulation. 5 Preferably, the protein antagonist has the amino acid sequence shown in SEQ ID NO:4. 2293065.1

Description

AUSTRALIA AUSTRALIA 2012202859 16 2012

Act Patents Act Patents

May COMPLETE SPECIFICATION COMPLETE SPECIFICATION (ORIGINAL) (ORIGINAL)

Class Class Int. Class Int. Class

Application Number: Application Number: Lodged: Lodged:

CompleteSpecification Complete Lodged: SpecificationLodged: Accepted: Accepted: Published: Published:

Priority Priority

Related Art: Related Art:

NameofofApplicant: Name Applicant:

Pharmaceuticals, RegeneronPharmaceuticals, Regeneron Inc. Inc.

Actual Inventor(s): Actual Inventor(s):

Dix, Kenneth Daniel Dix, Daniel S.Graham, Kenneth S. Frye,Eric KellyFrye, Graham,Kelly Furfine EricFurfine

for Service Addressfor Address Service and Correspondence: andCorrespondence:

PHILLIPS ORMONDE PHILLIPS FITZPATRICK ORMONDE FITZPATRICK and Trade Patent and Patent Attorneys MarkAttorneys TradeMark 367 Collins Street 367 Collins Street

Melbourne 3000 Melbourne AUSTRALIA 3000 AUSTRALIA

Title: InventionTitle: Invention

ANTAGONIST VEGF ANTAGONIST VEGF FORMULATIONS FORMULATIONS SUITABLE SUITABLE FOR INTRAVITREAL FOR INTRAVITREAL ADMINISTRATION ADMINISTRATION

Our Ref Our 941949 Ref: : 941949 POF POF Code: 134671/134671 Code: 134671/134671

The The following statement following statement is is a full a full description of of description this this invention, invention, including bestbest the the including method method of of performing it known performing it known to applicant(s): to applicant(s):

-1- - 1 - -

6006q

2012202859 16 2012

ANTAGONIST FORMULATIONS VEGF ANTAGONIST VEGF SUITABLE FORMULATIONSSUITABLE FOR INTRAVITREAL FOR ADMINISTRATION INTRAVITREALADMINISTRATION May

Thepresent The presentapplication a divisional is aisdivisional application application from from application Australian Australian patent application patent application number number 2007261536, 5 2007261536, 5 the entire the entire disclosure disclosure of which of which is incorporated is incorporated herein herein by reference. by reference.

BACKGROUNDOFOFINVENTION BACKGROUND INVENTION of the Field of Field the Invention Invention

[0001] The present invention is directed to pharmaceutical formulations suitable for

[0001] The present invention is directed to pharmaceutical formulations suitable for

10 intravitreal 10 agents comprisingagents administrationcomprising intravitrealadministration capable of of capable inhibiting vascular inhibiting growth endothelial growth vascularendothelial factor (VEGF), factor to methods and to (VEGF), and methodsfor makingand formaking using andusing such such formulations.TheThe formulations. invention invention

includes liquid pharmaceutical formulations having increased stability, as well as formulations includes liquid pharmaceutical formulations having increased stability, as well as formulations

maybebe that may that lyophilize andand lyophilize reconstituted reconstituted for intravitreal for intravitreal administration. administration.

15 Statement 15 of of Statement Related Related ArtArt

Vascularendothelial

[0002] Vascular

[0002] factor (VEGF) growthfactor endothelial growth (VEGF)expression is isnearly expression ubiquitousinin human nearlyubiquitous human cancer, with its consistent with cancer, consistent itsrole asas role a key a key mediatorofof mediator tumor neoangiogenesis. Blockade tumorneoangiogenesis. of Blockade of

VEGF bindingtoto the function,bybybinding VEGFfunction, moleculeoror its the molecule its VEGFR-2 VEGFR-2 receptor, inhibits growth receptor,inhibits of growth of

implanted tumor cells in multiple different xenograft models (see, for example, Gerber et al. implanted tumor cells in multiple different xenograft models (see, for example, Gerber et al.

20 (2000) 20 (2000) Cancer Cancer Res. Res. 60:6253-6258). 60:6253-6258). A soluble A soluble VEGF-specific VEGF-specific fusion fusion proteinprotein antagonist, antagonist,

termed aa"VEGF termed "VEGF trap"has trap" been hasbeen described described (Kim (Kim et al.(2002) et al. Proc.Nat]. (2002)Proc. Nat].Acad. Sci.USA Acad.Sci. USA 99:11399-404; Holashetetal. 99:11399-404;Holash (2002) Proc. al. (2002) NatI. Acad. Proc. Natl. Sci. USA Acad. Sci. 99:11393-8). USA 99:11393-8).

[0003] Ophthalmic

[0003] Ophthalmicformulations formulationsare known, areknown, seesee forfor example, example, U.S. U.S. 7,033,604 and and 7,033,604 6,777,429. 6,777,429.

An ophthalmic An formulationofof aa VEGF ophthalmicformulation VEGFantibody antibody is is describedininUSUS6,676,941. described 6,676,941. 25 [0004] 25 [0004] Lyophilization Lyophilization (freeze (freeze drying drying under under controlled controlled conditions) conditions) is is commonly commonly usedused for long for long-

term storage of proteins. The lyophilized protein is substantially resistant to degradation, term storage of proteins. The lyophilized protein is substantially resistant to degradation,

aggregation, oxidation, aggregation, oxidation, andand other other degenerative degenerative processes processes while while in the freeze-dried state (see, state (see, in the freeze-dried for example, for U.S. 6,436,897). example, U.S. 6,436,897).

30 30 BRIEF BRIEF SUMMARY SUMMARY OFOF THE THE INVENTION INVENTION

[0005] Stable

[0005] Stableformulations formulations of VEGF-specificfusion of aa VEGF-specific fusionprotein protein antagonist are provided. antagonist are provided. acceptableformulations Pharmaceuticallyacceptable Pharmaceutically formulationsare areprovided thatcomprise providedthat comprisea aVEGF VEGF "trap" "trap"

antagonist witha pharmaceutically antagonist with a pharmaceutically carrier.carrier. acceptable acceptable In specific In specific embodiments, liquid and liquid and embodiments, lyophilized formulations lyophilized formulationsareare provided. provided.

la la 2293065.1 2293065.1

2012202859 09 Sep 2014

[0005a] In one aspect, the invention provides An ophthalmic formulation of a vascular

endothelial growth factor (VEGF) antagonist, comprising

(a) about 1-100 mg/ml a VEGF antagonist comprising the amino acid sequence of

SEQ ID NO:4;

5 (b) 0.03% of polysorbate;

(c) 40 - 135 mM of sodium chloride; 2012202859

(d) 10 mM of sodium phosphate buffer; and

(e) 5% of sucrose, pH about 6.2-6.3.

[0005b] In a further aspect, the invention provides a lyophilizable formulation of a

10 vascular endothelial growth factor (VEGF) antagonist, comprising

(a) about 20 mg/ml of the VEGF antagonist comprising the amino acid sequence

of SEQ ID N0:4;

(b) about 5 - 10 mM of sodium phosphate buffer, pH about 5.8-7.0;

(c) about 0.015 0.03% polysorbate and about 0.1% polyethylene glycol (PEG);

15 (d) about 2.5% sucrose, pH about 6.2-6.3.

[0006] In a further 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 Ig domain 3 of a second VEGF receptor, and a multimerizing component (also

20 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 NO:2 or

SEQ ID NO:4. Preferably,

1b

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2012202859 16 May

the VEGF the VEGF antagonist antagonist is aisdimer a dimer comprising comprising two fusion two fusion proteins proteins of SEQ of IDSEQ NO:4.ID NO:4.

[0007] InIn one

[0007] oneaspect, aspect,a a stable stable liquidophthalmic liquid ophthalmic formulation formulation is provided is provided that that comprises comprises 1-100 1-100 mg/miVEGF-specific mg/ml VEGF-speclfic fusion fusion protein protein antagonist, antagonist, 0.01of-5% 0.01-5% one of or one moreor more co-solvent(s), organic organic co-solvent(s), 30-150mMmM 30-150 of one of one or more or more tonicity tonicity agent(s), agent(s), 5-40 5-40 mM ofmM of a buffering a buffering agent, agent, and optionally, and optionally, 1.0- 1.0 7.5%ofofaastabilizing 7.5% stabilizing agent, agent,pHpHbetween between about about 5.8-7.0. 5.8-7.0.

[0008] InInone

[0008] oneoror more more specific specific embodiments, embodiments, the organic the organic co-solvent co-solvent may be polysorbate, may be polysorbate, for for example,polysorbate example, polysorbate 20 polysorbate 20 or or polysorbate 80, polyethylene 80, polyethylene glycolglycol (PEG), (PEG), for example, for example, PEG 3350,PEG 3350, or propylene or glycol,oror aacombination propylene glycol, combination thereof; thereof; thethe tonicityagent tonicity agent maymay be, example, be, for for example, sodium sodium

chloride or chloride or potassium potassiumchloride; chloride;the thestabilizing stabilizingagent agent may may be sucrose, be sucrose, sorbitol, sorbitol, glycerol, glycerol,

trehalose, or trehalose, or mannitol; mannitol;and andthethebuffering bufferingagent agent maymay be, example, be, for for example, phosphate phosphate buffer. buffer. In a In a specific specific embodiment, embodiment, thethe phosphate phosphate buffer buffer is a is a sodium sodium phosphate phosphate buffer. buffer.

10009] InInvarious

[0009] variousembodiments, embodiments, the organic the organic co-solvent co-solvent is polysorbate is polysorbate and/or and/or PEG, thePEG, the stabilizing stabilizing agent is sucrose, agent is the buffering agent is phosphate buffer, and the tonicity agent sucrose, the buffering agent is phosphate buffer, and the tonicity agent is Is

sodium chloride. sodium chloride.

(0010] More

[0010] More specifically, specifically, thethe stable stable liquidophthalmic liquid ophthalmic formulation formulation comprises comprises about about 40-50 40-50 mg/ml mg/ml of the of theVEGF antagonist (SEQ VEGF antagonist ID NO:4), (SEQ ID NO:4), about about 10 10 mM mMphosphate phosphate buffer, 0.01-3% buffer, 0.01-3%polysorbate polysorbate and/or PEG, and/or PEG,40-135 40-135 mM sodium mM sodium chloride, chloride, and optionally and optionally 5.0% sucrose, 5.0% sucrose, pH about pH about 6.2-6.3. 6.2-6.3.

[0011] InIna aspecific

[0011] specificpreferred preferredembodiment, embodiment, the stable the stable liquid liquid ophthalmic ophthalmic formulation formulation comprises comprises

about 50 about 50 mg/mI of the mg/ml of the VEGF antagonist (SEQ VEGF antagonist (SEQ ID ID NO:4), NO:4), 10 10 mM mMsodium sodium phosphate phosphate buffer,5050 buffer, mM sodiumchloride, mM sodium chloride, 0.1% 0.1% polysorbate, polysorbate, and and 5% 5%sucrose, sucrose, pHpHabout about6.2-6.3. 6.2-6.3.

[0012] InIna aspecific

[0012] specificpreferred preferredembodiment, embodiment, the stable the stable liquid liquid ophthalmic ophthalmic formulation formulation comprises comprises

about 50 about mg/ml of 50 mg/ml of the the VEGF antagonist (SEQ VEGF antagonist (SEQIDID NO:4), NO:4), 10 10 mM mMsodium sodium phosphate phosphate buffer,5050 buffer, mMsodium mM sodiumchloride, chloride, 3% 3% PEG, PEG,and and 5%5% sucrose, sucrose, pH pH about about 6.2-6.3. 6.2-6.3.

[0013] InIna aspecific

[0013] specificpreferred preferredembodiment, embodiment, the stable the stable liquid liquid ophthalmic ophthalmic formulation formulation comprises comprises

about 40 about mg/ml of 40 mg/ml of the the VEGF antagonist (SEQ VEGF antagonist (SEQIDID NO:4), NO:4), 10 10 mM mMsodium sodium phosphate phosphate buffer,4040 buffer, mMsodium mM sodiumchloride, chloride, 0.03% 0.03%polysorbate, polysorbate, and and 5% 5%sucrose, sucrose,pHpHabout about6.2-6.3. 8.2-6.3.

[0014] InIna aspecific

[0014] specificpreferred preferredembodiment, embodiment, the stable the stable liquid liquid ophthalmic ophthalmic formulation formulation comprises comprises

about about 40 mg/ml of 40 mg/ml of the the VEGF antagonist (SEQ VEGF antagonist (SEQIDID NO:4), NO:4), 10 10 mM mMsodium sodium phosphate phosphate buffer,135 buffer, 135 mMsodium mM sodium chloride, chloride, and and 0.03% 0.03% polysorbate, polysorbate, pH6.2-6.3. pH about about 6.2-6.3.

[0015] Inanother

[0015] In anotheraspect, aspect, a stable a stable liquidophthalmic liquid ophthalmic formulation formulation is provided is provided that that comprises comprises 1- 1 100 mg/ml 100 mg/mlVEGF-specific VEGF-specific fusion fusion protein protein antagonist; antagonist; 0.01-5% 0.01-5% of one of one or more or more co- organic organic co solvent(s); 5-40 solvent(s); 5-40 mM mMof of a buffering a buffering agent; agent; andand optionally optionally 30-150 30-150 mM of mM one of or one more or more tonicity tonicity

agent(s) and/or1.0-7.5% agent(s) and/or 1.0-7.5% of of a stabilizingagent; a stabilizing agent; having having a pHa between pH between about about 5.8-7.0. 5.8-7.0.

[0016] InIn various

[0016] variousembodiments, embodiments, the VEGF antagonist (SEQ ID NO:4) is present at a the VEGF antagonist (SEQ ID NO:4) is present at a

concentration concentration ofofabout about1010 to to about about 80 80 mg/ml. mg/ml. In various In various embodiments, embodiments, the VEGF the VEGF antagonist antagonist

(SEQ (SEQ IDID NO:4) NO:4) is present is present at aatconcentration a concentration of about of about 10, about 10, about 20, about 20, about 30,40, 30, about about 40, about about

50, about 50, about60, 60,about about70, 70, or about 80 mg/mI. In a preferred embodiment, the VEGF antagonist or about 80 mg/ml. In a preferred embodiment, the VEGF antagonist

2

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(SEQ ID NO:4) is present (SEQ ID NO:4) at aatconcentration Is present of about of about a concentration 40 mg/m. 40 mg/ml.

[0017] In another embodiment, the stabilizing agent is selected agent is selected moreorofmore one orone from from of sucrose, sucrose,

[0017] In another embodiment, the stabilizing sorbitol, glycerol, sorbitol, trehalose, and glycerol,trehalose, mannitol. and mannitol.

[0018] In another embodiment, the organic co-solvent is selected

[0018] In another embodiment, the organic co-solvent is selected from one more or or one from of more of polysorbate, for example, polysorbate 20 or polysorbate 80, polyethylene glycol (PEG), for polysorbate, for example, polysorbate 20 or polysorbate 80, polyethylene glycol (PEG), for

example, PEG example, 3350,and PEG3350, glycol. propyleneglycol. andpropylene

[0019] In another embodiment, the buffer (0019] In another embodiment, is a is the buffer a phosphate phosphate buffer, buffer, for example, for example, sodium sodium

phosphate. phosphate.

[0020] In another embodiment, the tonicity agent is a is salt, for for a salt, example, example, sodium sodium chloride. chloride.

[0020] In another embodiment, the tonicity agent

[0021] In one embodiment, the stable liquid

[0021] in one embodiment, the stable ophthalmic liquid formulation formulation ophthalmic comprises comprises 10 mM 10 mM sodium sodium phosphate buffer, about 0.03 to about 0.1%0.1% phosphate buffer, about 0.03 to about polysorbate and/or and/or polysorbate about about 3% PEG or PEG or propylene 3%propylene glycol, about 40 mM sodium chloride, and about glycol, about 40 mM sodium chloride, and about 5% sucrose. In a specific In a specific 5% sucrose. embodiment, embodiment, the the liquid ophthalmic formulation comprises 10sodium stable liquid ophthalmic formulation comprises 10 mM stable mM sodium phosphate phosphate buffer, buffer, about about 0.03% 0.03%

polysorbate, about 40 mM sodium polysorbate, about 40 mM sodium chloride, chloride, and about about and 5% sucrose. In anotherIn 5% sucrose. another specific specific

embodiment, thethe embodiment, pH pH of the formulation of the is about is about formulation 6.2 to to about 6.2about 6.3. In In another 6.3.another specific specific

embodiment, the pH is achieved embodiment, the pH is by mixing achieved mono- mono- by mixing and dibasic sodium phosphate dibasicphosphate and sodium to to the desired the desired pH without pH acid/base withoutacid/base titration. titration.

[0022] In a specific embodiment, the the

[0022] In a specific embodiment, stable liquid stable ophthalmic ophthalmic liquid formulation formulation consists consists of essentially essentially of

a VEGF antagonist a VEGF antagonist(SEQ NO:4)atat40 (SEQIDIDNO:4) mg/ml,10 40mg/ml, sodium mMsodium 10mM phosphate phosphate buffer, polysorbate buffer,polysorbate at at 0.03%, sodium 0.03%, sodium chloride chloride mM, mM, 40 40 at at and sucrose and sucrose pH5%, at 5%, at pH 6.2-6.3. 6.2-6.3.

[0023] In another aspect, a stable liquid

[0023] In another aspect, a stable ophthalmic liquid formulation formulation ophthalmic is provided that that is provided comprises comprises

about about 10 to about 10 to about 80 80 mg/mI mg/ml VEGF VEGF antagonist, about 10 antagonist, about mMsodium 10 mM phosphate sodiumphosphate buffer, about buffer,about 0.03% 0.03%polysorbate, andand polysorbate, about about 135 mM mM sodium 135sodium toof pH of 6.2pH chloride,chloride, 6.2 6.3. to 6.3.

[0024] In various embodiments,

[0024] in various embodiments, the VEGF the VEGF antagonist (SEQ ID (SEQ antagonist NO:4) is NO:4) Is ID present at present a at a concentration of about concentration of about1010to to about 80 80 about mg/ml. mg/ml. In various In various embodiments, the VEGF the embodiments, VEGF antagonist antagonist

(SEQ ID NO:4) is present at a concentration of about 10, about 20, about 30, about 40, about (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 50, about 60, about 70, or 80 80 about mg/ml. In a In mg/mi. a specific specific embodiment, embodiment, the the VEGF VEGF antagonist antagonist

(SEQ IDID (SEQ NO:4) is present is present NO:4) at a a concentration atconcentration of about of about 40 mg/ml. 40 mg/ml.

[0025] In one embodiment, the stable liquid ophthalmic formulation comprises 40 mg/ml of

[0025] In one embodiment, the stable liquid ophthalmic formulation comprises 40 mg/ml of

VEGF VEGFantagonist antagonist(SEQ NO:4),1010mMmM (SEQIDIDNO:4), sodium sodium phosphate phosphate buffer, buffer, 0.03% 0.03% polysorbate, polysorbate, and 135135 and mM sodium chloride at pH mM sodium chloride at 6.2-6.3. In a In pH 6.2-6.3. a specific specific embodiment, embodiment, the stable liquid ophthalmic stable ophthalmic the liquid formulation consists essentially ofof4040 formulation consists essentially mg/ml of of mg/ml VEGF VEGF antagonist antagonist (SEQ (SEQ ID ID10NO:4), NO:4), 10 mM mM sodium sodium phosphate buffer, 0.03% polysorbate, phosphate buffer, 0.03% andmM135 and 135 polysorbate, sodium chloride sodium at mM chloride at pH pH 6.2-6.3. 6.2-6.3.

[0026] In another aspect, a lyophilizable formulation of a formulation a VEGF ofVEGF antagonist antagonist is provided, whereinwherein is provided,

[0026] In another aspect, a lyophilizable ophthalmic liquidophthalmic stableliquid reconstitution,a astable as as formulation upon lyophilization followedbybyreconstitution, upon lyophilization followed formulation

describedherein described obtained. hereinisisobtained.

[0027] In another

[0027] In anotheraspect, a lyophilizable aspect, formulation a lyophilizable a vascular of aofvascular formulation endothelial endothelial growth growth factor factor

3

WO 2007/149334 wo 2007/149334 PCT/US2007/014085 PCT/US2007/014085 2012202859 16 May 2012

(VEGF)-specific fusion (VEGF)-specific fusionprotein antagonist antagonist protein is provided, is provided, comprising 5-50 5-50 comprising of the of mg/ml mg/mi the VEGF VEGF antagonist, 5-25 antagonist, 5-25mMmM buffer, such buffer, as phosphate as phosphate such buffer, buffer, 0.01 0.01 to to 0.15% 0.15% of one or of more one or of more an of an organic co-solvent, suchas as organic co-solvent, such polysorbate, polysorbate, propylene propylene glycol glycol and/or and/or PEG, PEG. and optionally 1-10% of1-10% and optionally of a a stabilizing stabilizing agent sucrose, suchasassucrose, agent such sorbitol,trehalose, sorbitol, glycerol,orormannitol, trehalose,glycerol, pH pH mannitol, about about 5.8-7.0. 5.8-7.0.

In various embodiments, the the In various embodiments, VEGFVEGF antagonist antagonist NO:4) ID (SEQ ID(SEQ presentisat is NO:4) present atabout about 5, 5, about10, about 10, about 20, about 30,ororabout about 20, about 30, 40 40 about mg/ml. In a In mg/ml. a specific specific embodiment, embodiment, the lyophilizable the lyophilizable ophthalmic ophthalmic

formulation formulationofofthe theinvention comprises Inventioncomprises 20 mg/ml 20 mg/ml of the the VEGF of VEGF antagonist, antagonist, 10 mM 10 mM sodium sodium phosphate buffer, 0.03% phosphate buffer, 0.03% polysorbate, 0.1% PEG, polysorbate, 0.1% PEG, and 2.5%sucrose, and 2.5% pHabout sucrose, pH In 6.2-6.3. In about6.2-6.3. further embodiments, further embodiments, thethe lyophilizable lyophilizable formulation formulation further further comprises sodiumsodium comprises chloride. chloride. In a In a specific specific embodiment, embodiment, thethe sodium sodium chloride chloride is present at a at is present a concentration concentration of about 20 mM. 20 of about In mM. In another specific another embodiment, specificembodiment, the the sodium sodium chloride chloride is present is present at a concentration at a concentration of about about 67.5 of67.5 mM. mM.

[0028]

[0028] InIn another anotherspecific embodiment, specificembodiment, the lyophilizable the lyophilizable ophthalmic ophthalmic formulation of the of formulation the invention invention

comprises comprises 20 mg/ml of 20 mg/ml the VEGF of the antagonist, 55 mM VEGF antagonist, phosphate sodiumphosphate mMsodium buffer, 0.015% buffer,0.015% polysorbate,2020mMmM polysorbate, sodium sodium chloride, chloride, and 2.5% pH aboutpH 2.5% sucrose, and sucrose, about 6.2-6.3. 6.2-6.3.

[0029] InInanother

[0029] embodiment, anotherembodiment, the lyophilizable the lyophilizable ophthalmic ophthalmic formulation formulation comprises 10mg/ml, 10 5 mg/ml, 5 comprises mg/ml, or 40 mg/ml, or mg/ml VEGF 40 mg/mI antagonist, 55 mM VEGF antagonist, mMsodium phosphate sodiumphosphate buffer, 0.015% buffer,0.015% polysorbate, 20 polysorbate,20 mM sodium mM sodium chloride, 2.5% 2.5% and and chloride, sucrose, sucrose, at pH 6.2-6.3. at pH 6.2-6.3. In a specific In a specific embodiment, embodiment, the the lyophilizable lyophilizable ophthalmic formulation ophthalmicformulation consists consists essentially of of essentially 5 mg/ml, 5 mg/ml, 10 mg/ml, or 40 or 10 mg/ml, 40 mg/mI mg/ml

VEGF antagonist(SEQ VEGF antagonist NO:4),5 5mMmM (SEQID IDNO:4), sodium sodium phosphate phosphate buffer, buffer, 0.015% 0.015% polysorbate, polysorbate, 20 20 mMmM

sodium and chloride,and sodiumchloride, 2.5% 2.5% sucrose, sucrose, pH 6.2-6.3. at pHat6.2-6.3.

[0030] In another specific embodiment, the the lyophilizable

[0030] In another specific embodiment, lyophilizable ophthalmic ophthalmic formulation formulation comprises comprises 20 20 mg/ml mg/ml of the VEGF of the antagonist, 55 mM VEGF antagonist, mM sodium phosphate sodiumphosphate buffer, 0.015%polysorbate, buffer, 0.015% 67.5 and67.5 polysorbate, and sodium mMsodium mM chloride, chloride, pH about pH about 6.2-6.3. 6.2-6.3. In a more more specific In a specific embodiment, embodiment, the lyophilizable the lyophilizable

ophthalmic formulation ophthalmicformulation consists consists essentially essentially of 20 of 20 mg/ml mg/ml of the VEGF VEGF of the antagonist NO:4), ID (SEQ ID (SEQ antagonist NO:4), 5 mM 5 mM sodium phosphate sodiumphosphate buffer,0.015% buffer, polysorbate,and 0.015%polysorbate, 67.5mM and67.5 sodium mMsodium chloride, 6.2-6.3. chloride,pHpH6.2-6.3.

[0031]

[0031]InInanother anotherspecific embodiment, specificembodiment, the lyophilizable the lyophilizable ophthalmic ophthalmic formulation formulation comprises comprises 5 5 mg/ml, 10 mg/ml, mg/ml, 10 or 40 mglml, or mg/ml VEGF 40 mg/mI VEGF antagonist, sodium mMsodium antagonist, 55 mM phosphate phosphate buffer, 0.015% buffer,0.015% polysorbate, and polysorbate,and 67.5 67.5 mM mM sodium sodium chloride, chloride, pH about about 6.2-6.3. pH 6.2-6.3. In a more a more specific In specific embodiment, embodiment,

the the lyophilizable formulation ophthalmicformulation lyophilizable ophthalmic consists consists essentially essentially of 5 5 mg/ml, ofmg/ml, 10 mg/ml, or 40 or 10 mg/mi, 40 mg/ml mg/mI VEGF antagonist(SEQ VEGFantagonist NO:4),5 5mMmM (SEQIDIDNO:4), sodium sodium phosphate phosphate buffer, buffer, 0.015% 0.015% polysorbate, polysorbate, andand mM 67.5 mM 67.5 sodium sodium chloride, chloride, pH about pH about 6.2-6.3. 6.2-6.3.

(0032]Generally,

[0032] reconstituted Generally,thethereconstituted formulation formulation is about is about 2 times 2 times the concentration the concentration the of theofpre- pre lyophilized formulation, lyophilized 20 mg e.g., aa 20 formulation, e.g., pre-lyophilized protein/mlpre-lyophilized fusionprotein/ml mgfusion formulation formulation is is reconstitutedto reconstituted final formulation to aa final of 40 formulation of mgfusion 40 mg protein/mi. fusionprotein/ml.

[0033]

[0033] Generally, lyophilizedformulation Generally,thethelyophilized is is formulation reconstituted reconstituted with with sterile water sterilewater suitable forfor suitable injection. In one injection. In embodiment, one embodiment, the the reconstitution reconstitution liquid is is liquid bacteriostatic bacteriostatic water. water.

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a lyophilized

[0034] In another aspect, the invention features a method of producing a lyophilized formulation formulation 10034] In another aspect, the invention features a method of producing subjecting of a VEGF-specific fusion protein antagonist, comprising subjecting the lyophilizable the lyophilizable formulation formulation of a VEGF-specific fusion protein antagonist, comprising of the invention to lyophilization to generate a lyophilized formulation. The lyophilized of the invention to lyophilization to generate a lyophilized formulation. The lyophilized

formulation may be lyophilized by any method known in the art forart formulation may be lyophilized by any method known in the for lyophilizing lyophilizing a liquid. a liquid.

of producing

[0035] In another related aspect, the invention features a method of producing a reconstituted a reconstituted

[00351 In another related aspect, the invention features a method the lyophilized lyophilized formulation of a VEGF antagonist, comprising reconstituting lyophilized formulation of a VEGF antagonist, comprising reconstituting the lyophilized

oneone formulation of the invention to a reconstituted formulation. In In formulation of the invention to a reconstituted formulation. embodiment, embodiment, the reconstituted the reconstituted

of the formulation is twice the concentration of the pre-lyophilized formulation, e.g., the method 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 invention comprises: (a) producing a pre-lyophilized formulation of a VEGF-specific fusion

and protein antagonist, (b) subjecting the pre-lyophilized formulation of step (a) to lyophilization; protein antagonist, (b) subjecting the pre-lyophilized formulation of step (a) to lyophilization; and

(c) reconstituting lyophilized formulation the lyophilized (c) reconstituting the ofstep formulation of (b). step(b). in a pre-filled

[0036] The invention further features ophthalmic formulations provided in a pre-filled

[0036] The invention further features ophthalmic formulations provided syringe syringe or or vial, particularly suitable for intravitreal administration. vial, particularly suitable for intravitreal administration.

[0037] Other objects and advantages will become apparent from a review aofreview (0037] Other objects and advantages will become apparent from of the ensuing the ensuing

description. detailed description. detailed

DETAILED DESCRIPTION DETAILED THE INVENTION OF THE DESCRIPTION OF INVENTION and and methods,

[0038] The present invention is not limited to particular methods, experimental experimental conditions conditions

[0038] The present invention is not limited to particular described, as such methods and conditions may Itvary. described, as such methods and conditions may vary. It istoalso is also to be understood be understood the that the that

terminology used herein is for the purpose of describing particular embodiments only, and is not is not terminology used herein is for the purpose of describing particular embodiments only, and intended to be limiting unless indicated, since the scope of the present invention will be limited intended to be limiting unless indicated, since the scope of the present invention will be limited

only by the only by appended the appended claims. claims. used herein have

[0039] Unless stated otherwise, all technical and scientific terms and phrases

[0039] Unless stated otherwise, all technical and scientific terms and phrases used herein have

the same meaning as commonly understood the same meaning as commonly understood by one of by ordinary ordinary one ofskill skillartin to in the art tothewhich the thewhich invention belongs. Although any methods and materials similar or equivalent to those described 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 herein can be used in the practice or testing of the present invention, the preferred methods and

arenow materials are materials described. nowdescribed.

Description General Description General of formulations

[0040] Safe handling and administration of formulations comprising comprising proteins proteins represent represent (0040] Safe handling and administration Proteins significant challenges to pharmaceutical formulators. Proteins possess chemicalchemical unique unique possess and and significant challenges to pharmaceutical formulators. pathways exist for physical properties that present stability problems: a variety of degradation physical properties that present stability problems: a variety of degradation pathways exist for

proteins, implicating both chemical and physical instability. Chemical instability includes proteins, implicating both chemical and physical instability. Chemical instability includes

deamination, aggregation, clipping thethe deamination, aggregation, clipping of of peptide peptide backbone, backbone, and oxidation and oxidation of methionine of methionine

residues. Physical instability encompasses many many residues. Physical instability encompasses phenomena, including,including, phenomena, for example, for example,

and/or aggregationand/or aggregation precipitation. precipitation.

be promoted

[0041] Chemical and physical stability can be promoted by removing by removing water from the from water the protein. protein.

[0041] Chemical and physical stability can

2007/149334 WO2007/149334 wo PCT/US2007/014085 PCT/US2007/014085 2012202859 16 2012

May

under (freeze-dryingunder Lyophilization (freeze-drying Lyophilization controlled controlled conditions) is is conditions) commonly commonly used for for long-term usedlong-term storageofofproteins. storage The proteins. The lyophilized lyophilized protein is is protein substantiallyresistant substantially toto resistant degradation, degradation,

aggregation, aggregation,oxidation, and oxidation,and other other degenerative degenerative processes in the in while while processes freeze-dried state. state. the freeze-dried The The lyophilized protein may lyophilized protein reconstituted maybebereconstituted with with water water optionally optionally containing containing a bacteriostatic a bacteriostatic

preservative(e.g., preservative benzylalcohol) (e.g., benzyl priortoto administration. alcohol)prior administration.

Definitions Definitions

[0042] The

[0042] term Theterm "carrier" "carrier" includes includes a diluent, a diluent, adjuvant, adjuvant, excipient, or or excipient, vehicle vehicle with with which which a a administered. composition isisadministered. composition can can Carriers Carriers include include sterile sterile liquids, as, as, suchsuch liquids, for for example, waterwater example, oils, Including andoils, and including oils of petroleum, oils of petroleum, animal, vegetable animal,vegetable or or synthetic synthetic origin,such origin, forfor as,as, such example, example,

peanut oil, soybean peanutoil, oil, mineral soybean oil, oil, sesame mineraloil, sesame oiloiland and the the like. like.

term Theterm

[0043] The

[0043] "excipient" "excipient" includes includes a non-therapeutic a non-therapeutic added added agentagent to a pharmaceutical to a pharmaceutical

provide compositiontotoprovide composition a desired a desired consistency consistency or stabilizing or stabilizing effect. effect. Suitable Suitable pharmaceutical pharmaceutical

excipients include, excipients for example, Include, for example,starch, glucose, starch,glucose, lactose, lactose, sucrose, sucrose, gelatin, gelatin, malt, malt, rice, rice, flour,chalk, flour, chalk, gel,sodium silica gel, silica stearate, glycerol sodium stearate, monostearate, glycerol monostearate, talc, talc, sodium sodium chloride, chloride, dried dried milk,milk, skimskim

glycerol, propylene, glycerol, glycol, water, propylene,glycol, thethe and ethanoland water,ethanol like. like.

term Theterm

[0044]The

[0044] or or "lyophilized" "lyophilized" "freeze-dried" "freeze-dried" Includes includes a state a state ofsubstance of a that that a substance has has been been subjectedtotoaadrying subjected procedure dryingprocedure such such as lyophilization, as lyophilization, where where at least at least 90% 90% of of moisture moisture has has been been removed. removed.

Antagonists VEGFAntagonists VEGF

[0045] A A

[0045] VEGF VEGF antagonist antagonist is a compound capable capable is a compound of blocking of blocking or inhibiting or inhibiting the biological the biological action action of vascular of endothelialgrowth vascular endothelial factor growthfactor (VEGF), and and (VEGF), includes includes fusion fusion proteins proteins capable capable of trapping of trapping

VEGF. VEGF. a preferred In aInpreferred embodiment, embodiment, the VEGF VEGF antagonist theantagonist is theprotein is the fusion SEQ ID of fusionofprotein SEQ NO:2 ID NO:2 or 4; or 4; more more preferably, SEQ preferably,SEQ ID NO:4. ID NO:4. In specific In specific embodiments, the VEGFthe embodiments, VEGF antagonist antagonist is is mammalian expressed in ina amammalian expressed line line cell cell suchsuch CHOacell as a as be may mayand CHOandcell be modified modified post- post translationally. In translationally. In aa specific embodiment, thethe specific embodiment, fusion fusion protein protein comprises aminoamino comprises acids 27-457 acids 27-457 of of SEQIDID SEQ and and NO:4 NO:4 is glycosylated is glycosylated at Asn Asn residues at residues 62, 62, 94, 94,222 149, 149, 308.and and222 308. Preferably, Preferably, the the VEGF VEGF antagonist a dimer is aIsdimer antagonist composed of two of composed fusion proteinsproteins two fusion of SEQ ID SEQ of NO:4. ID NO:4. TheVEGF

[0046] The

[0046] VEGF antagonist the methods of theofmethods antagonist and formulations and formulations of the Invention of the Invention can be can be prepared prepared by any by method suitablemethod anysuitable known known in the the art, in art, or that or that comes comes be known. to betoknown. The The VEGF VEGF antagonist antagonist is is preferably substantiallyfree preferably substantially of protein free of contaminants protein contaminants at at thethe time used it itisisused time to to prepare thethe prepare acceptable pharmaceuticallyacceptable pharmaceutically formulation. formulation. By "substantially free free By "substantially of protein of protein contaminants" contaminants" is is preferably,that meant,preferably, meant, ofof least 9090% % thatatatleast the of of weight theweight protein of of protein the the VEGF-specific VEGF-specific fusion fusion protein protein

antagonist used preparationused antagonist preparation forfor making making a formulation a formulation is VEGF is VEGF proteinprotein fusion fusion antagonist protein,protein, antagonist preferablyatatleast morepreferably more 95%, least95%, most most preferably preferably at least 99%.99%. at least The fusion proteinprotein The fusion is preferably is preferably

substantially of aggregates. free of substantially free "Substantially aggregates. "Substantially free of of free aggregates" aggregates" means means that that at at least least 90% 90% of of

2007/149334 WO2007/149334 WO PCT/US2007/014085 PCT/US2007/014085 16 May 2012

the weightofoffusion the weight proteinisis not fusionprotein presentininananaggregate not present aggregate at the at the time time thethe fusion fusion protein protein is used is used

to prepare to thepharmaceutically prepare the pharmaceutically effective effective formulation. formulation. Unless Unless stated stated otherwise, otherwise, the phosphates the phosphates

employed areare employed sodium sodium phosphates phosphates and a desired and a desired buffering buffering pH is achieved by mixing by pH is achieved mixing appropriate appropriate

of mono- amountsof amounts mono-and anddibasic sodiumphosphate. dibasic sodium phosphate. 2012202859

Stable Liquid Stable Liquid Ophthalmic OphthalmicFormulations Formulations

[0047] InInone

[0047] one aspect, aspect, thethe invention invention provides provides a stable a stable pharmaceutically pharmaceutically acceptable acceptable formulation formulation

comprisinga aVEGF comprising VEGF antagonist, antagonist, wherein wherein the formulation the formulation is a liquid is a liquid formulation formulation suitable suitable for for ophthalmicuse. ophthalmic use.Preferably, Preferably, thethe liquid liquid formulation formulation comprises comprises a pharmaceutically a pharmaceutically effective effective

amountof amount of the the VEGF antagonist. The VEGF antagonist. Theformulation formulation can can also also comprise one or comprise one or more more pharmaceuticallyacceptable pharmaceutically acceptable carriers, carriers, buffers, buffers, tonicity tonicity agents, agents, stabilizers,and/or stabilizers, and/or exciplents. excipients. An An exampleofofa apharmaceutically example pharmaceutically acceptable acceptable liquidliquid formulation formulation comprises comprises a VEGF antagonist a VEGF antagonist in a in a pharmaceutically pharmaceutically effectiveamount, effective amount, a buffer, a buffer, an organic an organic co-solvent co-solvent such such as as polysorbate, polysorbate, a a tonicity agent tonicity suchasasNaCI, agent such NaCl,and and optionally, optionally, a stabilizer a stabilizer such such as sucrose as sucrose or trehalose. or trehalose.

[0048] Stabilityisis determined

[0048] Stability determined in in a a number number of ways of ways at specified at specified time points, time points, Including including

determinationofofpH, determination pH,visual visualinspection inspection of of color color and and appearance, appearance, determination determination of protein of total total protein content bybymethods content methods known known in art, in the the art, e.g., e.g., UV spectroscopy, UV spectroscopy, and purity and purity is determined is determined by, for by, for example, SDS-PAGE, example, SDS-PAGE, size-exclusion size-exclusion HPLC, bloassay HPLC, bloassay determination determination of activity,ofisoelectric activity, isoelectric focusing, and focusing, andisoaspartate isoaspartate quantification. quantification. In In oneone example example of a bioassay of a bioassay useful useful for determining for determining

VEGFantagonist VEGF antagonistactivity, activity, a aBAF/3 BAF/3VEGFR1/EPOR cellline VEGFR1/EPOR cell lineis is used used to to determine determine VEGF165 VEGF165 binding bythe binding by theVEGF VEGF antagonist antagonist of the of the invention. invention.

[0049] Liquid

[0049] Liquidformulations formulationscancan be stored be stored in oxygen-deprived in an an oxygen-deprive.d environment. environment. Oxygen-deprived Oxygen-deprived

environments environments cancan be generated be generated by storing by storing the formulations the formulations under under an inertan inert gas suchgas as,such for as, for example,nitrogen nitrogen or or argon. Liquid 0 example, argon. Liquid formulations formulations are preferably are preferably stored stored at about at about 5°C. 5 C.

OphthalmicLyophilized Ophthalmic LyophilizedFormulations Formulations

[0050] In one

[0050] In oneaspect aspectof of thethe invention, invention, an an ophthalmically ophthalmically acceptable acceptable formulation formulation comprising comprising a a VEGF VEGF antagonist antagonist is provided, is provided, wherein wherein the formulation the formulation is a lyophilizable is a lyophilizable formulation. formulation.

Lyophilizableformulations Lyophilizable formulationscancan be be reconstituted reconstituted intointo solutions, solutions, suspensions, suspensions, emulsions, emulsions, or any or any other suitable other suitable form formfor for administration administrationororuse. use.Lyophilizable Lyophilizable formulations formulations are typically are typically first first

preparedasasliquids, prepared liquids,then thenfrozen frozenandand lyophilized. lyophilized. The The total total liquid liquid volume volume before before lyophilization lyophilization

canbebeless, can less,equal equalto, to,oror more more than, than, the the finalreconstituted final reconstituted volume volume of the of the lyophilized lyophilized formulation. formulation.

Thelyophilization The lyophilizationprocess processisiswell wellknown known to those to those of ordinary of ordinary skill skill in In the the art,and art, andtypically typically Includessublimation includes sublimationofofwater water from from a frozen a frozen formulation formulation under under controlled controlled conditions. conditions.

[0051] Lyophilized

[0051] Lyophilized formulations formulations can can be stored be stored at a wide at a wide range range of temperatures. of temperatures. Lyophilized Lyophilized

formulationsmay formulations maybe be stored stored below below 25°C,25*C, for example, for example, refrigerated refrigerated at 2-8*C. at 2-8°C, or at or at room room temperature temperature (e.g.,approximately (e.g., approximately 25*C). 25°C). Preferably, Preferably, lyophilized lyophilized formulations formulations are stored are stored below below

2007/149334 WO2007/149334 wo PCT/US2007/014085 PCT/US2007/014085 May 2012

about 25°C, about 250C,more more preferably, preferably, at about at about 4-20*C; 4-20°C; belowbelow about about 4*C; about 4°C; below aboutabout- below-20°C; -20OC; about 2012202859 16

40*C; about 40°C; about-70°C, or or -70*C, about about -80*C. -80°C. Stability Stability of the of the lyophilized Iyophilized formulation formulation may may be be determined determined in in a number a number ofof ways ways known known to art, to the the art, for for example, example, by visual by visual appearance appearance of the of the cake cakebyand/or and/or by moisturecontent. moisture content.

[0052] Lyophilized

[0052] Lyophilized formulations formulations are are typically typically reconstituted reconstituted for for use use by addition by addition of anofaqueous an aqueous solution to solution to dissolve the lyophilized dissolve the lyophilized formulation. formulation. A Awide wide variety variety of of aqueous aqueous solutions solutions canused can be be used to reconstitute to a lyophilized reconstitute a lyophilized formulation. formulation. Preferably, lyophilizedformulations Preferably,lyophilized formulations areare reconstituted reconstituted

using water. using water.Lyophilized Lyophilized formulations formulations are are preferably preferably reconstituted reconstituted with with a solution a solution consisting consisting

essentially of essentially of water (e.g., USP water (e.g., WFI, USP WFI, or or water water forfor injection)ororbacteriostatic injection) bacteriostaticwater water (e.g.,USP (e.g., USP WFI WFI

with 0.9% with 0.9%benzyl benzyl alcohol).However, alcohol). However, solutions solutions comprising comprising buffersbuffers and/or and/or excipients excipients and/or and/or one one or or more pharmaceutically more pharmaceutically acceptable acceptable carries carries can be can also also be used. used.

(0053]Freeze-dried

[0053] Freeze-dried or lyophilized or lyophilized formulations formulations are typically are typically prepared prepared from from liquids, liquids, that that is, from is, from

solutions, suspensions, solutions, emulsions, suspensions, emulsions, and and the the like. like. Thus, Thus, the liquid the liquid thatthat is undergo is to to undergo freeze-drying freeze-drying

or lyophilization or lyophilization preferably comprisesallallcomponents preferably comprises components desired desired in a in a final final reconstituted reconstituted liquid liquid

formulation. AsAsa aresult, formulation. result,when when reconstituted, reconstituted, thethe freeze-dried freeze-dried or lyophilized or lyophilized formulation formulation will will

renderaadesired render desiredliquid liquidformulation formulationupon upon reconstitution. reconstitution.

EXAMPLES EXAMPLES

[0054] Before

[0054] Before thethe present present methods methods are described, are described, it isbetounderstood it is to be understood thatinvention that this this invention is is not limited not limited to to particular particular methods, andexperimental methods, and experimental conditions conditions described, described, as methods as such such methods and and conditions may conditions mayvary. vary.It Itisisalso alsototobebeunderstood understood that that thethe terminology terminology used used hereinherein is foristhe for the purposeofofdescribing purpose describing particularembodiments particular embodiments only, only, and and is notisintended not Intended to be limiting, to be limiting, sincesince the the

scopeofofthe scope thepresent presentinvention invention willbebelimited will limitedonly onlytotothe theappended appended claims. claims.

[0055]Unless

[0055] Unlessdefined defined otherwise, otherwise, all all technical technical and and scientific scientific terms terms usedused herein herein have have the the same same meaningasas meaning commonly commonly understood understood by ordinary by one of one of ordinary skill in skill in the the art to art to which which this invention this invention

belongs.Although belongs. Althoughanyany methods methods and materials and materials similarsimilar or equivalent or equivalent to thosetodescribed those described herein herein can beused can be usedininthe thepractice practice oror testingofofthe testing thepresent present invention, invention, thethe preferred preferred methods methods and and

materials arenow materials are nowdescribed. described.

Example1.1.Stability Example Stability of of 50 50 mg/ml VEGF mg/ml VEGF Trap Trap Liquid Liquid Formulation Formulation Stored Stored at 5*C at 5°C in 3inml 3 ml GlassVials. Glass Vials.

[0056] An

[0056] An ophthalmic ophthalmic liquid liquid formulation formulationcontaining containing50 50 mg/ml VEGF mg/ml VEGF Trap Trap (SEQ ID NO:4), (SEQ ID NO:4), 10 10 mMphosphate, mM phosphate,5050mMmM NaCl, NaCl, 0.1% 0.1% polysorbate polysorbate 20,20, 5% 5% sucrose, sucrose, andand pH pH 6.25, 6.25, waswas stored stored atat5 5 °C in 33 ml *C in ml glass glass vials vials and andsamples samples tested tested at 3, at 3, 6, 6, 9, 9, 12,18 18 12, andand 24 months. 24 months. Stability Stability was was determined by determined by SE-HPLC. SE-HPLC.TheThe resultsare results areshown shownininTable Table1.1. Turbidity Turbidity was measuredatatOD405 was measured OD 4 05 nm; andpercent nm; and percent recovered recovered protein protein and purity and purity by size by size exclusion exclusion HPLC. HPLC.

8

WO2007/149334 wo 2007/149334 PCTIUS2007/014085 PCT/US2007/014085 2012202859 16 May 2012

Table 1. Table 1. Stability Stability of of 50 50 mg/ml VEGFTrap mg/ml VEGF Trap Protein Protein (VGFT-SSO65) (VGFT-SS065)

Visual Visual Turbidity Turbidity % VEGF % Trap VEGF Trap %% VEGF Trap VEGFTrap Months Months pH Appearance Appearance (0D (OD4 nm) 405nm) PH Recovered Recovered Native Configuration Native Configuration 0 0 Pass Pass 0.00 0.00 6.2 6.2 100 100 98.8 98.8

3 3 Pass Pass 0.00 0.00 6.2 6.2 101 101 98.7 98.7

6 6 Pass Pass 0.01 0.01 6.3 6.3 100 100 98.3 98.3

9 9 Pass Pass 0.01 0.01 6.3 6.3 101 101 98.3 98.3 12 12 Pass Pass 0.01 0.01 6.3 6.3 104 104 98.4 98.4

18 18 Pass Pass 0.01 0.01 6.3 6.3 96 96 98.1 98.1

24 24 Pass Pass 0.01 0.01 6.3 6.3 105 105 98.1 98.1

Example2.2.Stability Example Stabilityofof50 50mg/ml mg/mlVEGF VEGF TrapTrap Liquid Liquid Formulation Formulation StoredStored in5"C at 5°Cat In 3 ml 3 mi GlassVials. Glass Vials.

[0057] AAliquid

[0057] liquid formulation formulation containing containing50 50mg/mI mg/ml VEGF Trap(SEQ VEGF Trap (SEQID ID NO:4), NO:4), 10 10 mM mM phosphate, phosphate,

50 mM 50 mMNaCI, NaCl,3%3% polyethylene polyethylene glycol3350, glycol 3350,5%5% sucrose, sucrose, andand pH pH 6.25, 6.25, waswas stored stored at at 5 *C 5 °C in in3 3nil nil glassvials glass vials and andsamples samples tested tested at 3,at 6,3,9,6,12, 9, 18 12,and 1824and 24 months. months. Stability Stability results results are shownare in shown in Table Table 2.2.Turbidity, Turbidity,percent percent recovered recovered protein protein and purity and purity was determined was determined as above. as described described above.

Table 2. Table 2. Stability Stability of of 50 50 mg/mI VEGFTrap mg/ml VEGF Trap Protein Protein (VGFT-SS065) (VGFT-SS065)

Months Visual Visual Turbidity pH % VEGF % Trap VEGFTrap % % VEGF Trap VEGFTrap Months Turbidity pH Appearance Appearance Recovered Recovered Native Configuration Native Configuration 0 0 Pass Pass 0.00 0.00 6.2 6.2 100 100 98.9 98.9 3 3 Pass Pass 0.00 0.00 6.1 6.1 104 104 98.5 98.5 6 6 Pass Pass 0.01 0.01 6.3 6.3 99 99 98.3 98.3 9 9 Pass Pass 0.00 0.00 6.3 6.3 102 102 97.6 97.6 12 12 Pass Pass 0.01 0.01 6.3 6.3 103 103 98.0 98.0 18 18 Pass Pass 0.00 0.00 6.3 6.3 113 113 97.7 97.7 24 24 Pass Pass 0.00 0.00 6.2 6.2 106 106 97.6 97.6

Example3.3.Stability Example Stability of of 40 mg/mIVEGF 40 mg/ml VEGF Trap Trap Uquid Liquid Formulation Formulation Stored Stored at 5°Catin 5"C in 3 ml 3 ml GlassVials. Glass Vials.

[0058] AAliquid

[00581 liquid formulation formulation containing containing40 40mg/mI mg/ml VEGF Trap(SEQ VEGF Trap (SEQID ID NO:4), NO:4), 10 10 mM mM phosphate, phosphate,

40 mM 40 mMNaCI, NaC,0.03% 0.03% polysorbate polysorbate 20,20, 5% 5% sucrose, sucrose, andand pH 6.3, pH 6.3, waswas stored stored at °C at 5 5 *C in in 3 3 mlmlglass glass vials and vials andsamples samples tested tested at 0.5, at 0.5, 1,3,2, and 1, 2, 3, and 4 months. 4 months. Stability Stability resultsresults areinshown are shown in Table 3. Table 3. Turbidity, percentrecovered Turbidity, percent recovered protein protein and purity and purity was determined was determined as described as described above. above.

2007/149334 WO2007/149334 WO PCT/US2007/014085 PCT/US2007/014085 2012202859 16 May 2012

Table 3. Stability Table 3. Stability of of40 40mg/ml mg/ml VEGF TrapProtein VEGF Trap Protein(VGFT-SS207) (VGFT-SS207)

Months Visual Visual Turbidity pH % Trap VEGF Trap % VEGF % Trap VEGFTrap % VEGF Months Turbidity pH Appearance Appearance Recovered Recovered Native Configuration Native Configuration

0 0 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.5 99.5

0.5 0.5 Pass Pass 0.00 0.00 6.3 6.3 99 99 99.4 99.4

1 1 Pass Pass 0.00 0.00 6.2 6.2 98 98 99.5 99.5 2 2 Pass Pass 0.00 0.00 6.2 6.2 95 95 99.2 99.2 3 3 Pass Pass 0.01 0.01 6.4 6.4

44 Pass Pass 0.01 0.01 6.3 6.3

Example Example 4.4. Stability Stability of of 40 40 mg/ml VEGFTrap mg/ml VEGF TrapLiquid LiquidFormulation Formulation Stored Stored at at 5°C5*C In in Pre-filled Pre-filled

Glass Syringe. Glass Syringe.

[0059] AA liquid

[0059] liquid formulation formulationcontaining 40 40 containing mg/ml VEGF mg/ml VEGF trap trap(SEQ (SEQ ID ID NO:4), NO:4), 10 10 mM phosphate, mM phosphate, 40 mM 40 mM NaCl, NaCI, 0.03% 0.03% polysorbate polysorbate 20, 5% 20, 5% sucrose, sucrose, and was and pH 6.3, pH stored 6.3, was at 5stored °C in at 5 *C in I ml 1 ml

prefilled luer prefilled luer glass glass syringe syringe with with 4D23/50 FluroTec 4023/50 FluroTec coated coated plunger plunger and samples and samples tested tested at 0.5, at 1,0.5, 1, 2, 3, 2, 3, and and 44 months. months.Stability Stabilityresults resultsare areshown shown in Table in Table 4. Turbidity, 4. Turbidity, percent percent recovered recovered protein protein

and purity and purity was wasdetermined determined as described as described above. above.

Table 4. Table 4. Stability Stability of of40 40mg/ml mg/ml VEGF TrapProtein VEGF Trap Protein(VGFT-SS207) (VGFT-SS207)

Months Visual Visual Turbidity pH % % VEGF Trap VEGF Trap % Trap VEGFTrap % VEGF Months Turbidity pH Appearance Appearance Recovered Recovered Native Configuration Native Configuration 0 0 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.4 99.4 0.5 0.5 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.3 99.3 I1 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.4 99.4 2 2 Pass Pass 0.00 0.00 6.3 6.3 97 97 99.1 99.1

3 3 Pass Pass 0.01 0.01 6.4 6.4

4 4 Pass Pass 0.01 0.01 6.3 6.3

Example Stability of Example 5.S.Stability of 40 40 mg/mI VEGFTrap mg/ml VEGF Trap Liquid Liquid Formulation Formulation Stored Stored at 60C at 5°C in 3Inml 3 ml GlassVials. Glass Vials.

[0060] AA liquid

[0060] liquid formulation formulationcontaining 4040 containing mg/ml VEGF mg/ml VEGF trap trap(SEQ (SEQ ID ID NO:4), NO:4), 10 10 mM phosphate, mM phosphate,

135 mM 135 mM NaCl, NaCI, 0.03% 0.03% polysorbate polysorbate 20, and20, pH and 6.3, pH was6.3, wasatstored stored at35ml 5 °C in in 3 ml *Cglass glass vials and vials and

samplestested samples testedat at0.5, 0.5,1,1,2,2,3,3,and and4 4months. months. Stability Stability results results areare shown shown in Table in Table 5. Turbidity, 5. Turbidity,

percentrecovered percent recovered protein protein andand purity purity waswas determined determined as described as described above. above.

10

WO2007/149334 wo 2007/149334 PCT[US2007/014085 PCT/US2007/014085 2012202859 16 May 2012

Table 5. Stability Table 5. Stability of of40 40mg/ml mg/ml VEGF TrapProtein VEGF Trap Protein(VGFT-SS203) (VGFT-SS203)

Visual Visual Trap VEGF Trap % VEGF % % VEGF Trap VEGFTrap Months Months Appearance Turbidity Turbidity pH % Recovered Native Native Appearance pH Recovered Configuration Configuration 0 0 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.3 99.3 0.5 0.5 Pass Pass 0.00 0.00 6.2 6.2 87 87 -99.2 99.2 1 1 Pass Pass 0.00 0.00 6.2 6.2 88 88 99.1 99.1

2 2 Pass Pass 0.00 0.00 6.3 6.3 103 103 99.2 99.2 33 Pass Pass 0.00 0.00 6.3 6.3 88 88 99.0 99.0 4 4 Pass Pass 0.00 0.00 6.2 6.2 85 85 98.9 98.9 5 5 Pass Pass 0.00 0.00 6.3 6.3 84 84 99.0 99.0

Example6.6.Stability Example Stability of of 40 40 mg/ml VEGFTrap mg/ml VEGF Trap Liquid Liquid Formulation Formulation Stored Stored at 5*C at 5°C in 1inml I ml Pre Pre-

filled filled Glass Syringe. Glass Syringe.

[00611 AAliquid

[0061] liquid formulation formulationcontaining containing40 40 mg/ml VEGF mg/ml VEGF trap trap(SEQ (SEQ ID ID NO:4), NO:4),10 10 mM phosphate, mM phosphate, 135 mM 135 mM NaCl, NaCi, 0.03% 0.03% polysorbate polysorbate 20, 20, and pHand 6.3,pH was6.3, wasatstored stored at 15ml 5 °C in in 1 ml prefilled *Cprefilled glass glass luer luer syringe with 4023/50 syringe with 4023/50 FluroTec Fluro coated Tec coated plunger plunger and samples and samples tested tested at 0.5, at 1,0.5, 1, 4, 2, 3, 2, 3, and4,5 and 5

months. Stabilityresults months. Stability resultsare areshown shown in Table in Table 6. Turbidity, 6. Turbidity, percent percent recovered recovered protein protein and purity and purity

was determined was determinedas as described described above. above.

Table 6. Table 6. Stability Stability of of40 40mg/mI mg/ml VEGF TrapProtein VEGF Trap Protein(VGFT-SS203) (VGFT-SS203)

Months Months Visual Visual Turbidity Turbidity pH % Trap VEOF Trap % VEGF % VEGF % Trap VEGF Trap Appearance Appearance pH Recovered Recovered Native Configuration Native Configuration 0 0 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.2 99.2 0.5 0.5 Pass Pass 0.01 0.01 6.3 6.3 101 101 99.2 99.2 1 1 Pass Pass 0.00 0.00 6.3 6.3 101 101 99.2 99.2 2 2 Pass Pass 0.00 0.00 6.3 6.3 - -

3 3 Pass Pass 0.01 0.01 6.3 6.3 102 102 99.1 99.1

4 4 Pass Pass 0.01 0.01 6.3 6.3 103 103 98.8 98.8 5 5 Pass Pass 0.00 0.00 6.3 6.3 99 99 98.9 98.9

Example7.7.Stability Example Stability of of Lyophilized 20 mg/ml Lyophilized 20 mg/mlVEGF VEGF Trap Trap Formulation Formulation Stored Stored at 5*C at 5°C in 3In 3 ml Glass Vials ml Glass Vials and and Reconstituted Reconstitutedtoto40 40mg/ml. mg/ml.

[0062] 0.8mlmlofofa aliquid

[0062] 0.8 liquidformulation formulationcontaining containing 20 20 mg/ml mg/ml VEGF VEGF trapID(SEQ trap (SEQ NO:4),ID5 NO:4), mM 5 mM phosphate,2020 phosphate, mM mM NaCl, NaCl, 0.015% polysorbate 20, 2.5% sucrose, and pH 6.3, were lyophilized in 0.015% polysorbate 20, 2.5% sucrose, and pH 6.3, were lyophilized in

3 ml 3 ml glass glass vials. vials. Samples Samples were were stored stored at 5°C 50C tested at and and tested at 1, at and1,2 and 2 months. months. VEGF VEGF trap was trap was reconstituted to aa final reconstituted to final concentration of40 concentration of 40mg/ml mg/ml VEGF VEGF Trap Trap (final(final volume volume of 0.4of 0.4 Stability ml). ml). Stability

11

2012202859 16 2012

results are shown results are shownInInTable Table 7 (t= =time 7 (t timein inmonths; months; = visual * = *visual appearance; appearance; **= reconstitution ** = reconstitution time). time).

Turbidity, Turbidity, percent recovered percent recovered protein protein andand purity purity waswas determined determined as described as described above. above. May

Table 7. Table 7. Stability Stability of ofLyophilized Lyophilized 20 mg/ml VEGF 20 mg/ml Trap VEGF Trap Protein Protein (VGFT-SS216) (VGFT-SS216)

Recon. Vis. App.* Vis. Recon. Vis. App.* % Trap % VEGF VEGF % VEOF Trap tt p.. Time" Reconst'd Reconst'd Turbidity Turbidity pH pH Trap % VEGF Native Trap Confg. App.* Time (min) (min) Liquid Liquid Recovered Native Config. Recovered 0 0 Pass Pass 0.6 0.6 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.5 99.5 I 1 Pass Pass 0.6 0.6 Pass Pass 0,01 0.01 6.3 6.3 106 106 99.4 99.4 2 2 Pass Pass 0.4 0.4 Pass Pass 0.01 0.01 6.2 6.2 103 103 99.3 99.3

Example Example 8.8.Stability Stability of of Lyophilized 20 mg/ml Lyophilized 20 mg/mIVEGF VEGF Trap Trap Formulation Formulation Stored Stored at 5*C at 5°C In 3In 3

Glass Vials. mi Glass ml Vials.

[00631 0.80.8mlml

[0063] of of a a liquidformulation liquid formulation containing containing 20 20 mg/ml mg/ml VEGF VEGF trapID(SEQ trap (SEQ NO:4),ID 5 NO:4), mM 6 mM phosphate, phosphate, 67.5 mM mM 67.6 NaCl,NaCl, 0.015% 0.015% polysorbate polysorbate 20.6.3, 20, and pH andwere pH 6.3. were lyophilized lyophilized in 3 ml glass In 3 ml glass vials. Sampleswere were stored 9C tested at 5and and tested at 1, at 2, 1, vials. Samples stored at 5°C and2,3and 3 months. months. VEGF VEGF trap was trap was reconstituted to aa final reconstituted to final concentration concentration of of4040mg/ml mg/mI VEGF VEGF trap trap (final (final volume volume of 0.4ofml). 0.4 Stability ml). Stability results are results showninInTable are shown Table 8 (t= =time 8 (t timeininmonths; months; = visual * =* visual appearance: appearance; = reconstitution ** - "reconstitution time). time).

Table 8. Stability Table 8. Stability of ofLyophilized Lyophilized 20 20 mg/mI mg/ml VEGF TrapProtein VEGF Trap Protein(VGFT-SS216) (VGFT-SS216) Recon. Recon. Vis. Vis. App. App. % VEGF VEOF % VEGF Trap t t Vis. A'., Time" Reconst'd Turbidity Turbidity pH % Trap % VEGF % VEGFia Trap Time** Reconst'd pH Trap App." (min) Liquid Recovered Native Config. Native Config. (min) Liquid Recovered O 0 Pass Pass 0.7 0.7 Pass Pass 0.00 0.00 6.3 6.3 100 100 99.0 99.0 1 1 Pass Pass 0.7 0.7 Pass Pass 0.01 0.01 6.2 6.2 105 105 98.9 98.9 2 2 Pass Pass 0.4 0.4 Pass Pass 0.01 0.01 6.2 6.2 103 103 98.9 98.9

Throughoutthe Throughout thedescription andclaims description and claimsofofthe specification, the the specification, theword word "comprise" and "comprise" and

variations of the word, such as "comprising" and "comprises", is not intended to exclude variations of the word, such as "comprising" and "comprises", is not intended to exclude

other additives, components, integers or steps. other additives, components, integers or steps.

A reference A reference herein herein to atopatent a patent document document or otherormatter otherwhich matter is which given asisprior givenartasisprior art is not to not to be be taken taken as as an an admission that that admission that that document ormatter document or matterwas known wasknown or or thatthe that the information information it itcontains containswas was part partofofthe common the general knowledge common general knowledgeas as at at thepriority the priority date date

of any of ofthe any of theclaims. claims.

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2293065.1 2293065.1

Claims (16)

Mar 2025 THE CLAIMS THE CLAIMSDEFINING DEFININGTHE THEINVENTION INVENTION ARE ARE ASAS FOLLOWS: FOLLOWS:

1. 1. An ophthalmic An ophthalmicformulation formulationofofa avascular vascularendothelial endothelialgrowth growth factor(VEGF) factor (VEGF) antagonist, antagonist, comprising comprising

2012202859 28

(a) (a) about 1-100mg/ml about 1-100 mg/mlofofaaVEGF VEGF antagonist antagonist comprising comprising thethe amino amino acidacid

sequence of SEQ sequence of ID NO:4; SEQ ID NO:4; (b) (b) 0.03% ofpolysorbate; 0.03% of polysorbate; 2012202859

(c) (c) 40-40- 135135 mM mM of sodium of sodium chloride; chloride; (d) (d) 10 10 mM mM of sodium of sodium phosphate phosphate buffer;and buffer; and (e) (e) 5% 5% of sucrose, of sucrose, pHpH about6.2-6.3. about 6.2-6.3.

2. 2. An ophthalmic An ophthalmicformulation formulationaccording accordingtotoclaim claim1,1,wherein whereinthe theformulation formulationisis suitable for intravitreal suitable for intravitreal administration. administration.

3. 3. An ophthalmic An ophthalmicformulation formulationaccording accordingtotoclaim claim1,1,wherein whereinthe theformulation formulationisis used forintravitreal used for intravitrealadministration. administration.

4. 4. An ophthalmic An ophthalmicformulation formulationaccording accordingtotoany anyone oneofofclaims claims11 to to 3, 3, comprising comprising

about 10-80mg/ml about 10-80 mg/mlofofthe theVEGF VEGF antagonist. antagonist.

5. 5. An ophthalmic formulation An ophthalmic formulation according according totoclaim claim4,4,comprising comprisingVEGF VEGF antagonist at a antagonist at a concentration selectedfrom concentration selected fromthe thegroup groupconsisting consistingofof1010mg/ml, mg/ml,20 20

mg/ml, 40mg/ml, mg/ml, 40 mg/ml,and and8080mg/ml. mg/ml.

6. 6. An ophthalmicformulation An ophthalmic formulationaccording accordingtotoany anyone oneofofclaims claims11 to to 5, 5, comprising comprising

10-80 mg/mlVEGF 10-80 mg/ml VEGF antagonist antagonist and and 135 135 mM sodium mM sodium chloride, chloride, pH6.2-6.3. pH about about 6.2-6.3.

7. 7. An ophthalmic An ophthalmicformulation formulationaccording accordingtotoany anyone oneofofclaims claims11 to to 6, 6, comprising comprising

about 40 mg/ml about 40 mg/mlofofthe theVEGF VEGF antagonist. antagonist.

8. 8. An ophthalmic An ophthalmicformulation formulationaccording accordingto to any any oneone of of claims claims 1 to 1 to 7, 7, wherein wherein

the formulation the formulation is is provided provided in ainvial. a vial.

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9. 9. A lyophilizable A lyophilizable formulation formulation of of aa vascular vascular endothelial endothelial growth growth factor factor (VEGF) (VEGF) 2012202859 28 Mar 2025

Mar 2025

antagonist, antagonist, comprising: comprising:

(a) (a) about about 20 mg/ml 20 mg/ml of VEGF of the the VEGF antagonist antagonist comprising comprising theacid the amino amino acid sequence of SEQ sequence of ID NO:4; SEQ ID NO:4; (b) (b) about about 5 - 5 10- mM 10 of mMsodium of sodium phosphate phosphate buffer,buffer, pH 5.8-7.0; pH about about 5.8-7.0; 2012202859 28

(c) about (c) about 0.015 0.015 -0.03% -0.03% polysorbate polysorbate and and aboutabout 0.1% 0.1% polyethylene polyethylene glycol (PEG); glycol (PEG);

(d) about (d) about 2.5% 2.5% sucrose, sucrose, pHpH about6.2-6.3. about 6.2-6.3. 2012202859

10. 10. A lyophilizable A lyophilizable formulation formulation according according to claim to claim 9, comprising 9, comprising aboutabout 5 mM 5 mM sodium phosphatebuffer, sodium phosphate buffer, about about 0.015% 0.015%polysorbate polysorbateand and furthercomprising further comprising sodium chlorideat sodium chloride at about about20 20mM, mM,pHpH about about 6.2-6.3. 6.2-6.3.

11. 11. A lyophilizable A lyophilizable formulation formulation according according to claim to claim 9, comprising 9, comprising aboutabout 5 mM 5 mM sodium phosphatebuffer, sodium phosphate buffer, about about 0.015% 0.015%polysorbate polysorbateand and furthercomprising further comprising sodium chlorideat sodium chloride at about about67.5 67.5mM, mM,pHpH about about 6.2-6.3. 6.2-6.3.

12. 12. A lyophilizable A lyophilizable formulation formulation according according to any to any one one of claims of claims 9 to9 11, to 11, wherein wherein

the formulation the formulation can can be reconstituted be reconstituted suitable suitable for intravitreal for intravitreal administration. administration.

13. 13. A lyophilizable A lyophilizable formulation formulation according according to any to any one one of claims of claims 9 to9 12, to 12, wherein wherein

the formulation the formulation can bereconstituted can be reconstituted to to aa final final concentration concentration of of about about 40 40 mg/ml of mg/ml of

the VEGF the antagonist. VEGF antagonist.

14. 14. A pre-filled A pre-filled syringe syringe suitable suitable forfor intravitrealadministration intravitreal administrationcomprising comprisingthethe

formulationofofany formulation any oneone of claims of claims 1 to 1 7.to 7.

15. 15. An ophthalmic An ophthalmic formulation formulation according according to 1, to claim claim 1, substantially substantially as as herein herein described with reference described with referenceto to any any of of the the Examples and/orFigures. Examples and/or Figures.

16. 16. A lyophilizable A lyophilizable formulation formulation according according to claim to claim 9, substantially 9, substantially as herein as herein

described with reference described with referenceto to any any of of the the Examples and/orFigures. Examples and/or Figures.

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